The Genotypic Population Structure of Mycobacterium tuberculosis Complex from Moroccan Patients Reveals a Predominance of Euro-American Lineages

PubMed Central

Lahlou, Ouafae; Millet, Julie; Chaoui, Imane; Sabouni, Radia; Filali-Maltouf, Abdelkarim; Akrim, Mohammed; El Mzibri, Mohammed; Rastogi, Nalin; El Aouad, Rajae

2012-01-01

Background Tuberculosis (TB) remains a major health problem in Morocco. Characterization of circulating Mycobacterium tuberculosis genotypic lineages, important to understand the dynamic of the disease, was hereby addressed for the first time at a national level. Methodology/Principal Findings Spoligotyping was performed on a panel of 592 M. tuberculosis complex strains covering a 2-year period (2004–2006). It identified 129 patterns: 105 (n = 568 strains) corresponded to a SIT number in the SITVIT2 database, while 24 patterns were labeled as orphan. A total of 523 (88.3%) strains were clustered vs. 69 or 11.7% unclustered. Classification of strains within 3 large phylogenetical groups was as follows: group 1– ancestral/TbD1+/PGG1 (EAI, Bovis, Africanum), group 2– modern/TbD1−/PGG1 group (Beijing, CAS), group 3– evolutionary recent/TbD1−/PGG2/3 (Haarlem, X, S, T, LAM; alternatively designated as the Euro-American lineage). As opposed to group 3 strains (namely LAM, Haarlem, and T) that predominated (86.5% of all isolates), 6 strains belonged to group 2 (Beijing n = 5, CAS n = 1), and 3 strains (BOV_1 n = 2, BOV_4-CAPRAE) belonged to ancestral group 1 (EAI and AFRI lineage strains were absent). 12-loci MIRU-VNTR typing of the Casablanca subgroup (n = 114 strains) identified 71 patterns: 48 MITs and 23 orphan patterns; it allowed to reduce the clustering rate from 72.8% to 29.8% and the recent transmission rate from 64% to 20.2%. Conclusion The M. tuberculosis population structure in Morocco is highly homogeneous, and is characterized by the predominance of the Euro-American lineages, namely LAM, Haarlem, and T, which belong to the “evolutionary recent” TbD1−/PGG2/3 phylogenetic group. The combination of spoligotyping and MIRUs decreased the clustering rate significantly, and should now be systematically applied in larger studies. The methods used in this study appear well suited to monitor the M. tuberculosis population structure for an enhanced TB management program in Morocco. PMID:23077552

[Differentiation of species within the Mycobacterium tuberculosis complex by molecular techniques].

PubMed

Herrera-León, Laura; Pozuelo-Díaz, Rodolfo; Molina Moreno, Tamara; Valverde Cobacho, Azucena; Saiz Vega, Pilar; Jiménez Pajares, María Soledad

2009-11-01

The Mycobacterium tuberculosis complex includes the following species: Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium bovis-BCG, Mycobacterium microti, Mycobacterium caprae, Mycobacterium pinnipedii, and Mycobacterium canettii. These species cause tuberculosis in humans and animals. Identification of mycobacterial strains has classically been performed by phenotype study. Over the last years, laboratories have developed several molecular techniques to differentiate between these species. The aim of this study is to evaluate these methods and develop a simple, fast, identification scheme. We analyzed 251 strains randomly obtained from the strains studied in 2004, and 797 strains received by the Reference Laboratory between 2005 and 2007. Phenotype characterization of 4183 strains isolated during that period was done by studying the colony morphology, characteristics in culture, nitrate reduction, niacin accumulation, and growth in the presence of thiophen-2-carboxylic acid hydrazide 10 microg/mL and pyrazinamide 50 microg/mL. The molecular identification scheme designed was as follows: 1) gyrB PCR-RFLP with RsaI, TaqI or SacII and hsp65 RFLP/PCR with HhaI., and 2) multiplex-PCR to determine the presence/absence of the RD9 and RD1 regions. The results showed 100% agreement between phenotype study and the molecular scheme. This molecular identification scheme is a simple and fast method, with 100% sensitivity and specificity, that can be implemented in most clinical laboratories at a low cost.

A genomic library-based amplification approach (GL-PCR) for the mapping of multiple IS6110 insertion sites and strain differentiation of Mycobacterium tuberculosis.

PubMed

Namouchi, Amine; Mardassi, Helmi

2006-11-01

Evidence suggests that insertion of the IS6110 element is not without consequence to the biology of Mycobacterium tuberculosis complex strains. Thus, mapping of multiple IS6110 insertion sites in the genome of biomedically relevant clinical isolates would result in a better understanding of the role of this mobile element, particularly with regard to transmission, adaptability and virulence. In the present paper, we describe a versatile strategy, referred to as GL-PCR, that amplifies IS6110-flanking sequences based on the construction of a genomic library. M. tuberculosis chromosomal DNA is fully digested with HincII and then ligated into a plasmid vector between T7 and T3 promoter sequences. The ligation reaction product is transformed into Escherichia coli and selective PCR amplification targeting both 5' and 3' IS6110-flanking sequences are performed on the plasmid library DNA. For this purpose, four separate PCR reactions are performed, each combining an outward primer specific for one IS6110 end with either T7 or T3 primer. Determination of the nucleotide sequence of the PCR products generated from a single ligation reaction allowed mapping of 21 out of the 24 IS6110 copies of two 12 banded M. tuberculosis strains, yielding an overall sensitivity of 87,5%. Furthermore, by simply comparing the migration pattern of GL-PCR-generated products, the strategy proved to be as valuable as IS6110 RFLP for molecular typing of M. tuberculosis complex strains. Importantly, GL-PCR was able to discriminate between strains differing by a single IS6110 band.

Discriminatory Power and Reproducibility of Novel DNA Typing Methods for Mycobacterium tuberculosis Complex Strains

PubMed Central

Kremer, Kristin; Arnold, Catherine; Cataldi, Angel; Gutiérrez, M. Cristina; Haas, Walter H.; Panaiotov, Stefan; Skuce, Robin A.; Supply, Philip; van der Zanden, Adri G. M.; van Soolingen, Dick

2005-01-01

In recent years various novel DNA typing methods have been developed which are faster and easier to perform than the current internationally standardized IS6110 restriction fragment length polymorphism typing method. However, there has been no overview of the utility of these novel typing methods, and it is largely unknown how they compare to previously published methods. In this study, the discriminative power and reproducibility of nine recently described PCR-based typing methods for Mycobacterium tuberculosis were investigated using the strain collection of the interlaboratory study of Kremer et al. (J. Clin. Microbiol. 37:2607-2618, 1999). This strain collection contains 90 M. tuberculosis complex and 10 non-M. tuberculosis complex mycobacterial strains, as well as 31 duplicated DNA samples to assess reproducibility. The highest reproducibility was found with variable numbers of tandem repeat typing using mycobacterial interspersed repetitive units (MIRU VNTR) and fast ligation-mediated PCR (FLiP), followed by second-generation spoligotyping, ligation-mediated PCR (LM-PCR), VNTR typing using five repeat loci identified at the Queens University of Belfast (QUB VNTR), and the Amadio speciation PCR. Poor reproducibility was associated with fluorescent amplified fragment length polymorphism typing, which was performed in three different laboratories. The methods were ordered from highest discrimination to lowest by the Hunter-Gaston discriminative index as follows: QUB VNTR typing, MIRU VNTR typing, FLiP, LM-PCR, and spoligotyping. We conclude that both VNTR typing methods and FLiP typing are rapid, highly reliable, and discriminative epidemiological typing methods for M. tuberculosis and that VNTR typing is the epidemiological typing method of choice for the near future. PMID:16272496

High Throughput Phenotypic Analysis of Mycobacterium tuberculosis and Mycobacterium bovis Strains' Metabolism Using Biolog Phenotype Microarrays

PubMed Central

Khatri, Bhagwati; Fielder, Mark; Jones, Gareth; Newell, William; Abu-Oun, Manal; Wheeler, Paul R.

2013-01-01

Tuberculosis is a major human and animal disease of major importance worldwide. Genetically, the closely related strains within the Mycobacterium tuberculosis complex which cause disease are well-characterized but there is an urgent need better to understand their phenotypes. To search rapidly for metabolic differences, a working method using Biolog Phenotype MicroArray analysis was developed. Of 380 substrates surveyed, 71 permitted tetrazolium dye reduction, the readout over 7 days in the method. By looking for ≥5-fold differences in dye reduction, 12 substrates differentiated M. tuberculosis H37Rv and Mycobacterium bovis AF2122/97. H37Rv and a Beijing strain of M. tuberculosis could also be distinguished in this way, as could field strains of M. bovis; even pairs of strains within one spoligotype could be distinguished by 2 to 3 substrates. Cluster analysis gave three clear groups: H37Rv, Beijing, and all the M. bovis strains. The substrates used agreed well with prior knowledge, though an unexpected finding that AF2122/97 gave greater dye reduction than H37Rv with hexoses was investigated further, in culture flasks, revealing that hexoses and Tween 80 were synergistic for growth and used simultaneously rather than in a diauxic fashion. Potential new substrates for growth media were revealed, too, most promisingly N-acetyl glucosamine. Osmotic and pH arrays divided the mycobacteria into two groups with different salt tolerance, though in contrast to the substrate arrays the groups did not entirely correlate with taxonomic differences. More interestingly, these arrays suggested differences between the amines used by the M. tuberculosis complex and enteric bacteria in acid tolerance, with some hydrophobic amino acids being highly effective. In contrast, γ-aminobutyrate, used in the enteric bacteria, had no effect in the mycobacteria. This study proved principle that Phenotype MicroArrays can be used with slow-growing pathogenic mycobacteria and already has generated interesting data worthy of further investigation. PMID:23326347

Revisiting Host Preference in the Mycobacterium tuberculosis Complex: Experimental Infection Shows M. tuberculosis H37Rv to Be Avirulent in Cattle

PubMed Central

Whelan, Adam O.; Coad, Michael; Cockle, Paul J.; Hewinson, Glyn; Vordermeier, Martin; Gordon, Stephen V.

2010-01-01

Experiments in the late 19th century sought to define the host specificity of the causative agents of tuberculosis in mammals. Mycobacterium tuberculosis, the human tubercle bacillus, was independently shown by Smith, Koch, and von Behring to be avirulent in cattle. This finding was erroneously used by Koch to argue the converse, namely that Mycobacterium bovis, the agent of bovine tuberculosis, was avirulent for man, a view that was subsequently discredited. However, reports in the literature of M. tuberculosis isolation from cattle with tuberculoid lesions suggests that the virulence of M. tuberculosis for cattle needs to be readdressed. We used an experimental bovine infection model to test the virulence of well-characterized strains of M. tuberculosis and M. bovis in cattle, choosing the genome-sequenced strains M. tuberculosis H37Rv and M. bovis 2122/97. Cattle were infected with approximately 106 CFU of M. tuberculosis H37Rv or M. bovis 2122/97, and sacrificed 17 weeks post-infection. IFN-γ and tuberculin skin tests indicated that both M. bovis 2122 and M. tuberculosis H37Rv were equally infective and triggered strong cell-mediated immune responses, albeit with some indication of differential antigen-specific responses. Postmortem examination revealed that while M. bovis 2122/97–infected animals all showed clear pathology indicative of bovine tuberculosis, the M. tuberculosis–infected animals showed no pathology. Culturing of infected tissues revealed that M. tuberculosis was able to persist in the majority of animals, albeit at relatively low bacillary loads. In revisiting the early work on host preference across the M. tuberculosis complex, we have shown M. tuberculosis H37Rv is avirulent for cattle, and propose that the immune status of the animal, or genotype of the infecting bacillus, may have significant bearing on the virulence of a strain for cattle. This work will serve as a baseline for future studies into the genetic basis of host preference, and in particular the molecular basis of virulence in M. bovis. PMID:20049086

The Guinea-Bissau Family of Mycobacterium tuberculosis Complex Revisited

PubMed Central

Groenheit, Ramona; Ghebremichael, Solomon; Svensson, Jenny; Rabna, Paulo; Colombatti, Raffaella; Riccardi, Fabio; Couvin, David; Hill, Véronique; Rastogi, Nalin; Koivula, Tuija; Källenius, Gunilla

2011-01-01

The Guinea-Bissau family of strains is a unique group of the Mycobacterium tuberculosis complex that, although genotypically closely related, phenotypically demonstrates considerable heterogeneity. We have investigated 414 M. tuberculosis complex strains collected in Guinea-Bissau between 1989 and 2008 in order to further characterize the Guinea-Bissau family of strains. To determine the strain lineages present in the study sample, binary outcomes of spoligotyping were compared with spoligotypes existing in the international database SITVIT2. The major circulating M. tuberculosis clades ranked in the following order: AFRI (n = 195, 47.10%), Latin-American-Mediterranean (LAM) (n = 75, 18.12%), ill-defined T clade (n = 53, 12.8%), Haarlem (n = 37, 8.85%), East-African-Indian (EAI) (n = 25, 6.04%), Unknown (n = 12, 2.87%), Beijing (n = 7, 1.68%), X clade (n = 4, 0.96%), Manu (n = 4, 0.97%), CAS (n = 2, 0.48%). Two strains of the LAM clade isolated in 2007 belonged to the Cameroon family (SIT61). All AFRI isolates except one belonged to the Guinea-Bissau family, i.e. they have an AFRI_1 spoligotype pattern, they have a distinct RFLP pattern with low numbers of IS6110 insertions, and they lack the regions of difference RD7, RD8, RD9 and RD10, RD701 and RD702. This profile classifies the Guinea-Bissau family, irrespective of phenotypic biovar, as part of the M. africanum West African 2 lineage, or the AFRI_1 sublineage according to the spoligtyping nomenclature. Guinea-Bissau family strains display a variation of biochemical traits classically used to differentiate M. tuberculosis from M. bovis. Yet, the differential expression of these biochemical traits was not related to any genes so far investigated (narGHJI and pncA). Guinea-Bissau has the highest prevalence of M. africanum recorded in the African continent, and the Guinea-Bissau family shows a high phylogeographical specificity for Western Africa, with Guinea-Bissau being the epicenter. Trends over time however indicate that this family of strains is waning in most parts of Western Africa, including Guinea-Bissau (p = 0.048). PMID:21533101

First insights into circulating Mycobacterium tuberculosis complex lineages and drug resistance in Guinea

PubMed Central

Ejo, Mebrat; Gehre, Florian; Barry, Mamadou Dian; Sow, Oumou; Bah, Nene Mamata; Camara, Mory; Bah, Boubacar; Uwizeye, Cecile; Nduwamahoro, Elie; Fissette, Kristina; Rijk, Pim De; Merle, Corinne; Olliaro, Piero; Burgos, Marcos; Lienhardt, Christian; Rigouts, Leen; de Jong, Bouke C.

2015-01-01

In this study we assessed first-line anti-tuberculosis drug resistance and the genotypic distribution of Mycobacterium tuberculosis complex (MTBC) isolates that had been collected from consecutive new tuberculosis patients enrolled in two clinical trials conducted in Guinea between 2005 and 2010. Among the total 359 MTBC strains that were analyzed in this study, 22.8% were resistant to at least one of the first line anti-tuberculosis drugs, including 2.5% multidrug resistance and 17.5% isoniazid resistance, with or without other drugs. In addition, further characterization of isolates from a subset of the two trials (n = 184) revealed a total of 80 different spoligotype patterns, 29 “orphan” and 51 shared patterns. We identified the six major MTBC lineages of human relevance, with predominance of the Euro-American lineage. In total, 132 (71.7%) of the strains were genotypically clustered, and further analysis (using the DESTUS model) suggesting significantly faster spread of LAM10_CAM family (p = 0.00016). In conclusion, our findings provide a first insight into drug resistance and the population structure of the MTBC in Guinea, with relevance for public health scientists in tuberculosis control programs. PMID:26004194

First insights into circulating Mycobacterium tuberculosis complex lineages and drug resistance in Guinea.

PubMed

Ejo, Mebrat; Gehre, Florian; Barry, Mamadou Dian; Sow, Oumou; Bah, Nene Mamata; Camara, Mory; Bah, Boubacar; Uwizeye, Cecile; Nduwamahoro, Elie; Fissette, Kristina; De Rijk, Pim; Merle, Corinne; Olliaro, Piero; Burgos, Marcos; Lienhardt, Christian; Rigouts, Leen; de Jong, Bouke C

2015-07-01

In this study we assessed first-line anti-tuberculosis drug resistance and the genotypic distribution of Mycobacterium tuberculosis complex (MTBC) isolates that had been collected from consecutive new tuberculosis patients enrolled in two clinical trials conducted in Guinea between 2005 and 2010. Among the total 359 MTBC strains that were analyzed in this study, 22.8% were resistant to at least one of the first line anti-tuberculosis drugs, including 2.5% multidrug resistance and 17.5% isoniazid resistance, with or without other drugs. In addition, further characterization of isolates from a subset of the two trials (n = 184) revealed a total of 80 different spoligotype patterns, 29 "orphan" and 51 shared patterns. We identified the six major MTBC lineages of human relevance, with predominance of the Euro-American lineage. In total, 132 (71.7%) of the strains were genotypically clustered, and further analysis (using the DESTUS model) suggesting significantly faster spread of LAM10_CAM family (p = 0.00016). In conclusion, our findings provide a first insight into drug resistance and the population structure of the MTBC in Guinea, with relevance for public health scientists in tuberculosis control programs. Copyright © 2015. Published by Elsevier B.V.

Direct detection of Mycobacterium tuberculosis complex in bovine and bubaline tissues through nested-PCR.

PubMed

Araújo, Cristina P; Osório, Ana Luiza A R; Jorge, Klaudia S G; Ramos, Carlos A N; Souza Filho, Antonio F; Vidal, Carlos E S; Vargas, Agueda P C; Roxo, Eliana; Rocha, Adalgiza S; Suffys, Philip N; Fonseca, Antônio A; Silva, Marcio R; Barbosa Neto, José D; Cerqueira, Valíria D; Araújo, Flábio R

2014-01-01

Post-mortem bacterial culture and specific biochemical tests are currently performed to characterize the etiologic agent of bovine tuberculosis. Cultures take up to 90 days to develop. A diagnosis by molecular tests such as PCR can provide fast and reliable results while significantly decreasing the time of confirmation. In the present study, a nested-PCR system, targeting rv2807, with conventional PCR followed by real-time PCR, was developed to detect Mycobacterium tuberculosis complex (MTC) organisms directly from bovine and bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other Actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. Regarding the analytical sensitivity, DNA of the M. bovis AN5 strain was detected up to 1.5 pg by nested-PCR, whereas DNA of M. tuberculosis H37Rv strain was detected up to 6.1 pg. The nested-PCR system showed 100% analytical specificity for MTC when tested with DNA of reference strains of non-tuberculous mycobacteria and closely-related Actinomycetales. A clinical sensitivity level of 76.7% was detected with tissues samples positive for MTC by means of the culture and conventional PCR. A clinical specificity of 100% was detected with DNA from tissue samples of cattle with negative results in the comparative intradermal tuberculin test. These cattle exhibited no visible lesions and were negative in the culture for MTC. The use of the nested-PCR assay to detect M. tuberculosis complex in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.

Direct detection of Mycobacterium tuberculosis complex in bovine and bubaline tissues through nested-PCR

PubMed Central

Araújo, Cristina P.; Osório, Ana Luiza A.R.; Jorge, Klaudia S.G.; Ramos, Carlos A.N.; Souza Filho, Antonio F.; Vidal, Carlos E.S.; Vargas, Agueda P.C.; Roxo, Eliana; Rocha, Adalgiza S.; Suffys, Philip N.; Fonseca, Antônio A.; Silva, Marcio R.; Barbosa Neto, José D.; Cerqueira, Valíria D.; Araújo, Flábio R.

2014-01-01

Post-mortem bacterial culture and specific biochemical tests are currently performed to characterize the etiologic agent of bovine tuberculosis. Cultures take up to 90 days to develop. A diagnosis by molecular tests such as PCR can provide fast and reliable results while significantly decreasing the time of confirmation. In the present study, a nested-PCR system, targeting rv2807, with conventional PCR followed by real-time PCR, was developed to detect Mycobacterium tuberculosis complex (MTC) organisms directly from bovine and bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other Actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. Regarding the analytical sensitivity, DNA of the M. bovis AN5 strain was detected up to 1.5 pg by nested-PCR, whereas DNA of M. tuberculosis H37Rv strain was detected up to 6.1 pg. The nested-PCR system showed 100% analytical specificity for MTC when tested with DNA of reference strains of non-tuberculous mycobacteria and closely-related Actinomycetales. A clinical sensitivity level of 76.7% was detected with tissues samples positive for MTC by means of the culture and conventional PCR. A clinical specificity of 100% was detected with DNA from tissue samples of cattle with negative results in the comparative intradermal tuberculin test. These cattle exhibited no visible lesions and were negative in the culture for MTC. The use of the nested-PCR assay to detect M. tuberculosis complex in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis. PMID:25242951

The Cyclic Peptide Ecumicin Targeting ClpC1 Is Active against Mycobacterium tuberculosis In Vivo

PubMed Central

Gao, Wei; Kim, Jin-Yong; Anderson, Jeffrey R.; Akopian, Tatos; Hong, Seungpyo; Jin, Ying-Yu; Kandror, Olga; Kim, Jong-Woo; Lee, In-Ae; Lee, Sun-Young; McAlpine, James B.; Mulugeta, Surafel; Sunoqrot, Suhair; Wang, Yuehong; Yang, Seung-Hwan; Yoon, Tae-Mi; Goldberg, Alfred L.; Pauli, Guido F.; Cho, Sanghyun

2014-01-01

Drug-resistant tuberculosis (TB) has lent urgency to finding new drug leads with novel modes of action. A high-throughput screening campaign of >65,000 actinomycete extracts for inhibition of Mycobacterium tuberculosis viability identified ecumicin, a macrocyclic tridecapeptide that exerts potent, selective bactericidal activity against M. tuberculosis in vitro, including nonreplicating cells. Ecumicin retains activity against isolated multiple-drug-resistant (MDR) and extensively drug-resistant (XDR) strains of M. tuberculosis. The subcutaneous administration to mice of ecumicin in a micellar formulation at 20 mg/kg body weight resulted in plasma and lung exposures exceeding the MIC. Complete inhibition of M. tuberculosis growth in the lungs of mice was achieved following 12 doses at 20 or 32 mg/kg. Genome mining of lab-generated, spontaneous ecumicin-resistant M. tuberculosis strains identified the ClpC1 ATPase complex as the putative target, and this was confirmed by a drug affinity response test. ClpC1 functions in protein breakdown with the ClpP1P2 protease complex. Ecumicin markedly enhanced the ATPase activity of wild-type (WT) ClpC1 but prevented activation of proteolysis by ClpC1. Less stimulation was observed with ClpC1 from ecumicin-resistant mutants. Thus, ClpC1 is a valid drug target against M. tuberculosis, and ecumicin may serve as a lead compound for anti-TB drug development. PMID:25421483

[Epidemiology of resistance to antituberculosis drugs in Mycobacterium tuberculosis complex strains isolated from adenopathies in Djibouti. Prospective study carried out in 1999].

PubMed

Koeck, J L; Bernatas, J J; Gerome, P; Fabre, M; Houmed, A; Herve, V; Teyssou, R

2002-01-01

Tuberculosis is a major cause of death in the Republic of Djibouti. Tuberculous lymphadenitis represents about 25% of the clinical forms of tuberculosis in this country. Between January 1999 and April 1999, 196 lymph node specimens were consecutively collected from 153 patients living in Djibouti. Testing of susceptibility to the major anti-tuberculosis drugs was performed by the proportion method. Growth of Mycobacterium tuberculosis complex strains was obtained from specimens of 85 patients including 9 with prior treatment. Strains were identified as Mycobacterium tuberculosis in 78 cases, Mycobacterium canetti in 3, Mycobacterium africanum in 3, and Mycobacterium bovis in 1. Prevalence of HIV infection was 15%. Assessment of primary resistance demonstrated that the overall resistance rate, i.e., resistance to 1 or more drugs, was 18 (21.2%). Results showed resistance to isoniazid (H) in 6 cases (7.1%), rifampicin (R) in 3 (3.5%), ethambutol (E) in 1 (1.2%), streptomycin (S) in 13 (15.3%) and pyrazinamide (Z) in 1 (1.2%). Multidrug resistance (MDR) was found in 2 cases (2.4%). Assessment of acquired resistance demonstrated resistance to H in 4 cases (44%), R in 2 (22%), S in 2 (22%), E in 0, Z in 0 and MDR in 1 (11%). These findings were not significantly different from data obtained from sputum samples analysed between 1997 and 2000 or from those described in a study conducted in 1985.

Modern laboratory diagnosis of tuberculosis.

PubMed

Drobniewski, F A; Caws, M; Gibson, A; Young, D

2003-03-01

One-third of the global population is believed to be infected with bacteria of the Mycobacterium tuberculosis complex, the causative agent of tuberculosis. More than 8 million new cases of tuberculosis occur annually leading to 2 million deaths. Mortality is particularly high in those coinfected with HIV and where the bacteria are multiple-drug-resistant strains--ie, strains resistant to at least isoniazid and rifampicin. Early diagnosis of tuberculosis and drug resistance improves survival and by identifying infectious cases promotes contact tracing, implementation of institutional cross-infection procedures, and other public-health actions. This review addresses significant advances made in the diagnosis of infection, clinical disease, and drug resistance over the past decade. It proposes operational criteria for a modern diagnostic service in the UK (as a model of a low-incidence country) and explores some of the economic issues surrounding the use of these techniques.

Differential Virulence and Disease Progression following Mycobacterium tuberculosis Complex Infection of the Common Marmoset (Callithrix jacchus)

PubMed Central

Via, Laura E.; Weiner, Danielle M.; Schimel, Daniel; Lin, Philana Ling; Dayao, Emmanuel; Tankersley, Sarah L.; Cai, Ying; Coleman, M. Teresa; Tomko, Jaime; Paripati, Praveen; Orandle, Marlene; Kastenmayer, Robin J.; Tartakovsky, Michael; Rosenthal, Alexander; Portevin, Damien; Eum, Seok Yong; Lahouar, Saher; Gagneux, Sebastien; Young, Douglas B.; Flynn, JoAnne L.

2013-01-01

Existing small-animal models of tuberculosis (TB) rarely develop cavitary disease, limiting their value for assessing the biology and dynamics of this highly important feature of human disease. To develop a smaller primate model with pathology similar to that seen in humans, we experimentally infected the common marmoset (Callithrix jacchus) with diverse strains of Mycobacterium tuberculosis of various pathogenic potentials. These included recent isolates of the modern Beijing lineage, the Euro-American X lineage, and M. africanum. All three strains produced fulminant disease in this animal with a spectrum of progression rates and clinical sequelae that could be monitored in real time using 2-deoxy-2-[18F]fluoro-d-glucose (FDG) positron emission tomography (PET)/computed tomography (CT). Lesion pathology at sacrifice revealed the entire spectrum of lesions observed in human TB patients. The three strains produced different rates of progression to disease, various extents of extrapulmonary dissemination, and various degrees of cavitation. The majority of live births in this species are twins, and comparison of results from siblings with different infecting strains allowed us to establish that the infection was highly reproducible and that the differential virulence of strains was not simply host variation. Quantitative assessment of disease burden by FDG-PET/CT provided an accurate reflection of the pathology findings at necropsy. These results suggest that the marmoset offers an attractive small-animal model of human disease that recapitulates both the complex pathology and spectrum of disease observed in humans infected with various M. tuberculosis strain clades. PMID:23716617

High Genetic Diversity Revealed by Variable-Number Tandem Repeat Genotyping and Analysis of hsp65 Gene Polymorphism in a Large Collection of “Mycobacterium canettii” Strains Indicates that the M. tuberculosis Complex Is a Recently Emerged Clone of “M. canettii”

PubMed Central

Fabre, Michel; Koeck, Jean-Louis; Le Flèche, Philippe; Simon, Fabrice; Hervé, Vincent; Vergnaud, Gilles; Pourcel, Christine

2004-01-01

We have analyzed, using complementary molecular methods, the diversity of 43 strains of “Mycobacterium canettii” originating from the Republic of Djibouti, on the Horn of Africa, from 1998 to 2003. Genotyping by multiple-locus variable-number tandem repeat analysis shows that all the strains belong to a single but very distant group when compared to strains of the Mycobacterium tuberculosis complex (MTBC). Thirty-one strains cluster into one large group with little variability and five strains form another group, whereas the other seven are more diverged. In total, 14 genotypes are observed. The DR locus analysis reveals additional variability, some strains being devoid of a direct repeat locus and others having unique spacers. The hsp65 gene polymorphism was investigated by restriction enzyme analysis and sequencing of PCR amplicons. Four new single nucleotide polymorphisms were discovered. One strain was characterized by three nucleotide changes in 441 bp, creating new restriction enzyme polymorphisms. As no sequence variability was found for hsp65 in the whole MTBC, and as a single point mutation separates M. tuberculosis from the closest “M. canettii” strains, this diversity within “M. canettii” subspecies strongly suggests that it is the most probable source species of the MTBC rather than just another branch of the MTBC. PMID:15243089

Molecular characterisation of Mycobacterium caprae strains isolated in Poland.

PubMed

Krajewska-Wędzina, Monika; Kozińska, Monika; Orłowska, Blanka; Weiner, Marcin; Szulowski, Krzysztof; Augustynowicz-Kopeć, Ewa; Anusz, Krzysztof; Smith, Noel H

2018-03-10

Bovine tuberculosis (bovine TB, bTB) is caused by bovine bacilli: Mycobacterium bovis and M caprae The studies conducted in Poland, in the National Bovine Tuberculosis Reference Laboratory in the Department of Microbiology of the National Veterinary Research Institute in Pulawy, show that animal tuberculosis in Poland is also caused by M caprae We here describe the identification and genotypic assessment of 52 isolates of M caprae obtained from Polish cattle and wild animals over the last five years. We show that strains isolated from bison have significant genotypic diversity and are distinct compared with the genotypes of strains isolated from cattle. Similarly, isolates from cattle herds can be highly genotypically variable. Formal designation of the members of the Mycobacterium tuberculosis complex is controversial in Poland; there is a gap in veterinary legislation with regard to bTB and no explicit mention of M caprae causing tuberculosis in animal. © British Veterinary Association (unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

[Immunobiologic characteristics of a recombinant Listeria monocytogenes expressing Mycobacterium tuberculosis antigens].

PubMed

Yin, Yuelan; Zhao, Dan; Kang, Meiqin; Tan, Weijun; Lian, Kai; Hu, Maozhi; Chen, Xiang; Pan, Zhiming; Jiao, Xin'an

2013-12-04

Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis complex. Hence, novel vaccines against TB are urgently needed and important to the public health. Immunobiologic characteristics of a recombinant attenuated Listeria monocytogenes strain LMdeltahly: :Ag85b-esat-6 was evaluated. LMdeltahly: :Ag85b-esat-6 had lost the hemolytic activity. It was completely cleared from the livers and spleens of mice 5 days after inoculation via intravenous route. Furthermore, the LD50 of the recombinant strain increased by 4 Logs comparing to that of the parent strain. Histopathology reveals no obvious pathological changes following administration of the recombinant strain to mice, indicating its safety. In addition, the potential protective immune response was evaluated on C57BL/6 mice via intravenous immunization route. The results indicate that the antigen delivered by the recombination LM could induce Th1 type immune response and elicit strong cytotoxic lymphocyte effect against Ag85B-ESAT-6. Thus, LMdeltahly::Ag85b-esat-6 had high safety to mice, and could be used as a novel vaccines candidate for preventing tuberculosis.

Rapid and simultaneous detection of Mycobacterium tuberculosis complex and Beijing/W genotype in sputum by an optimized DNA extraction protocol and a novel multiplex real-time PCR.

PubMed

Leung, Eric T Y; Zheng, L; Wong, Rity Y K; Chan, Edward W C; Au, T K; Chan, Raphael C Y; Lui, Grace; Lee, Nelson; Ip, Margaret

2011-07-01

Rapid diagnosis and genotyping of Mycobacterium tuberculosis by molecular methods are often limited by the amount and purity of DNA extracted from body fluids. In this study, we evaluated 12 DNA extraction methods and developed a highly sensitive protocol for mycobacterial DNA extraction directly from sputa using surface-coated magnetic particles. We have also developed a novel multiplex real-time PCR for simultaneous identification of M. tuberculosis complex and the Beijing/W genotype (a hypervirulent sublineage of M. tuberculosis) by using multiple fluorogenic probes targeting both the M. tuberculosis IS6110 and the Rv0927c-pstS3 intergenic region. With reference strains and clinical isolates, our real-time PCR accurately identified 20 non-Beijing/W and 20 Beijing/W M. tuberculosis strains from 17 different species of nontuberculosis Mycobacterium (NTM). Further assessment of our DNA extraction protocol and real-time PCR with 335 nonduplicate sputum specimens correctly identified all 74 M. tuberculosis culture-positive specimens. In addition, 15 culture-negative specimens from patients with confirmed tuberculosis were also identified. No cross-reactivity was detected with NTM specimens (n = 31). The detection limit of the assay is 10 M. tuberculosis bacilli, as determined by endpoint dilution analysis. In conclusion, an optimized DNA extraction protocol coupled with a novel multiprobe multiplex real-time PCR for the direct detection of M. tuberculosis, including Beijing/W M. tuberculosis, was found to confer high sensitivity and specificity. The combined procedure has the potential to compensate for the drawbacks of conventional mycobacterial culture in routine clinical laboratory setting, such as the lengthy incubation period and the limitation to viable organisms.

Controlled fire use in early humans might have triggered the evolutionary emergence of tuberculosis.

PubMed

Chisholm, Rebecca H; Trauer, James M; Curnoe, Darren; Tanaka, Mark M

2016-08-09

Tuberculosis (TB) is caused by the Mycobacterium tuberculosis complex (MTBC), a wildly successful group of organisms and the leading cause of death resulting from a single bacterial pathogen worldwide. It is generally accepted that MTBC established itself in human populations in Africa and that animal-infecting strains diverged from human strains. However, the precise causal factors of TB emergence remain unknown. Here, we propose that the advent of controlled fire use in early humans created the ideal conditions for the emergence of TB as a transmissible disease. This hypothesis is supported by mathematical modeling together with a synthesis of evidence from epidemiology, evolutionary genetics, and paleoanthropology.

Identification of a New DNA Region Specific for Members of Mycobacterium tuberculosis Complex

PubMed Central

Magdalena, Juana; Vachée, Anne; Supply, Philip; Locht, Camille

1998-01-01

The successful use of DNA amplification for the detection of tuberculous mycobacteria crucially depends on the choice of the target sequence, which ideally should be present in all tuberculous mycobacteria and absent from all other bacteria. In the present study we developed a PCR procedure based on the intergenic region (IR) separating two genes encoding a recently identified mycobacterial two-component system named SenX3-RegX3. The senX3-regX3 IR is composed of a novel type of repetitive sequence, called mycobacterial interspersed repetitive units (MIRUs). In a survey of 116 Mycobacterium tuberculosis strains characterized by different IS6110 restriction fragment length polymorphisms, 2 Mycobacterium africanum strains, 3 Mycobacterium bovis strains (including 2 BCG strains), and 1 Mycobacterium microti strain, a specific PCR fragment was amplified in all cases. This collection included M. tuberculosis strains that lack IS6110 or mtp40, two target sequences that have previously been used for the detection of M. tuberculosis. No PCR fragment was amplified when DNA from other organisms was used, giving a sensitivity of 100% and a specificity of 100% in the confidence limit of this study. The numbers of MIRUs were found to vary among strains, resulting in six different groups of strains on the basis of the size of the amplified PCR fragment. However, the vast majority of the strains (approximately 90%) fell within the same group, containing two 77-bp MIRUs followed by one 53-bp MIRU. PMID:9542912

Development of a Quantitative Sandwich Enzyme-Linked Immunosorbent Assay for Detecting the MPT64 Antigen of Mycobacterium tuberculosis

PubMed Central

Ji, Mijung; Cho, Byungki; Cho, Young Shik; Park, Song-Yong; Cho, Sang-Nae

2014-01-01

Purpose Tuberculosis (TB) is a major infectious disease and is responsible for two million deaths annually. For the identification and quantitation of Mycobacterium tuberculosis (M. tuberculosis), a causative agent of TB, a sandwich enzyme-linked immunosorbent assay (ELISA) against the MPT64 protein of M. tuberculosis, an antigen marker of the M. tuberculosis complex, was developed. Materials and Methods The MPT64 protein was expressed, and anti-MPT64 monoclonal antibodies were prepared. A sandwich ELISA was established using recombinant MPT64 protein and anti-MPT64 monoclonal antibodies. The sandwich MPT64 ELISA was evaluated using reference and clinical mycobacterial strains. Results The sandwich MPT64 ELISA detected MPT64 protein from 2.1 ng/mL to 250 ng/mL (equivalent to 1.7×104 CFU/mL and 2.0×106 CFU/mL). All 389 clinical M. tuberculosis isolates tested positive in the sandwich MPT64 ELISA (sensitivity, 100%), and the assay showed no cross reactivity to any tested nontuberculous mycobacterial strain (specificity, 100%). Conclusion The sandwich MPT64 ELISA is a highly sensitive and quantitative test for MPT64 protein, which can identify M. tuberculosis. PMID:24719143

Development of a quantitative sandwich enzyme-linked immunosorbent assay for detecting the MPT64 antigen of Mycobacterium tuberculosis.

PubMed

Ji, Mijung; Cho, Byungki; Cho, Young Shik; Park, Song-Yong; Cho, Sang-Nae; Jeon, Bo-Young; Yoon, Byoung-Su

2014-05-01

Tuberculosis (TB) is a major infectious disease and is responsible for two million deaths annually. For the identification and quantitation of Mycobacterium tuberculosis (M. tuberculosis), a causative agent of TB, a sandwich enzyme-linked immunosorbent assay (ELISA) against the MPT64 protein of M. tuberculosis, an antigen marker of the M. tuberculosis complex, was developed. The MPT64 protein was expressed, and anti-MPT64 monoclonal antibodies were prepared. A sandwich ELISA was established using recombinant MPT64 protein and anti-MPT64 monoclonal antibodies. The sandwich MPT64 ELISA was evaluated using reference and clinical mycobacterial strains. The sandwich MPT64 ELISA detected MPT64 protein from 2.1 ng/mL to 250 ng/mL (equivalent to 1.7×10⁴ CFU/mL and 2.0×10⁶ CFU/mL). All 389 clinical M. tuberculosis isolates tested positive in the sandwich MPT64 ELISA (sensitivity, 100%), and the assay showed no cross reactivity to any tested nontuberculous mycobacterial strain (specificity, 100%). The sandwich MPT64 ELISA is a highly sensitive and quantitative test for MPT64 protein, which can identify M. tuberculosis.

Microwell hybridization assay for detection of PCR products from Mycobacterium tuberculosis complex and the recombinant Mycobacterium smegmatis strain 1008 used as an internal control.

PubMed Central

Kox, L F; Noordhoek, G T; Kunakorn, M; Mulder, S; Sterrenburg, M; Kolk, A H

1996-01-01

A microwell hybridization assay was developed for the detection of the PCR products from both Mycobacterium tuberculosis complex bacteria and the recombinant Mycobacterium smegmatis strain 1008 that is used as an internal control to monitor inhibition in the PCR based on the M. tuberculosis complex-specific insertion sequence IS6110. The test is based on specific detection with digoxigenin-labeled oligonucleotide probes of biotinylated PCR products which are captured in a microtiter plate coated with streptavidin. The captured PCR products are hybridized separately with two probes, one specific for the PCR product from IS6110 from M. tuberculosis complex and the other specific for the PCR fragment from the modified IS6110 fragment from the recombinant M. smegmatis 1008. The microwell hybridization assay discriminates perfectly between the two types of amplicon. The amount of PCR product that can be detected by this assay is 10 times less than that which can be detected by agarose gel electrophoresis. The test can be performed in 2 h. It is much faster and less laborious than Southern blot hybridization. Furthermore, the interpretation of results is objective. The assay was used with 172 clinical samples in a routine microbiology laboratory, and the results were in complete agreement with those of agarose gel electrophoresis and Southern blot hybridization. PMID:8862568

Rapid molecular assays for detection of tuberculosis.

PubMed

Eddabra, Rkia; Ait Benhassou, Hassan

2018-01-01

Tuberculosis (TB) is an infectious disease that remains an important public health problem at the global level. It is one of the main causes of morbidity and mortality, due to the emergence of antibiotic resistant Mycobacterium strains and HIV co-infection. Over the past decade, important progress has been made for better control of the disease. While microscopy and culture continue to be indispensible for laboratory diagnosis of tuberculosis, the range of several molecular diagnostic tests, including the nucleic acid amplification test (NAAT) and whole-genome sequencing (WGS), have expanded tremendously. They are becoming more accessible not only for detection and identification of Mycobacterium tuberculosis complex in clinical specimens, but now extend to diagnosing multi-drug resistant strains. Molecular diagnostic tests provide timely results useful for high-quality patient care, low contamination risk, and ease of performance and speed. This review focuses on the current diagnostic tests in use, including emerging technologies used for detection of tuberculosis in clinical specimens. The sensitivity and specificity of these tests have also been taken into consideration.

Evolution of Mycobacterium tuberculosis.

PubMed

Behr, Marcel A

2013-01-01

Genomic studies have provided a refined understanding of the genetic diversity within the Mycobacterium genus, and more specifically within Mycobacterium tuberculosis. These results have informed a new perspective on the macro- and micro-evolution of the tubercle bacillus. In the first step, a M. kansasii-like opportunistic pathogen acquired new genes, through horizontal gene transfer, that enabled it to better exploit an intracellular niche and ultimately evolve into a professional pathogen. In the second step, different subspecies and strains of the M. tuberculosis complex emerged through mutation and deletion of unnecessary DNA. Understanding the differences between M. tuberculosis and related less pathogenic mycobacteria is expected to reveal key bacterial virulence mechanisms and provide opportunities to understand host resistance to mycobacterial infection. Understanding differences within the M. tuberculosis complex and the evolutionary forces shaping these differences is important for investigating the basis of its success as both a symbiont and a pathogen.

DNA Replication Fidelity in the Mycobacterium tuberculosis Complex.

PubMed

Warner, Digby F; Rock, Jeremy M; Fortune, Sarah M; Mizrahi, Valerie

2017-01-01

Mycobacterium tuberculosis is genetically isolated, with no evidence for horizontal gene transfer or the acquisition of episomal genetic information in the modern evolution of strains of the Mycobacterium tuberculosis complex. When considered in the context of the specific features of the disease M. tuberculosis causes (e.g., transmission via cough aerosol, replication within professional phagocytes, subclinical persistence, and stimulation of a destructive immune pathology), this implies that to understand the mechanisms ensuring preservation of genomic integrity in infecting mycobacterial populations is to understand the source of genetic variation, including the emergence of microdiverse sub-populations that may be linked to the acquisition of drug resistance. In this chapter, we focus on mechanisms involved in maintaining DNA replication fidelity in M. tuberculosis, and consider the potential to target components of the DNA replication machinery as part of novel therapeutic regimens designed to curb the emerging threat of drug-resistance.

Whole Genome Sequencing Demonstrates Limited Transmission within Identified Mycobacterium tuberculosis Clusters in New South Wales, Australia

PubMed Central

Gurjav, Ulziijargal; Outhred, Alexander C.; Jelfs, Peter; McCallum, Nadine; Wang, Qinning; Hill-Cawthorne, Grant A.; Marais, Ben J.; Sintchenko, Vitali

2016-01-01

Australia has a low tuberculosis incidence rate with most cases occurring among recent immigrants. Given suboptimal cluster resolution achieved with 24-locus mycobacterium interspersed repetitive unit (MIRU-24) genotyping, the added value of whole genome sequencing was explored. MIRU-24 profiles of all Mycobacterium tuberculosis culture-confirmed tuberculosis cases diagnosed between 2009 and 2013 in New South Wales (NSW), Australia, were examined and clusters identified. The relatedness of cases within the largest MIRU-24 clusters was assessed using whole genome sequencing and phylogenetic analyses. Of 1841 culture-confirmed TB cases, 91.9% (1692/1841) had complete demographic and genotyping data. East-African Indian (474; 28.0%) and Beijing (470; 27.8%) lineage strains predominated. The overall rate of MIRU-24 clustering was 20.1% (340/1692) and was highest among Beijing lineage strains (35.7%; 168/470). One Beijing and three East-African Indian (EAI) clonal complexes were responsible for the majority of observed clusters. Whole genome sequencing of the 4 largest clusters (30 isolates) demonstrated diverse single nucleotide polymorphisms (SNPs) within identified clusters. All sequenced EAI strains and 70% of Beijing lineage strains clustered by MIRU-24 typing demonstrated distinct SNP profiles. The superior resolution provided by whole genome sequencing demonstrated limited M. tuberculosis transmission within NSW, even within identified MIRU-24 clusters. Routine whole genome sequencing could provide valuable public health guidance in low burden settings. PMID:27737005

Tuberculosis in Antelopes in a Zoo in Poland--Problem of Public Health.

PubMed

Krajewska, Monika; Załuski, Michał; Zabost, Anna; Orłowska, Blanka; Augustynowicz-Kopeć, Ewa; Anusz, Krzysztof; Lipiec, Marek; Weiner, Marcin; Szulowski, Krzysztof

2015-01-01

Bovine tuberculosis is an infectious disease that occurs in many species of both domestic and wild animals, as well as those held in captivity. The etiological factor is the acid resistant bacillus (Mycobacterium bovis or Mycobacterium caprae), which is characterized by the major pathogenicity among mycobacteria belonging to the Mycobacterium tuberculosis complex. The material from 8 antelopes from the zoo, suspected for tuberculosis were examined, and M. bovis strains were isolated from 6 of them. The spoligotyping method showing spoligo pattern 676763777777600. In Poland, this spoligotype has not been observed so far.

LPA or GeneXpert in the diagnosis of multidrug-resistant tuberculosis.

PubMed

Mindru, Roxana; Spinu, Victor; Popescu, Oana

2016-01-01

Facing a constant increase of multidrug-resistant tuberculosis (MDR-TB), there is large need for routine use of new diagnostic tests, based on molecular techniques that allow both a rapid diagnosis for TB complex and rapid identification of resistance mutations. The resistances are due to genetic factors: accumulation of changes within the genome structure, acquisition or loss of genes, spontaneous mutations in chromosomal genes, and changes that induce selection of resistant strains during a suboptimal treatment. The bacteriology laboratory plays a crucial role in the making of the diagnosis, monitoring and preventing TB transmission. World Health Organization offers consistent recommendations in favour of use of Xpert MTB/RIF, GeneXpert platform, as initial diagnostic test in adults and children suspected of TB, because it can simultaneously determine the presence of Mycobacterium tuberculosis and the Rifampicin resistance, which is a surrogate marker of MDR strains. The very high sensibility and specificity, also in the smear negative samples, as well as the short time needed for the results, make Xpert MTB/RIF a valuable tool in the fight against TB. Other recommended tests are: LPA, which identifies M. Tuberculosis complex, the Rifancim and Isoniazid resistance; MTBDR plus or, for second line anti-TB drugs, the MTBDRsl.

Novel Multiplex Real-Time PCR Diagnostic Assay for Identification and Differentiation of Mycobacterium tuberculosis, Mycobacterium canettii, and Mycobacterium tuberculosis Complex Strains▿†

PubMed Central

Reddington, Kate; O'Grady, Justin; Dorai-Raj, Siobhan; Maher, Majella; van Soolingen, Dick; Barry, Thomas

2011-01-01

Tuberculosis (TB) in humans is caused by members of the Mycobacterium tuberculosis complex (MTC). Rapid detection of the MTC is necessary for the timely initiation of antibiotic treatment, while differentiation between members of the complex may be important to guide the appropriate antibiotic treatment and provide epidemiological information. In this study, a multiplex real-time PCR diagnostics assay using novel molecular targets was designed to identify the MTC while simultaneously differentiating between M. tuberculosis and M. canettii. The lepA gene was targeted for the detection of members of the MTC, the wbbl1 gene was used for the differentiation of M. tuberculosis and M. canettii from the remainder of the complex, and a unique region of the M. canettii genome, a possible novel region of difference (RD), was targeted for the specific identification of M. canettii. The multiplex real-time PCR assay was tested using 125 bacterial strains (64 MTC isolates, 44 nontuberculosis mycobacteria [NTM], and 17 other bacteria). The assay was determined to be 100% specific for the mycobacteria tested. Limits of detection of 2.2, 2.17, and 0.73 cell equivalents were determined for M. tuberculosis/M. canettii, the MTC, and M. canettii, respectively, using probit regression analysis. Further validation of this diagnostics assay, using clinical samples, should demonstrate its potential for the rapid, accurate, and sensitive diagnosis of TB caused by M. tuberculosis, M. canettii, and the other members of the MTC. PMID:21123525

Virulence and Immune Response Induced by Mycobacterium avium Complex Strains in a Model of Progressive Pulmonary Tuberculosis and Subcutaneous Infection in BALB/c Mice

PubMed Central

González-Pérez, Mónica; Mariño-Ramírez, Leonardo; Parra-López, Carlos Alberto; Murcia, Martha Isabel; Marquina, Brenda; Mata-Espinoza, Dulce; Rodriguez-Míguez, Yadira; Baay-Guzman, Guillermina J.; Huerta-Yepez, Sara

2013-01-01

The genus Mycobacterium comprises more than 150 species, including important pathogens for humans which cause major public health problems. The vast majority of efforts to understand the genus have been addressed in studies with Mycobacterium tuberculosis. The biological differentiation between M. tuberculosis and nontuberculous mycobacteria (NTM) is important because there are distinctions in the sources of infection, treatments, and the course of disease. Likewise, the importance of studying NTM is not only due to its clinical significance but also due to the mechanisms by which some species are pathogenic while others are not. Mycobacterium avium complex (MAC) is the most important group of NTM opportunistic pathogens, since it is the second largest medical complex in the genus after the M. tuberculosis complex. Here, we evaluated the virulence and immune response of M. avium subsp. avium and Mycobacterium colombiense, using experimental models of progressive pulmonary tuberculosis and subcutaneous infection in BALB/c mice. Mice infected intratracheally with a high dose of MAC strains showed high expression of tumor necrosis factor alpha (TNF-α) and inducible nitric oxide synthase with rapid bacillus elimination and numerous granulomas, but without lung consolidation during late infection in coexistence with high expression of anti-inflammatory cytokines. In contrast, subcutaneous infection showed high production of the proinflammatory cytokines TNF-α and gamma interferon with relatively low production of anti-inflammatory cytokines such as interleukin-10 (IL-10) or IL-4, which efficiently eliminate the bacilli but maintain extensive inflammation and fibrosis. Thus, MAC infection evokes different immune and inflammatory responses depending on the MAC species and affected tissue. PMID:23959717

Proteogenomic Analysis of Polymorphisms and Gene Annotation Divergences in Prokaryotes using a Clustered Mass Spectrometry-Friendly Database*

PubMed Central

de Souza, Gustavo A.; Arntzen, Magnus Ø.; Fortuin, Suereta; Schürch, Anita C.; Målen, Hiwa; McEvoy, Christopher R. E.; van Soolingen, Dick; Thiede, Bernd; Warren, Robin M.; Wiker, Harald G.

2011-01-01

Precise annotation of genes or open reading frames is still a difficult task that results in divergence even for data generated from the same genomic sequence. This has an impact in further proteomic studies, and also compromises the characterization of clinical isolates with many specific genetic variations that may not be represented in the selected database. We recently developed software called multistrain mass spectrometry prokaryotic database builder (MSMSpdbb) that can merge protein databases from several sources and be applied on any prokaryotic organism, in a proteomic-friendly approach. We generated a database for the Mycobacterium tuberculosis complex (using three strains of Mycobacterium bovis and five of M. tuberculosis), and analyzed data collected from two laboratory strains and two clinical isolates of M. tuberculosis. We identified 2561 proteins, of which 24 were present in M. tuberculosis H37Rv samples, but not annotated in the M. tuberculosis H37Rv genome. We were also able to identify 280 nonsynonymous single amino acid polymorphisms and confirm 367 translational start sites. As a proof of concept we applied the database to whole-genome DNA sequencing data of one of the clinical isolates, which allowed the validation of 116 predicted single amino acid polymorphisms and the annotation of 131 N-terminal start sites. Moreover we identified regions not present in the original M. tuberculosis H37Rv sequence, indicating strain divergence or errors in the reference sequence. In conclusion, we demonstrated the potential of using a merged database to better characterize laboratory or clinical bacterial strains. PMID:21030493

In vitro effects of citrus oils against Mycobacterium tuberculosis and non-tuberculous Mycobacteria of clinical importance.

PubMed

Crandall, Philip G; Ricke, Steven C; O'Bryan, Corliss A; Parrish, Nicole M

2012-01-01

We evaluated the in vitro activity of citrus oils against Mycobacterium tuberculosis and other non-tuberculous Mycobacterium species. Citrus essential oils were tested against a variety of Mycobacterium species and strains using the BACTEC radiometric growth system. Cold pressed terpeneless Valencia oil (CPT) was further tested using the Wayne model of in vitro latency. Exposure of M. tuberculosis and M. bovis BCG to 0.025 % cold pressed terpeneless Valencia orange oil (CPT) resulted in a 3-log decrease in viable counts versus corresponding controls. Inhibition of various clinical isolates of the M. avium complex and M. abscessus ranged from 2.5 to 5.2-logs. Some species/strains were completely inhibited in the presence of CPT including one isolate each of the following: the M. avium complex, M. chelonae and M. avium subsp. paratuberculosis. CPT also inhibited the growth of BCG more than 99 % in an in vitro model of latency which mimics anaerobic dormancy thought to occur in vivo. The activity of CPT against drug-resistant strains of the M. avium complex and M. abscessus suggest that the mechanism of action for CPT is different than that of currently available drugs. Inhibition of latently adapted bacilli offers promise for treatment of latent infections of MTB. These results suggest that the antimycobacterial properties of CPT warrant further study to elucidate the specific mechanism of action and clarify the spectrum of activity.

Proposal of a Consensus Set of Hypervariable Mycobacterial Interspersed Repetitive-Unit–Variable-Number Tandem-Repeat Loci for Subtyping of Mycobacterium tuberculosis Beijing Isolates

PubMed Central

Allix-Béguec, Caroline; Wahl, Céline; Hanekom, Madeleine; Nikolayevskyy, Vladyslav; Drobniewski, Francis; Maeda, Shinji; Campos-Herrero, Isolina; Mokrousov, Igor; Niemann, Stefan; Kontsevaya, Irina; Rastogi, Nalin; Samper, Sofia; Sng, Li-Hwei; Warren, Robin M.

2014-01-01

Mycobacterium tuberculosis Beijing strains represent targets of special importance for molecular surveillance of tuberculosis (TB), especially because they are associated with spread of multidrug resistance in some world regions. Standard 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing lacks resolution power for accurately discriminating closely related clones that often compose Beijing strain populations. Therefore, we evaluated a set of 7 additional, hypervariable MIRU-VNTR loci for better resolution and tracing of such strains, using a collection of 535 Beijing isolates from six world regions where these strains are known to be prevalent. The typeability and interlaboratory reproducibility of these hypervariable loci were lower than those of the 24 standard loci. Three loci (2163a, 3155, and 3336) were excluded because of their redundant variability and/or more frequent noninterpretable results compared to the 4 other markers. The use of the remaining 4-locus set (1982, 3232, 3820, and 4120) increased the number of types by 52% (from 223 to 340) and reduced the clustering rate from 58.3 to 36.6%, when combined with the use of the standard 24-locus set. Known major clonal complexes/24-locus-based clusters were all subdivided, although the degree of subdivision varied depending on the complex. Only five single-locus variations were detected among the hypervariable loci of an additional panel of 92 isolates, representing 15 years of clonal spread of a single Beijing strain in a geographically restricted setting. On this calibrated basis, we propose this 4-locus set as a consensus for subtyping Beijing clonal complexes and clusters, after standard typing. PMID:24172154

Proposal of a consensus set of hypervariable mycobacterial interspersed repetitive-unit-variable-number tandem-repeat loci for subtyping of Mycobacterium tuberculosis Beijing isolates.

PubMed

Allix-Béguec, Caroline; Wahl, Céline; Hanekom, Madeleine; Nikolayevskyy, Vladyslav; Drobniewski, Francis; Maeda, Shinji; Campos-Herrero, Isolina; Mokrousov, Igor; Niemann, Stefan; Kontsevaya, Irina; Rastogi, Nalin; Samper, Sofia; Sng, Li-Hwei; Warren, Robin M; Supply, Philip

2014-01-01

Mycobacterium tuberculosis Beijing strains represent targets of special importance for molecular surveillance of tuberculosis (TB), especially because they are associated with spread of multidrug resistance in some world regions. Standard 24-locus mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing lacks resolution power for accurately discriminating closely related clones that often compose Beijing strain populations. Therefore, we evaluated a set of 7 additional, hypervariable MIRU-VNTR loci for better resolution and tracing of such strains, using a collection of 535 Beijing isolates from six world regions where these strains are known to be prevalent. The typeability and interlaboratory reproducibility of these hypervariable loci were lower than those of the 24 standard loci. Three loci (2163a, 3155, and 3336) were excluded because of their redundant variability and/or more frequent noninterpretable results compared to the 4 other markers. The use of the remaining 4-locus set (1982, 3232, 3820, and 4120) increased the number of types by 52% (from 223 to 340) and reduced the clustering rate from 58.3 to 36.6%, when combined with the use of the standard 24-locus set. Known major clonal complexes/24-locus-based clusters were all subdivided, although the degree of subdivision varied depending on the complex. Only five single-locus variations were detected among the hypervariable loci of an additional panel of 92 isolates, representing 15 years of clonal spread of a single Beijing strain in a geographically restricted setting. On this calibrated basis, we propose this 4-locus set as a consensus for subtyping Beijing clonal complexes and clusters, after standard typing.

A First Insight on the Population Structure of Mycobacterium tuberculosis Complex as Studied by Spoligotyping and MIRU-VNTRs in Santiago, Chile

PubMed Central

Balcells, María Elvira; García, Patricia; Meza, Paulina; Peña, Carlos; Cifuentes, Marcela; Couvin, David; Rastogi, Nalin

2015-01-01

Tuberculosis (TB) remains a significant public health problem worldwide, but the ecology of the prevalent mycobacterial strains, and their transmission, can vary depending on country and region. Chile is a country with low incidence of TB, that has a geographically isolated location in relation to the rest of South American countries due to the Andes Mountains, but recent migration from neighboring countries has changed this situation. We aimed to assess the genotypic diversity of Mycobacterium tuberculosis complex (MTBC) strains in Santiago, Chile, and compare with reports from other Latin-American countries. We analyzed MTBC isolates from pulmonary tuberculosis cases collected between years 2008 and 2013 in Central Santiago, using two genotyping methods: spoligotyping and 12-loci mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTRs). Data obtained were analyzed and compared to the SITVIT2 database. Mean age of the patients was 47.5 years and 61% were male; 11.6% were migrants. Of 103 strains (1 isolate/patient) included, there were 56 distinct spoligotype patterns. Of these, 16 strains (15.5%) corresponded to orphan strains in the SITVIT2 database, not previously reported. Latin American and Mediterranean (LAM) (34%) and T (33%) lineages were the most prevalent strains, followed by Haarlem lineage (16.5%). Beijing family was scarcely represented with only two cases (1.9%), one of them isolated from a Peruvian migrant. The most frequent clustered spoligotypes were SIT33/LAM3 (10.7%), SIT53/T1 (8.7%), SIT50/H3 (7.8%), and SIT37/T3 (6.8%). We conclude that LAM and T genotypes are the most prevalent genotypes of MTBC in Santiago, Chile, and together correspond to almost two thirds of analyzed strains, which is similar to strain distribution reported from other countries of Latin America. Nevertheless, the high proportion of SIT37/T3, which was rarely found in other Latin American countries, may underline a specific history or demographics of Chile related to probable human migrations and evolutions. PMID:25671320

A first insight on the population structure of Mycobacterium tuberculosis complex as studied by spoligotyping and MIRU-VNTRs in Santiago, Chile.

PubMed

Balcells, María Elvira; García, Patricia; Meza, Paulina; Peña, Carlos; Cifuentes, Marcela; Couvin, David; Rastogi, Nalin

2015-01-01

Tuberculosis (TB) remains a significant public health problem worldwide, but the ecology of the prevalent mycobacterial strains, and their transmission, can vary depending on country and region. Chile is a country with low incidence of TB, that has a geographically isolated location in relation to the rest of South American countries due to the Andes Mountains, but recent migration from neighboring countries has changed this situation. We aimed to assess the genotypic diversity of Mycobacterium tuberculosis complex (MTBC) strains in Santiago, Chile, and compare with reports from other Latin-American countries. We analyzed MTBC isolates from pulmonary tuberculosis cases collected between years 2008 and 2013 in Central Santiago, using two genotyping methods: spoligotyping and 12-loci mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTRs). Data obtained were analyzed and compared to the SITVIT2 database. Mean age of the patients was 47.5 years and 61% were male; 11.6% were migrants. Of 103 strains (1 isolate/patient) included, there were 56 distinct spoligotype patterns. Of these, 16 strains (15.5%) corresponded to orphan strains in the SITVIT2 database, not previously reported. Latin American and Mediterranean (LAM) (34%) and T (33%) lineages were the most prevalent strains, followed by Haarlem lineage (16.5%). Beijing family was scarcely represented with only two cases (1.9%), one of them isolated from a Peruvian migrant. The most frequent clustered spoligotypes were SIT33/LAM3 (10.7%), SIT53/T1 (8.7%), SIT50/H3 (7.8%), and SIT37/T3 (6.8%). We conclude that LAM and T genotypes are the most prevalent genotypes of MTBC in Santiago, Chile, and together correspond to almost two thirds of analyzed strains, which is similar to strain distribution reported from other countries of Latin America. Nevertheless, the high proportion of SIT37/T3, which was rarely found in other Latin American countries, may underline a specific history or demographics of Chile related to probable human migrations and evolutions.

Molecular identification of Mycobacterium tuberculosis in cattle.

PubMed

Sweetline Anne, N; Ronald, B S M; Kumar, T M A Senthil; Kannan, P; Thangavelu, A

2017-01-01

Bovine tuberculosis continued to be a re-emerging problem in some countries especially in endemic areas due to the fact that human and animal health surveillance system is not adopted to diagnose the infection. This crisis can be attributed due to sharing of the same habitat especially in rural areas. In the present study, a total of 148 samples were collected from cattle for isolation over a period of 3 years from cattle with and without lesions, of which 67 isolates were obtained by culture. Fifty one isolates were identified as Mycobacterium tuberculosis complex (MTBC) by IS6110 PCR of which 43 (84.3%) were identified as M. tuberculosis and 08 (15.6%) were identified as M. bovis by using 12.7kb fragment multiplex PCR. Among this, 31 isolates which were positive for IS6110 PCR were subjected to spoligotyping and revealed 28 isolates belonging to MANU1 strain of M. tuberculosis. This study clearly indicates that high prevalence of M. tuberculosis than M. bovis in bovine was identified by means of culture and by molecular methods M. tuberculosis can affect cattle producing lesion in contradiction to the earlier thoughts. This study speculates that M. tuberculosis MANU1 strain infection in cattle could be due to spill over from human or other non specific hosts in tuberculosis endemic areas. Though bovine tuberculosis due to M. tuberculosis in cattle is not considered a serious threat worldwide, in countries where human TB is endemic, M. tuberculosis infection of cattle needs to be considered. Copyright © 2016 Elsevier B.V. All rights reserved.

Diversity and Evolution of Mycobacterium tuberculosis: Moving to Whole-Genome-Based Approaches

PubMed Central

Niemann, Stefan; Supply, Philip

2014-01-01

Genotyping of clinical Mycobacterium tuberculosis complex (MTBC) strains has become a standard tool for epidemiological tracing and for the investigation of the local and global strain population structure. Of special importance is the analysis of the expansion of multidrug (MDR) and extensively drug-resistant (XDR) strains. Classical genotyping and, more recently, whole-genome sequencing have revealed that the strains of the MTBC are more diverse than previously anticipated. Globally, several phylogenetic lineages can be distinguished whose geographical distribution is markedly variable. Strains of particular (sub)lineages, such as Beijing, seem to be more virulent and associated with enhanced resistance levels and fitness, likely fueling their spread in certain world regions. The upcoming generalization of whole-genome sequencing approaches will expectedly provide more comprehensive insights into the molecular and epidemiological mechanisms involved and lead to better diagnostic and therapeutic tools. PMID:25190252

Animal-adapted members of the Mycobacterium tuberculosis complex endemic to the southern African subregion.

PubMed

Clarke, Charlene; Van Helden, Paul; Miller, Michele; Parsons, Sven

2016-04-26

Members of the Mycobacterium tuberculosis complex (MTC) cause tuberculosis (TB) in both animals and humans. In this article, three animal-adapted MTC strains that are endemic to the southern African subregion - that is, Mycobacterium suricattae, Mycobacterium mungi, and the dassie bacillus - are reviewed with a focus on clinical and pathological presentations, geographic distribution, genotyping methods, diagnostic tools and evolution. Moreover, factors influencing the transmission and establishment of TB pathogens in novel host populations, including ecological, immunological and genetic factors of both the host and pathogen, are discussed. The risks associated with these infections are currently unknown and further studies will be required for greater understanding of this disease in the context of the southern African ecosystem.

Animal-adapted members of the Mycobacterium tuberculosis complex endemic to the southern African subregion.

PubMed

Clarke, Charlene; Van Helden, Paul; Miller, Michele; Parsons, Sven

2016-04-26

Members of the Mycobacterium tuberculosis complex (MTC) cause tuberculosis (TB) in both animals and humans. In this article, three animal-adapted MTC strains that are endemic to the southern African subregion - that is, Mycobacterium suricattae, Mycobacterium mungi, and the dassie bacillus - are reviewed with a focus on clinical and pathological presentations, geographic distribution, genotyping methods, diagnostic tools and evolution. Moreover, factors influencing the transmission and establishment of TB pathogens in novel host populations, including ecological, immunological and genetic factors of both the host and pathogen, are discussed. The risks associated with these infections are currently unknown and further studies will be required for greater understanding of this disease in the context of the southern African ecosystem.

Methods for determining the antimicrobial susceptibility of mycobacteria.

PubMed

Alcaide, Fernando; Esteban, Jaime; González-Martin, Julià; Palacios, Juan-José

2017-10-01

Mycobacteria are a large group of microorganisms, multiple species of which are major causes of morbidity and mortality, such as tuberculosis and leprosy. At present, the emergence and spread of multidrug-resistant strains of Mycobacterium tuberculosis complex are one of the most serious health problems worldwide. Furthermore, in contrast to M. tuberculosis and Mycobacterium leprae, non-tuberculous mycobacteria (NTM) are more frequently isolated and, in many cases, treatment is based on drug susceptibility testing. This article is a review of the different methods to determine the in vitro drug susceptibility of M. tuberculosis complex and the most relevant NTM isolates. The molecular techniques currently used for rapid detection of resistance of clinical specimens are also analysed. Copyright © 2016 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

Genetic diversity of the Mycobacterium tuberculosis complex in San Luis Potosí, México.

PubMed

López-Rocha, Estela; Juárez-Álvarez, Julio; Riego-Ruiz, Lina; Enciso-Moreno, Leonor; Ortega-Aguilar, Francisco; Hernández-Nieto, Julián; Enciso-Moreno, José A; López-Revilla, Rubén

2013-05-01

Although epidemiologic and socioeconomic criteria and biomedical risk factors indicate high-priority for tuberculosis (TB) control in Mexico, molecular epidemiology studies of the disease in the country are scarce. Complete sociodemographic and clinical data were obtained from 248 of the 432 pulmonary TB (PTB) cases confirmed from 2006 to 2010 on the population under epidemiological surveillance in the state of San Luis Potosí, México. From most PTB cases with complete data Mycobacterium tuberculosis complex (MTC) isolates were recovered and their spoligotypes, lineages and families, geographic distribution and drug resistance determined. Pulmonary tuberculosis incidence ranged from 2.4 to 33.4 (cases per 100,000 inhabitants) in the six state sanitary jurisdictions that were grouped in regions of low (jurisdictions I-II-III), intermediate (jurisdictions IV-V) and high incidence (jurisdiction VI) with 6.2, 17.3 and 33.4 rates, respectively. Most patients were poor, 50-years-median-age males and housewives. Among the 237 MTC spoligotyped isolates, 232 corresponded to M. tuberculosis (104 spoligotypes in 24 clusters) and five to M. bovis. The predominant Euro-American lineage was distributed all over the state, the East-Asian lineage (Beijing family) in the capital city, the Indo-Oceanic (Manila family) in eastern localities, and M. bovis in rural localities. In San Luis Potosí TB affects mainly poor male adults and is caused by M. tuberculosis and to a minor extent by M. bovis. There is great genotypic diversity among M. tuberculosis strains, the Euro-American lineage being much more prevalent than the Indo-Oceanic and East-Asian lineages. The frequency of resistant strains is relatively low and not associated to any particular lineage.

Genetic diversity of the Mycobacterium tuberculosis Complex in San Luis Potosí, México

PubMed Central

2013-01-01

Background Although epidemiologic and socioeconomic criteria and biomedical risk factors indicate high-priority for tuberculosis (TB) control in Mexico, molecular epidemiology studies of the disease in the country are scarce. Methods Complete sociodemographic and clinical data were obtained from 248 of the 432 pulmonary TB (PTB) cases confirmed from 2006 to 2010 on the population under epidemiological surveillance in the state of San Luis Potosí, México. From most PTB cases with complete data Mycobacterium tuberculosis complex (MTC) isolates were recovered and their spoligotypes, lineages and families, geographic distribution and drug resistance determined. Results Pulmonary tuberculosis incidence ranged from 2.4 to 33.4 (cases per 100,000 inhabitants) in the six state sanitary jurisdictions that were grouped in regions of low (jurisdictions I-II-III), intermediate (jurisdictions IV-V) and high incidence (jurisdiction VI) with 6.2, 17.3 and 33.4 rates, respectively. Most patients were poor, 50-years-median-age males and housewives. Among the 237 MTC spoligotyped isolates, 232 corresponded to M. tuberculosis (104 spoligotypes in 24 clusters) and five to M. bovis. The predominant Euro-American lineage was distributed all over the state, the East-Asian lineage (Beijing family) in the capital city, the Indo-Oceanic (Manila family) in eastern localities, and M. bovis in rural localities. Conclusions In San Luis Potosí TB affects mainly poor male adults and is caused by M. tuberculosis and to a minor extent by M. bovis. There is great genotypic diversity among M. tuberculosis strains, the Euro-American lineage being much more prevalent than the Indo-Oceanic and East-Asian lineages. The frequency of resistant strains is relatively low and not associated to any particular lineage. PMID:23635381

Transmission of Mycobacterium tuberculosis between Farmers and Cattle in Central Ethiopia

PubMed Central

Ameni, Gobena; Tadesse, Konjit; Hailu, Elena; Deresse, Yohannes; Medhin, Girmay; Aseffa, Abraham; Hewinson, Glyn; Vordermeier, Martin; Berg, Stefan

2013-01-01

Background Transmission of Mycobacterium tuberculosis (M. tuberculosis) complex could be possible between farmers and their cattle in Ethiopia. Methodology/Principal Findings A study was conducted in mixed type multi-purposes cattle raising region of Ethiopia on 287 households (146 households with case of pulmonary tuberculosis (TB) and 141 free of TB) and 287 herds consisting of 2,033 cattle belonging to these households to evaluate transmission of TB between cattle and farmers. Interview, bacteriological examinations and molecular typing were used for human subjects while comparative intradermal tuberculin (CIDT) test, post mortem and bacteriological examinations, and molecular typing were used for animal studies. Herd prevalence of CIDT reactors was 9.4% and was higher (p<0.01) in herds owned by households with TB than in herds owned by TB free households. Animal prevalence was 1.8% and also higher (p<0.01) in cattle owned by households with TB case than in those owned by TB free households. All mycobacteria (141) isolated from farmers were M. tuberculosis, while only five of the 16 isolates from cattle were members of the M. tuberculosis complex (MTC) while the remaining 11 were members of non-tuberculosis mycobacteria (NTM). Further speciation of the five MTC isolates showed that three of the isolates were M. bovis (strain SB1176), while the remaining two were M. tuberculosis strains (SIT149 and SIT53). Pathology scoring method described by “Vordermeier et al. (2002)” was applied and the average severity of pathology in two cattle infected with M. bovis, in 11 infected with NTM and two infected with M. tuberculosis were 5.5, 2.1 and 0.5, respectively. Conclusions/Significance The results showed that transmission of TB from farmers to cattle by the airborne route sensitizes the cows but rarely leads to TB. Similarly, low transmission of M. bovis between farmers and their cattle was found, suggesting requirement of ingestion of contaminated milk from cows with tuberculous mastitis. PMID:24130804

Transmission of Mycobacterium tuberculosis between farmers and cattle in central Ethiopia.

PubMed

Ameni, Gobena; Tadesse, Konjit; Hailu, Elena; Deresse, Yohannes; Medhin, Girmay; Aseffa, Abraham; Hewinson, Glyn; Vordermeier, Martin; Berg, Stefan

2013-01-01

Transmission of Mycobacterium tuberculosis (M. tuberculosis) complex could be possible between farmers and their cattle in Ethiopia. A study was conducted in mixed type multi-purposes cattle raising region of Ethiopia on 287 households (146 households with case of pulmonary tuberculosis (TB) and 141 free of TB) and 287 herds consisting of 2,033 cattle belonging to these households to evaluate transmission of TB between cattle and farmers. Interview, bacteriological examinations and molecular typing were used for human subjects while comparative intradermal tuberculin (CIDT) test, post mortem and bacteriological examinations, and molecular typing were used for animal studies. Herd prevalence of CIDT reactors was 9.4% and was higher (p<0.01) in herds owned by households with TB than in herds owned by TB free households. Animal prevalence was 1.8% and also higher (p<0.01) in cattle owned by households with TB case than in those owned by TB free households. All mycobacteria (141) isolated from farmers were M. tuberculosis, while only five of the 16 isolates from cattle were members of the M. tuberculosis complex (MTC) while the remaining 11 were members of non-tuberculosis mycobacteria (NTM). Further speciation of the five MTC isolates showed that three of the isolates were M. bovis (strain SB1176), while the remaining two were M. tuberculosis strains (SIT149 and SIT53). Pathology scoring method described by "Vordermeier et al. (2002)" was applied and the average severity of pathology in two cattle infected with M. bovis, in 11 infected with NTM and two infected with M. tuberculosis were 5.5, 2.1 and 0.5, respectively. The results showed that transmission of TB from farmers to cattle by the airborne route sensitizes the cows but rarely leads to TB. Similarly, low transmission of M. bovis between farmers and their cattle was found, suggesting requirement of ingestion of contaminated milk from cows with tuberculous mastitis.

Transmission of tuberculosis among people living in the border areas of Poland, the Czech Republic, and Slovakia.

PubMed

Kozińska, Monika; Zientek, Jerzy; Augustynowicz-Kopeć, Ewa; Zwolska, Zofia; Kozielski, Jerzy

2016-01-01

In 2007, Poland, the Czech Republic, and Slovakia joined the Schengen Agreement, abolishing restrictions on people crossing the borders. Currently, these areas are places of population movements for economic, family, and touristic reasons. This favors the transmission of infectious diseases, including tuberculosis, and requires enhanced control over the spread of the source of infection in the population of patients living in the border areas. The aim of this study was to investigate the genetic relatedness among Mycobacterium tuberculosis complex strains isolated from patients living in 3 border areas: Poland, the Czech Republic, and Slovakia. PATIENTS AND METHODS The study group consisted of 209 patients with tuberculosis diagnosed and treated between 2007 and 2011 in health care facilities in the Silesia Province in Poland (121 patients [58%]), Žilina in Slovakia (57 [27%]), and the Moravian-Silesian Region in the Czech Republic (31 [15%]). Genotyping of strains was performed using spoligotyping and IS6110-Mtb1-Mtb2 polymerase chain reaction. Among 209 strains, 23 molecular families (clusters) were identified. Seventeen clusters were identified as national. Six international clusters consisted of 30 strains isolated from patients of various nationalities. We identified 6 potential outbreaks of tuberculosis transmission between patients of different nationalities. The circumstances favorable to potential contacts of patients included mainly travelling to the neighboring countries, hospital stays, and addictions. However, there was no evidence of an epidemiological link between these patients, so it may be assumed that if they had come in contact with one another, it was accidental. We observed that the greater incidence of tuberculosis on the Polish territory did not affect the incidence in the Czech Republic or Slovakia over the analysis period.

Mycobacterium smegmatis strain for detection of Mycobacterium tuberculosis by PCR used as internal control for inhibition of amplification and for quantification of bacteria.

PubMed Central

Kolk, A H; Noordhoek, G T; de Leeuw, O; Kuijper, S; van Embden, J D

1994-01-01

For the detection of Mycobacterium tuberculosis by PCR, the IS6110 sequence was used. A modified target was constructed by insertion of 56 nucleotides in the IS6110 insertion element of Mycobacterium bovis BCG. This modified insertion sequence was integrated into the genome of Mycobacterium smegmatis, a mycobacterium species which does not contain the IS6110 element. When DNA from the modified M. smegmatis 1008 strain was amplified with IS6110-specific primers INS1 and INS2, a band of 301 bp was seen on agarose gel, whereas the PCR product of M. tuberculosis complex DNA was a 245-bp fragment with these primers. The addition of a small number of M. smegmatis 1008 cells to clinical samples before DNA purification enables the detection of problems which may be due to the loss of DNA in the isolation procedure or to the presence of inhibitors. The presence of inhibitors of the amplification reaction can be confirmed by the addition of M. smegmatis 1008 DNA after the DNA isolation procedure. Furthermore, competition between the different target DNAs of M. smegmatis 1008 DNA and M. tuberculosis complex DNA enables the estimation of the number of IS6110 elements in the clinical sample. Images PMID:8051267

In Vitro Activity and MIC of Sitafloxacin against Multidrug-Resistant and Extensively Drug-Resistant Mycobacterium tuberculosis Isolated in Thailand

PubMed Central

Leechawengwongs, Manoon; Prammananan, Therdsak; Jaitrong, Sarinya; Billamas, Pamaree; Makhao, Nampueng; Thamnongdee, Nongnard; Thanormchat, Arirat; Phurattanakornkul, Arisa; Rattanarangsee, Somcharn; Ratanajaraya, Chate; Disratthakit, Areeya

2017-01-01

ABSTRACT New fluoroquinolones (FQs) have been shown to be more active against drug-resistant Mycobacterium tuberculosis strains than early FQs, such as ofloxacin. Sitafloxacin (STFX) is a new fluoroquinolone with in vitro activity against a broad range of bacteria, including M. tuberculosis. This study aimed to determine the in vitro activity of STFX against all groups of drug-resistant strains, including multidrug-resistant M. tuberculosis (MDR M. tuberculosis), MDR M. tuberculosis with quinolone resistance (pre-XDR), and extensively drug-resistant (XDR) strains. A total of 374 drug-resistant M. tuberculosis strains were tested for drug susceptibility by the conventional proportion method, and 95 strains were randomly submitted for MIC determination using the microplate alamarBlue assay (MABA). The results revealed that all the drug-resistant strains were susceptible to STFX at a critical concentration of 2 μg/ml. Determination of the MIC90s of the strains showed different MIC levels; MDR M. tuberculosis strains had a MIC90 of 0.0625 μg/ml, whereas pre-XDR and XDR M. tuberculosis strains had identical MIC90s of 0.5 μg/ml. Common mutations within the quinolone resistance-determining region (QRDR) of gyrA and/or gyrB did not confer resistance to STFX, except that double mutations of GyrA at Ala90Val and Asp94Ala were found in strains with a MIC of 1.0 μg/ml. The results indicated that STFX had potent in vitro activity against all the groups of drug-resistant M. tuberculosis strains and should be considered a new repurposed drug for treatment of multidrug-resistant and extensively drug-resistant TB. PMID:29061759

Diagnosis and Treatment of Drug-Resistant Tuberculosis.

PubMed

Caminero, José A; Cayla, Joan A; García-García, José-María; García-Pérez, Francisco J; Palacios, Juan J; Ruiz-Manzano, Juan

2017-09-01

In the last 2 decades, drug-resistant tuberculosis has become a threat and a challenge to worldwide public health. The diagnosis and treatment of these forms of tuberculosis are much more complex and prognosis clearly worsens as the resistance pattern intensifies. Nevertheless, it is important to remember that with the appropriatesystematic clinical management, most of these patients can be cured. These guidelines itemize the basis for the diagnosis and treatment of all tuberculosis patients, from those infected by strains that are sensitive to all drugs, to those who are extensively drug-resistant. Specific recommendations are given forall cases. The current and future role of new molecular methods for detecting resistance, shorter multi-drug-resistant tuberculosis regimens, and new drugs with activity against Mycobacterium tuberculosis are also addressed. Copyright © 2017 SEPAR. Publicado por Elsevier España, S.L.U. All rights reserved.

Oligonucleotide (GTG)5 as a marker for Mycobacterium tuberculosis strain identification.

PubMed Central

Wiid, I J; Werely, C; Beyers, N; Donald, P; van Helden, P D

1994-01-01

Culture of Mycobacterium tuberculosis provides no information on the identity of a strain or the distribution of such a strain in the community. Strain identification of M. tuberculosis can help to address important epidemiological questions, e.g., the origin of an infection in a patient's household or community, whether reactivation of infection is endogenous or exogenous in origin, and the spread and early detection of organisms with acquired antibiotic resistance. To research this problem, strain identification must be reliable and accurate. Although genetic identification techniques already exist, it is valuable to have genetic identification techniques based on a number of genetic markers to improve the accurate identification of M. tuberculosis strains. We show that oligonucleotide (GTG)5 can be successfully applied to the identification of M. tuberculosis strains. This technique may be particularly useful in cases in which M. tuberculosis strains have few or no insertion elements (e.g., IS6110) or in identifying other strains of mycobacteria when informative probes are lacking. Images PMID:7914207

Advances in Mycobacterium tuberculosis therapeutics discovery utlizing structural biology

PubMed Central

Chim, Nicholas; Owens, Cedric P.; Contreras, Heidi; Goulding, Celia W.

2013-01-01

In 2012, tuberculosis (TB) remains a global health threat and is exacerbated both by the emergence of drug resistant Mycobacterium tuberculosis strains and its synergy with HIV infection. The waning effectiveness of current treatment regimens necessitates the development of new or repurposed anti-TB therapeutics for improved combination therapies against the disease. Exploiting atomic resolution structural information of proteins in complex with their substrates and/or inhibitors can facilitate structure-based rational drug design. Since our last review in 2009, there has been a wealth of new M. tuberculosis protein structural information. Once again, we have compiled the most promising structures with regards to potential anti-TB drug development and present them in this updated review. PMID:23167715

[Mycobacterium tuberculosis strain transmission caused by migratory processes in the Russian Federation (in case of populational migration from the Caucasian Region to Moscow and the Moscow Region)].

PubMed

Andreevskaia, S N; Chernousova, L N; Smirnova, T G; Larionova, E E; Kuz'min, A V

2006-01-01

The investigation was carried out on 134 M. tuberculosis isolated from 134 patients treated at the Central Research Institute of Tuberculosis, Russian Academy of Medical Sciences. The patients were divided into 2 groups: 1) those who were natives of Moscow and the Moscow Region (MR patients); 2) those who were migrants to the Moscow Region from Azerbaijan, Daghestan, Chechnya, Ingushetia, Karachai-Cherkessia, North Ossetia (the Caucasian Region) (CR patients) who had fallen in the place of birth. Genotyping by the polymorphism of lengths of the restriction fragments containing the insertion sequence IS6110 revealed a genetic diversity of M. tuberculosis strains. The examined M. tuberculosis strains belonged to 13 genotypic families. The W and AI families were prevalent. The family W M. tuberculosis strains isolated from the Caucasians were highly clustered, as confirmed by the overwhelming predominance of the strain variant W148 (19.7%). The spectrum of the strain variants of the W family, and those of the AI family in particular, greatly differed in MR and CR patients. Only one strain variant AI12 occurring both in MR and CR patients was detected. A study of the transmission activity coefficient (TAC) of the families W and AI indicated that the transmission activity of W strains was significantly higher than that of M. tuberculosis strains of the AI family. A comparative analysis of the TAC of M. tuberculosis strains of the AI family demonstrated that the transmission activity of the strains of this family was identical no matter where a patient had fallen ill (1.59 and 1.41% in the Moscow and Caucasian Regions, respectively). Unlike M. tuberculosis strains of the AI family, the TAC of W strains isolated from the patients infected in the Moscow Region (28.17 and 19.05%, respectively), which suggests the more intensive transmission of the pathogen M. tuberculosis of the W family in the Caucasian Region.

Rapid identification of Mycobacterium avium ssp paratuberculosis laboratory strains by IS900-Nested polymerase chain reaction.

PubMed

Taheri, Mohammad Mohammad; Mosavari, Nader; Feizabadi, Mohammad Mehdi; Tadayon, Keyvan; Keshavarz, Rouholah; Pajoohi, Reza Aref; Soleimani, Kioomars; Pour, Shojaat Dashti

2016-12-01

Mycobacterium avium ssp paratuberculosis (MAP) causes paratuberculosis (Johne's disease) in ruminants. As a species, M. avium comprises M. avium subsp. hominissuis and a number of clones that are known to have evolved from this subspecies, namely M. avium subsp. avium (MAA), M. avium subsp. silvaticum, and MAP. Despite the very high genomic similarity of MAP and MAA, the insertion sequence IS900, which is 1,451-bp long, is now understood to be exclusively present in 10-20 copies in the genome of MAP. In the present study, a multidiscipline polymerase chain reaction (PCR)-based algorithm targeting16SrRNA, IS6110, IS901, IS1245, and IS900 markers has been employed to differentiate between six laboratory strains of M. avium complex (including MAP 316F, III&V, and 2e plus MAA D4), Mycobacterium tuberculosis DT, and Mycobacterium bovis AN5 strains used at the Razi Institute (Tehran, Iran) for the preparation of paratuberculin, avian, human, and bovine tuberculin, respectively. Three laboratory strains of III&V, 2e, and 316F were subcultured on Herrold's egg yolk medium, whereas the MAA strain of D4 along with M. bovis AN5 and M. tuberculosis DT were subcultured on Lowenstein-Jensen slopes. All the inoculated culture tubes were incubated for 8weeks at 37°C. Eventually, their genomic DNA was extracted according to the method of van Soolingen. Five individual PCRs were conducted on these templates to amplify 16SrRNA (genus-specific marker shared by all mycobacteria), IS900 (MAP-specific marker), IS901 (MAA-specific marker), IS1245 (M. avium complex (MAC)-specific marker), and IS6110 (M. tuberculosis complex (MTC)-specific marker) loci. Consequently, a 543-bp amplicon was amplified by all the six strains in PCR against 16SrRNA, an indication of their identity as members of Mycobacterium genus. A 245-bp fragment was detected in only IS6110-PCR with M. bovis AN5 as well as M. tuberculosis DT. In the IS1245 assessment, the MAA strain of D4 produced a 427-bp amplicon, whereas none of the other studied strains produced this amplicon. A 1,108-bp amplicon fragment of the IS901 marker was successfully produced by MAA strain, whereas no PCR product was achieved in amplification of all the three MAP strains. In IS900-nested PCR, the three MAP strains produced the expected 400-bp and 298-bp fragments CONCLUTION: However, no amplification was observed with other strains. Two main achievements of this work are the development of an efficient means of differentiation between the six Razi laboratory mycobacterial strains and characterization of the genomic profile of these strains, a capability that is vital when cross contamination is potentially an important concern. Copyright © 2016.

First insights into the genetic diversity of Mycobacterium tuberculosis isolates from HIV-infected Mexican patients and mutations causing multidrug resistance

PubMed Central

2010-01-01

Background The prevalence of infections with Mycobacterium tuberculosis (MTb) and nontuberculous mycobacteria (NTM) species in HIV-infected patients in Mexico is unknown. The aims of this study were to determine the frequency of MTb and NTM species in HIV-infected patients from Mexico City, to evaluate the genotypic diversity of the Mycobacterium tuberculosis complex strains, to determine their drug resistance profiles by colorimetric microplate Alamar Blue assay (MABA), and finally, to detect mutations present in katG, rpoB and inhA genes, resulting in isoniazid (INH) and rifampin (RIF) resistance. Results Of the 67 mycobacterial strains isolated, 48 were identified as MTb, 9 as M. bovis, 9 as M. avium and 1 as M. intracellulare. IS6110-RFLP of 48 MTb strains showed 27 profiles. Spoligotyping of the 48 MTb strains yielded 21 patterns, and 9 M. bovis strains produced 7 patterns. Eleven new spoligotypes patterns were found. A total of 40 patterns were produced from the 48 MTb strains when MIRU-VNTR was performed. Nineteen (39.6%) MTb strains were resistant to one or more drugs. One (2.1%) multidrug-resistant (MDR) strain was identified. A novel mutation was identified in a RIF-resistant strain, GAG → TCG (Glu → Ser) at codon 469 of rpoB gene. Conclusions This is the first molecular analysis of mycobacteria isolated from HIV-infected patients in Mexico, which describe the prevalence of different mycobacterial species in this population. A high genetic diversity of MTb strains was identified. New spoligotypes and MIRU-VNTR patterns as well as a novel mutation associated to RIF-resistance were found. This information will facilitate the tracking of different mycobacterial species in HIV-infected individuals, and monitoring the spread of these microorganisms, leading to more appropriate measures for tuberculosis control. PMID:20236539

Direct Detection by the Xpert MTB/RIF Assay and Characterization of Multi and Poly Drug-Resistant Tuberculosis in Guinea-Bissau, West Africa

PubMed Central

Ponce, Gema; Sanca, Lilica; Mané, Morto; Armada, Ana; Machado, Diana; Vieira, Fina; Gomes, Victor F.; Martins, Elisabete; Colombatti, Raffaella; Riccardi, Fabio; Perdigão, João; Sotero, Joana; Portugal, Isabel; Couto, Isabel; Atouguia, Jorge; Rodrigues, Amabélia; Viveiros, Miguel

2015-01-01

Background This study aimed to evaluate the usefulness of the Xpert MTB/RIF assay for the rapid direct detection of M. tuberculosis complex (MTBC) strains and rifampicin resistance associated mutations in a resource-limited setting such as Guinea-Bissau and its implications in the management of tuberculosis (TB) and drug resistant tuberculosis, complementing the scarce information on resistance and genotypic diversity of MTBC strains in this West African country. Methods and Results This cross-sectional prospective study included 100 consecutive TB patients with positive acid-fast smears at two months of anti-tuberculosis treatment or in a re-treatment situation, between May and December 2012. Resistance to rifampicin was detected using the GeneXpert system and the Xpert MTB/RIF assay. MTBC isolates obtained with the BACTEC MGIT 960 system were tested for susceptibility to first- and second-line anti-tuberculosis drugs. Overall, the prevalence of multidrug-resistant tuberculosis (MDR-TB) was found to be 9 cases. Of these, 67% (6 patients) of confirmed MDR-TB cases had no past history of TB treatment and 33% (3 patients) were previously treated cases. Extensively drug-resistant TB was not found. Molecular typing of the MDR-TB strains revealed recent transmission patterns of imported MDR strains. Conclusions The Xpert MTB/RIF assay was reliable for the detection of rifampicin resistant MTBC strains directly from sputum samples of patients undergoing first-line treatment for two months, being more trustworthy than the simple presence of acid-fast bacilli in the smear. Its implementation is technically simple, does not require specialized laboratory infrastructures and is suitable for resource-limited settings when a regular source of electricity and maintenance is available as well as financial and operation sustainability is guaranteed by the health authorities. A high prevalence of MDR-TB among patients at risk of MDR-TB after two months of first-line treatment was found, in support of the WHO recommendations for its use in the management of this risk group. PMID:26017968

[Molecular-genetic characteristics of Mycobacterium tuberculosis strains isolated from patients with tuberculous spondylitis].

PubMed

Viazovaia, A A; Solov'eva, N S; Zhuravlev, V Iu; Mokrousov, I V; Manicheva, O A; Vishnevskiĭ, B I; Narvskaia, O V

2013-01-01

Molecular-genetic characteristic of M. tuberculosis strains isolated from operation material of patients with tuberculous spondylitis. 107 strains of M. tuberculosis isolated in 2007 - 2011 from patients with spine tuberculosis were studied by methods of spoligotyping and MIRU-VNTR by 12 and 24 loci. Strains of genetic family Beijing dominated (n = 80), 78% of those had multiple drug resistance (MDR). Strains of genetic families T, H3 (Ural), LAM, Manu, H4 and S were also detected. Differentiating of 80 strains of Beijing genotype by MIRU-VNTR method by 24 loci revealed 24 variants (HGI = 0.83) including 7 clusters, the largest of those (100-32) included 23 strains (87% MDR). The leading role of Beijing genotype M. tuberculosis strains in development of tuberculous spondylitis with multiple drug resistance of the causative agent is shown.

Strain-based HLA association analysis identified HLA-DRB1*09:01 associated with modern strain tuberculosis.

PubMed

Toyo-Oka, L; Mahasirimongkol, S; Yanai, H; Mushiroda, T; Wattanapokayakit, S; Wichukchinda, N; Yamada, N; Smittipat, N; Juthayothin, T; Palittapongarnpim, P; Nedsuwan, S; Kantipong, P; Takahashi, A; Kubo, M; Sawanpanyalert, P; Tokunaga, K

2017-09-01

Tuberculosis (TB) occurs as a result of complex interactions between the host immune system and pathogen virulence factors. Human leukocyte antigen (HLA) class II molecules play an important role in the host immune system. However, no study has assessed the association between HLA class II genes and susceptibility to TB caused by specific strains. This study investigated the possible association of HLA class II genes with TB caused by modern and ancient Mycobacterium tuberculosis (MTB). The study included 682 patients with TB and 836 control subjects who were typed for HLA-DRB1 and HLA-DQB1 alleles. MTB strains were classified using a large sequence polymorphism typing method. Association analysis was performed using common HLA alleles and haplotypes in different MTB strains. HLA association analysis of patients infected with modern MTB strains showed significant association for HLA-DRB1*09:01 (odds ratio [OR] = 1.82; P-value = 9.88 × 10 -4 ) and HLA-DQB1*03:03 alleles (OR = 1.76; P-value = 1.31 × 10 -3 ) with susceptibility to TB. Haplotype analysis confirmed that these alleles were in strong linkage disequilibrium and did not exert an interactive effect. Thus, the results of this study showed an association between HLA class II genes and susceptibility to TB caused by modern MTB strains, suggesting the importance of strain-specific analysis to determine susceptibility genes associated with TB. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Use of the VNTR typing technique to determine the origin of Mycobacterium tuberculosis strains isolated from Filipino patients in Korea.

PubMed

Lee, Jihye; Tupasi, Thelma E; Park, Young Kil

2014-05-01

With increasing international interchange of personnel, international monitoring is necessary to decrease tuberculosis incidence in the world. This study aims to develop a new tool to determine origin of Mycobacterium tuberculosis strains isolated from Filipino patients living in Korea. Thirty-two variable number tandem repeat (VNTR) loci were used for discrimination of 50 Filipino M. tuberculosis strains isolated in the Philippines, 317 Korean strains isolated in Korea, and 8 Filipino strains isolated in Korea. We found that the VNTR loci 0580, 0960, 2531, 2687, 2996, 0802, 2461, 2163a, 4052, 0424, 1955, 2074, 2347, 2401, 3171, 3690, 2372, 3232, and 4156 had different mode among copy numbers or exclusively distinct copy number in VNTR typing between Filipino and Korean M. tuberculosis strains. When these differences of the VNTR loci were applied to 8 Filipino M. tuberculosis strains isolated in Korea, 6 of them revealed Filipino type while 2 of them had Korean type. Using the differences of mode or repeated number of VNTR loci were very useful in distinguishing the Filipino strain from Korean strain.

Molecular characterization of Mycobacterium tuberculosis complex (MTBC) isolated from cattle in northeast and northwest China.

PubMed

Du, Yanfen; Qi, Yingfang; Yu, Lu; Lin, Jingkai; Liu, Siguo; Ni, Hongbo; Pang, Hai; Liu, Huifang; Si, Wei; Zhao, Hailing; Wang, Chunlai

2011-06-01

We studied throat swabs and corresponding serum samples collected from 1067 protein purified derivative (PPD)-tuberculin skin test (TST) positive cattle from different regions of China. The 1067 throat swabs were inoculated onto modified Löwenstein-Jensen medium for the isolation and culture of Mycobacteria. Acid-fast bacilli were identified using traditional biochemical methods, polymerase chain reaction (PCR) amplification and multiplex PCR. They were distinguished as Mycobacterium tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM) strains. An indirect Enzyme-Linked Immunosorbent Assay (ELISA) was applied to detect specific antibodies against bovine TB (bTB). Correlations among the ELISA, bacteriology and TST were analyzed and compared. Spoligotyping and variable number tandem repeats-mycobacterial interspersed repetitive unit (VNTR-MIRU) analysis were used to genotype the MTBC. In total, 111 strains of Mycobacteria were cultured from the 1067 throat swab samples, including 43 stains of MTBC (14 strains of Mycobacterium bovis and 29 of Mycobacterium tuberculosis) and 68 strains of NTM. Thirty-eight MTBC strains and four NTM strains were isolated from 72 throat swab samples that the ELISA determined were antibody positive; five MTBC strains and 64 NTM strains were isolated from 995 throat swab samples that were antibody negative on the ELISA. The positive isolation rates of MTBC and NTM were 38.7% (43/111) and 61.3% (68/111), respectively. The concordance rate of cultured MTBC with a positive result on the indirect ELISA for antibody was 52.8% (38/72), which was much higher than the positive rate for TST (4.0%; 43/1067). Genotyping of the 43 strains of MTBC isolated, using spoligotyping and VNTR-MIRU, showed that the 43 isolates had 26 genotypes; 16 strains had a unique genotype. Two groups of six strains and two strains, respectively, showed the same spoligotyping pattern, and belonged to the Beijing family and Beijing-like family, respectively. Combined application of spoligotyping and VNTR-MIRU typing would improve the molecular epidemiological investigation and monitoring of the etiology of bTB in China. Copyright © 2010 Elsevier Ltd. All rights reserved.

Genotyping and drug resistance patterns of M. tuberculosis strains in Pakistan.

PubMed

Tanveer, Mahnaz; Hasan, Zahra; Siddiqui, Amna R; Ali, Asho; Kanji, Akbar; Ghebremicheal, Solomon; Hasan, Rumina

2008-12-24

The incidence of tuberculosis in Pakistan is 181/100,000 population. However, information about transmission and geographical prevalence of Mycobacterium tuberculosis strains and their evolutionary genetics as well as drug resistance remains limited. Our objective was to determine the clonal composition, evolutionary genetics and drug resistance of M. tuberculosis isolates from different regions of the country. M. tuberculosis strains isolated (2003-2005) from specimens submitted to the laboratory through collection units nationwide were included. Drug susceptibility was performed and strains were spoligotyped. Of 926 M. tuberculosis strains studied, 721(78%) were grouped into 59 "shared types", while 205 (22%) were identified as "Orphan" spoligotypes. Amongst the predominant genotypes 61% were Central Asian strains (CAS ; including CAS1, CAS sub-families and Orphan Pak clusters), 4% East African-Indian (EAI), 3% Beijing, 2% poorly defined TB strains (T), 2% Haarlem and LAM (0.2). Also TbD1 analysis (M. tuberculosis specific deletion 1) confirmed that CAS1 was of "modern" origin while EAI isolates belonged to "ancestral" strain types.Prevalence of CAS1 clade was significantly higher in Punjab (P < 0.01, Pearsons Chi-square test) as compared with Sindh, North West Frontier Province and Balochistan provinces. Forty six percent of isolates were sensitive to five first line antibiotics tested, 45% were Rifampicin resistant, 50% isoniazid resistant. MDR was significantly associated with Beijing strains (P = 0.01, Pearsons Chi-square test) and EAI (P = 0.001, Pearsons Chi-square test), but not with CAS family. Our results show variation of prevalent M. tuberculosis strain with greater association of CAS1 with the Punjab province. The fact that the prevalent CAS genotype was not associated with drug resistance is encouraging. It further suggests a more effective treatment and control programme should be successful in reducing the tuberculosis burden in Pakistan.

The Evolutionary History, Demography, and Spread of the Mycobacterium tuberculosis Complex.

PubMed

Barbier, Maxime; Wirth, Thierry

2016-08-01

With the advent of next-generation sequencing technology, the genotyping of clinical Mycobacterium tuberculosis strains went through a major breakup that dramatically improved the field of molecular epidemiology but also revolutionized our deep understanding of the M. tuberculosis complex evolutionary history. The intricate paths of the pathogen and its human host are reflected by a common geographical origin in Africa and strong biogeographical associations that largely reflect the past migration waves out of Africa. This long coevolutionary history is cardinal for our understanding of the host-pathogen dynamic, including past and ongoing demographic components, strains' genetic background, as well as the immune system genetic architecture of the host. Coalescent- and Bayesian-based analyses allowed us to reconstruct population size changes of M. tuberculosis through time, to date the most recent common ancestor and the several phylogenetic lineages. This information will ultimately help us to understand the spread of the Beijing lineage, the rise of multidrug-resistant sublineages, or the fall of others in the light of socioeconomic events, antibiotic programs, or host population densities. If we leave the present and go through the looking glass, thanks to our ability to handle small degraded molecules combined with targeted capture, paleomicrobiology covering the Pleistocene era will possibly unravel lineage replacements, dig out extinct ones, and eventually ask for major revisions of the current model.

Impact of fgd1 and ddn Diversity in Mycobacterium tuberculosis Complex on In Vitro Susceptibility to PA-824 ▿ ‡

PubMed Central

Feuerriegel, Silke; Köser, Claudio U.; Baù, Davide; Rüsch-Gerdes, Sabine; Summers, David K.; Archer, John A. C.; Marti-Renom, Marc A.; Niemann, Stefan

2011-01-01

PA-824 is a promising drug candidate for the treatment of tuberculosis (TB). It is in phase II clinical trials as part of the first newly designed regimen containing multiple novel antituberculosis drugs (PA-824 in combination with moxifloxacin and pyrazinamide). However, given that the genes involved in resistance against PA-824 are not fully conserved in the Mycobacterium tuberculosis complex (MTBC), this regimen might not be equally effective against different MTBC genotypes. To investigate this question, we sequenced two PA-824 resistance genes (fgd1 [Rv0407] and ddn [Rv3547]) in 65 MTBC strains representing major phylogenetic lineages. The MICs of representative strains were determined using the modified proportion method in the Bactec MGIT 960 system. Our analysis revealed single-nucleotide polymorphisms in both genes that were specific either for several genotypes or for individual strains, yet none of these mutations significantly affected the PA-824 MICs (≤0.25 μg/ml). These results were supported by in silico modeling of the mutations identified in Fgd1. In contrast, “Mycobacterium canettii” strains displayed a higher MIC of 8 μg/ml. In conclusion, we found a large genetic diversity in PA-824 resistance genes that did not lead to elevated PA-824 MICs. In contrast, M. canettii strains had MICs that were above the plasma concentrations of PA-824 documented so far in clinical trials. As M. canettii is also intrinsically resistant against pyrazinamide, new regimens containing PA-824 and pyrazinamide might not be effective in treating M. canettii infections. This finding has implications for the design of multiple ongoing clinical trials. PMID:21930879

Evaluation of a new rapid kit, BD MGIT TBc identification test for confirmation of Mycobacterium tuberculosis complex.

PubMed

Kandhakumari, Gandhi; Stephen, Selvaraj

2017-01-01

At present, three rapid kits are available globally for the confirmation of Mycobacterium tuberculosis complex (MTBC) in cultures by MPT64 antigen (MPT64 Ag) detection. These include Capilia TB, SD Bioline, and BD MGIT TBc Identification (TBcID). The third kit is yet to be validated in India. We have tested this kit and compared with SD Bioline using conventional tests as gold standard. Seventy-one MTBC (70 M. tuberculosis and one Mycobacterium bovis) and four nontuberculous mycobacteria (NTM) were isolated from 649 clinical specimens in MGIT 960 and/or Lowenstein-Jensen slants (LJ). MPT64 Ag was detected by both TBcID and SD Bioline kits in all the 71 clinical isolates and the reference strain M. tuberculosis H37Rv. All NTM species tested were negative by the two different kits. Thus, TBcID kit showed 100% concordance in terms of sensitivity and specificity. Rapid kits confirm MTBC cultures within 15 min in contrast to several weeks' time required by conventional techniques.

The Evolution of Strain Typing in the Mycobacterium tuberculosis Complex.

PubMed

Merker, Matthias; Kohl, Thomas A; Niemann, Stefan; Supply, Philip

2017-01-01

Tuberculosis (TB) is a contagious disease with a complex epidemiology. Therefore, molecular typing (genotyping) of Mycobacterium tuberculosis complex (MTBC) strains is of primary importance to effectively guide outbreak investigations, define transmission dynamics and assist global epidemiological surveillance of the disease. Large-scale genotyping is also needed to get better insights into the biological diversity and the evolution of the pathogen. Thanks to its shorter turnaround and simple numerical nomenclature system, mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) typing, based on 24 standardized plus 4 hypervariable loci, optionally combined with spoligotyping, has replaced IS6110 DNA fingerprinting over the last decade as a gold standard among classical strain typing methods for many applications. With the continuous progress and decreasing costs of next-generation sequencing (NGS) technologies, typing based on whole genome sequencing (WGS) is now increasingly performed for near complete exploitation of the available genetic information. However, some important challenges remain such as the lack of standardization of WGS analysis pipelines, the need of databases for sharing WGS data at a global level, and a better understanding of the relevant genomic distances for defining clusters of recent TB transmission in different epidemiological contexts. This chapter provides an overview of the evolution of genotyping methods over the last three decades, which culminated with the development of WGS-based methods. It addresses the relative advantages and limitations of these techniques, indicates current challenges and potential directions for facilitating standardization of WGS-based typing, and provides suggestions on what method to use depending on the specific research question.

Complete annotated genome sequence of Mycobacterium tuberculosis (Zopf) Lehmann and Neumann (ATCC35812) (Kurono).

PubMed

Miyoshi-Akiyama, Tohru; Satou, Kazuhito; Kato, Masako; Shiroma, Akino; Matsumura, Kazunori; Tamotsu, Hinako; Iwai, Hiroki; Teruya, Kuniko; Funatogawa, Keiji; Hirano, Takashi; Kirikae, Teruo

2015-01-01

We report the completely annotated genome sequence of Mycobacterium tuberculosis (Zopf) Lehmann and Neumann (ATCC35812) (Kurono), which is a used for virulence and/or immunization studies. The complete genome sequence of M. tuberculosis Kurono was determined with a length of 4,415,078 bp and a G+C content of 65.60%. The chromosome was shown to contain a total of 4,340 protein-coding genes, 53 tRNA genes, one transfer messenger RNA for all amino acids, and 1 rrn operon. Lineage analysis based on large sequence polymorphisms indicated that M. tuberculosis Kurono belongs to the Euro-American lineage (lineage 4). Phylogenetic analysis using whole genome sequences of M. tuberculosis Kurono in addition to 22 M. tuberculosis complex strains indicated that H37Rv is the closest relative of Kurono based on the results of phylogenetic analysis. These findings provide a basis for research using M. tuberculosis Kurono, especially in animal models. Copyright © 2014 Elsevier Ltd. All rights reserved.

Prevalence of Mycobacterium tuberculosis strain genotypes in Taiwan reveals a close link to ethnic and population migration.

PubMed

Dou, Horng-Yunn; Chen, Yih-Yuan; Kou, Shu-Chen; Su, Ih-Jen

2015-06-01

Taiwan is a relatively isolated island, serving as a mixing vessel for colonization by different waves of ethnic and migratory groups over the past 4 centuries. The potential transmission pattern of Mycobacterium tuberculosis in different ethnic and migratory populations remains to be elucidated. By using mycobacterial tandem repeat sequences as genetic markers, the prevalence of M. tuberculosis strains in Taiwan revealed a close link to the historical migration. Interestingly, the M. tuberculosis strain in the aborigines of Eastern and Central Taiwan had a dominance of the Haarlem (Dutch) strain while those in Southern Taiwan had a dominance of the East-African Indian (EAI) strain. The prevalence of different M. tuberculosis strains in specific ethnic populations suggests that M. tuberculosis transmission is limited and restricted to close contact. The prevalence of the Beijing modern strain in the young population causes a concern for M. tuberculosis control, because of high virulence and drug resistance. Furthermore, our data using molecular genotyping should provide valuable information on the historical study of the origin and migration of aborigines in Taiwan. Copyright © 2014. Published by Elsevier B.V.

Molecular Characteristics of Mycobacterium tuberculosis Strains Isolated from Cutaneous Tuberculosis Patients in China.

PubMed

Jiang, Haiqin; Jin, Yali; Vissa, Varalakshmi; Zhang, Liangfen; Liu, Weijun; Qin, Lianhua; Wan, Kanglin; Wu, Xiaocui; Wang, Hongsheng; Liu, Weida; Wang, Baoxi

2017-04-06

Cutaneous tuberculosis (CTB) is probably underreported due to difficulties in detection and diagnosis. To address this issue, genotypes of Mycobacterium tuberculosis strains isolated from 30 patients with CTB were mapped at multiple loci, namely, RD105 deletions, spacer oligonucleotides, and Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeats (MIRU-VNTRs). Fifty-eight strains of pulmonary tuberculosis (PTB) were mapped as experimental controls. Drug resistance-associated gene mutations were determined by amplicon sequencing of target regions within 7 genes. Beijing family isolates were the most prevalent strains in CTB and PTB. MIRU-VNTR typing separated the Beijing strains from the non-Beijing strains, and the majority of CTB could be separated from PTB counterparts. Drug resistance determining regions showed only one CTB strain expressing isomazid resistance. Thus, while the CTB strains belonged to the same phylogenetic lineages and sub-lineages as the PTB strains, they differed at the level of several MIRU-VNTRs and in the proportion of drug resistance.

Evolutionary landscape of the Mycobacterium tuberculosis complex from the viewpoint of PhoPR: implications for virulence regulation and application to vaccine development.

PubMed

Broset, Esther; Martín, Carlos; Gonzalo-Asensio, Jesús

2015-10-20

Different members of the Mycobacterium genus have evolved to cause tuberculosis in diverse human populations and in a variety of animal species. Our cumulative knowledge of mycobacterial genomes indicates that mutations in the PhoPR two-component virulence system were acquired not only during the natural evolution of mycobacterial species but also during in vitro subculture, which has given rise to the attenuated reference strain H37Ra or to different daughter strains of Mycobacterium bovis BCG. PhoPR is a well-known regulator of pathogenic phenotypes, including secretion of the virulence factor ESAT-6, biosynthesis of acyltrehalose-based lipids, and modulation of antigen export, in members of the Mycobacterium tuberculosis complex (MTBC). Evolutionarily conserved polymorphisms in PhoPR from Mycobacterium africanum, M. bovis, or M. tuberculosis H37Ra result in loss of functional phenotypes. Interestingly, some members of the MTBC have acquired compensatory mutations to counteract these polymorphisms and, probably, to maintain their pathogenic potential. Some of these compensatory mutations include the insertion of the IS6110 element upstream from phoPR in a particular M. bovis strain that is able to transmit between humans or polymorphisms in M. africanum and M. bovis that affect the regulatory region of the espACD operon, allowing PhoPR-independent ESAT-6 secretion. This review highlights the increasing knowledge of the significance of PhoPR in the evolution of the MTBC and its potential application in the construction of new attenuated vaccines based on phoPR inactivation. In this context, the live attenuated vaccine MTBVAC, based on a phoP fadD26 deletion mutant of M. tuberculosis, is the first vaccine of this kind to successfully enter into clinical development, representing a historic milestone in the field of human vaccinology. Copyright © 2015 Broset et al.

Evolutionary Landscape of the Mycobacterium tuberculosis Complex from the Viewpoint of PhoPR: Implications for Virulence Regulation and Application to Vaccine Development

PubMed Central

Broset, Esther

2015-01-01

ABSTRACT Different members of the Mycobacterium genus have evolved to cause tuberculosis in diverse human populations and in a variety of animal species. Our cumulative knowledge of mycobacterial genomes indicates that mutations in the PhoPR two-component virulence system were acquired not only during the natural evolution of mycobacterial species but also during in vitro subculture, which has given rise to the attenuated reference strain H37Ra or to different daughter strains of Mycobacterium bovis BCG. PhoPR is a well-known regulator of pathogenic phenotypes, including secretion of the virulence factor ESAT-6, biosynthesis of acyltrehalose-based lipids, and modulation of antigen export, in members of the Mycobacterium tuberculosis complex (MTBC). Evolutionarily conserved polymorphisms in PhoPR from Mycobacterium africanum, M. bovis, or M. tuberculosis H37Ra result in loss of functional phenotypes. Interestingly, some members of the MTBC have acquired compensatory mutations to counteract these polymorphisms and, probably, to maintain their pathogenic potential. Some of these compensatory mutations include the insertion of the IS6110 element upstream from phoPR in a particular M. bovis strain that is able to transmit between humans or polymorphisms in M. africanum and M. bovis that affect the regulatory region of the espACD operon, allowing PhoPR-independent ESAT-6 secretion. This review highlights the increasing knowledge of the significance of PhoPR in the evolution of the MTBC and its potential application in the construction of new attenuated vaccines based on phoPR inactivation. In this context, the live attenuated vaccine MTBVAC, based on a phoP fadD26 deletion mutant of M. tuberculosis, is the first vaccine of this kind to successfully enter into clinical development, representing a historic milestone in the field of human vaccinology. PMID:26489860

Interleukin-1 or tumor necrosis factor-alpha augmented the cytotoxic effect of mycobacteria on human fibroblasts: application to evaluation of pathogenesis of clinical isolates of Mycobacterium tuberculosis and M. avium complex.

PubMed

Takii, T; Abe, C; Tamura, A; Ramayah, S; Belisle, J T; Brennan, P J; Onozaki, K

2001-03-01

Mycobacteria-induced in vitro events reflecting human tuberculosis can contribute to the evaluation of the pathogenesis of Mycobacterium tuberculosis (MTB). In this study, we propose such an in vitro method based on live mycobacteria-induced cytotoxicity to human cell lines. When human lung-derived normal fibroblast cell line MRC-5 was infected with various strains of mycobacteria (M. tuberculosis H(37)Rv and H(37) Ra, Mycobacterium avium 427S and 2151SmO, and Mycobacterium bovis BCG Pasteur and Tokyo), the fibroblasts were killed by mycobacteria according to the degree of virulence. Other human originated macrophage (U-937, THP-1), myeloid (HL-60), and epithelial carcinoma (A549) cell lines exhibited a similar cytotoxic response to virulent mycobacteria. MRC-5 was most susceptible to virulent mycobacteria among various human cell lines examined. The cytotoxicity was enhanced by the proinflammatory cytokines, interleukin-1 (IL-1) and tumor necrosis factor-a (TNF-alpha), which in the absence of mycobacteria stimulate the growth of normal human fibroblasts. This in vitro evaluation system was applied to clinical isolates of drug-sensitive MTB (DS-MTB), drug-resistant MTB (DR-MTB) including multidrug-resistant (MDR-MTB), and M. avium complex (MAC). MTB strains (n = 24) exhibited strong cytotoxic activity, but MAC strains (n = 5) had only weak activity. Furthermore, there was no significant difference in cytotoxicity between DS-MTB (n = 11) and DR-MTB (n = 13). Collectively, these results suggest that this new in vitro system is useful for evaluating the pathogenesis of mycobacteria and that there was no difference in the pathogenesis between drug-susceptible and drug-resistant clinical isolates.

Predominance of modern Mycobacterium tuberculosis strains and active transmission of Beijing sublineage in Jayapura, Indonesia Papua.

PubMed

Chaidir, Lidya; Sengstake, Sarah; de Beer, Jessica; Oktavian, Antonius; Krismawati, Hana; Muhapril, Erfin; Kusumadewi, Inri; Annisa, Jessi; Anthony, Richard; van Soolingen, Dick; Achmad, Tri Hanggono; Marzuki, Sangkot; Alisjahbana, Bachti; van Crevel, Reinout

2016-04-01

Mycobacterium tuberculosis genotype distribution is different between West and Central Indonesia, but there are no data on the most Eastern part, Papua. We aimed to identify the predominant genotypes of M. tuberculosis responsible for tuberculosis in coastal Papua, their transmission, and the association with patient characteristics. A total of 199 M. tuberculosis isolates were collected. Spoligotyping was applied to describe the population structure of M. tuberculosis, lineage identification was performed using a combination of lineage-specific markers, and genotypic clusters were identified using a combination of 24-locus-MIRU-VNTR and spoligotyping. A high degree of genetic diversity was observed among isolates based on their spoligopatterns. Strains from modern lineage 4 made up almost half of strains (46.9%), being more abundant than the ancient lineage 1 (33.7%), and modern lineage 2 (19.4%). Thirty-five percent of strains belonged to genotypic clusters, especially strains in the Beijing genotype. Previous TB treatment and mutations associated with drug resistance were more common in patients infected with strains of the Beijing genotype. Papua shows a different distribution of M. tuberculosis genotypes compared to other parts of Indonesia. Clustering and drug resistance of modern strains recently introduced to Papua may contribute to the high tuberculosis burden in this region. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification of proteins from Mycobacterium tuberculosis missing in attenuated Mycobacterium bovis BCG strains.

PubMed

Mattow, J; Jungblut, P R; Schaible, U E; Mollenkopf, H J; Lamer, S; Zimny-Arndt, U; Hagens, K; Müller, E C; Kaufmann, S H

2001-08-01

A proteome approach, combining high-resolution two-dimensional electrophoresis (2-DE) with mass spectrometry, was used to compare the cellular protein composition of two virulent strains of Mycobacterium tuberculosis with two attenuated strains of Mycobacterium bovis Bacillus Calmette-Guerin (BCG), in order to identify unique proteins of these strains. Emphasis was given to the identification of M. tuberculosis specific proteins, because we consider these proteins to represent putative virulence factors and interesting candidates for vaccination and diagnosis of tuberculosis. The genome of M. tuberculosis strain H37Rv comprises nearly 4000 predicted open reading frames. In contrast, the separation of proteins from whole mycobacterial cells by 2-DE resulted in silver-stained patterns comprising about 1800 distinct protein spots. Amongst these, 96 spots were exclusively detected either in the virulent (56 spots) or in the attenuated (40 spots) mycobacterial strains. Fifty-three of these spots were analyzed by mass spectrometry, of which 41 were identified, including 32 M. tuberculosis specific spots. Twelve M. tuberculosis specific spots were identified as proteins, encoded by genes previously reported to be deleted in M. bovis BCG. The remaining 20 spots unique for M. tuberculosis were identified as proteins encoded by genes that are not known to be missing in M. bovis BCG.

Admixed Phylogenetic Distribution of Drug Resistant Mycobacterium tuberculosis in Saudi Arabia

PubMed Central

Varghese, Bright; Supply, Philip; Allix-Béguec, Caroline; Shoukri, Mohammed; Al-Omari, Ruba; Herbawi, Mais; Al-Hajoj, Sahal

2013-01-01

Background The phylogeographical structure of Mycobacterium tuberculosis is generally bimodal in low tuberculosis (TB) incidence countries, where genetic lineages of the isolates generally differ with little strain clustering between autochthonous and foreign-born TB patients. However, less is known on this structure in Saudi Arabia—the most important hub of human migration as it hosts a total population of expatriates and pilgrims from all over the world which is equal to that of its citizens. Methodology We explored the mycobacterial phylogenetic structure and strain molecular clustering in Saudi Arabia by genotyping 322 drug-resistant clinical isolates collected over a 12-month period in a national drug surveillance survey, using 24 locus-based MIRU-VNTR typing and spoligotyping. Principal Findings In contrast to the cosmopolitan population of the country, almost all the known phylogeographic lineages of M. tuberculosis complex (with noticeable exception of Mycobacterium africanum/West-African 1 and 2) were detected, with Delhi/CAS (21.1%), EAI (11.2%), Beijing (11.2%) and main branches of the Euro-American super-lineage such as Ghana (14.9%), Haarlem (10.6%) and Cameroon (7.8%) being represented. Statistically significant associations of strain lineages were observed with poly-drug resistance and multi drug resistance especially among previously treated cases (p value of < = 0.001 for both types of resistance), with relative over-representation of Beijing strains in the latter category. However, there was no significant difference among Saudi and non-Saudi TB patients regarding distribution of phylogenetic lineages (p = 0.311). Moreover, 59.5% (22/37) of the strain molecular clusters were shared between the Saudi born and immigrant TB patients. Conclusions Specific distribution of M. tuberculosis phylogeographic lineages is not observed between the autochthonous and foreign-born populations. These observations might reflect both socially favored ongoing TB transmission between the two population groups, and historically deep-rooted, prolonged contacts and trade relations of the peninsula with other world regions. More vigorous surveillance and strict adherence to tuberculosis control policies are urgently needed in the country. PMID:23383340

Influence of Stress and Antibiotic Resistance on Cell-Length Distribution in Mycobacterium tuberculosis Clinical Isolates

PubMed Central

Vijay, Srinivasan; Vinh, Dao N.; Hai, Hoang T.; Ha, Vu T. N.; Dung, Vu T. M.; Dinh, Tran D.; Nhung, Hoang N.; Tram, Trinh T. B.; Aldridge, Bree B.; Hanh, Nguyen T.; Thu, Do D. A.; Phu, Nguyen H.; Thwaites, Guy E.; Thuong, Nguyen T. T.

2017-01-01

Mycobacterial cellular variations in growth and division increase heterogeneity in cell length, possibly contributing to cell-to-cell variation in host and antibiotic stress tolerance. This may be one of the factors influencing Mycobacterium tuberculosis persistence to antibiotics. Tuberculosis (TB) is a major public health problem in developing countries, antibiotic persistence, and emergence of antibiotic resistance further complicates this problem. We wanted to investigate the factors influencing cell-length distribution in clinical M. tuberculosis strains. In parallel we examined M. tuberculosis cell-length distribution in a large set of clinical strains (n = 158) from ex vivo sputum samples, in vitro macrophage models, and in vitro cultures. Our aim was to understand the influence of clinically relevant factors such as host stresses, M. tuberculosis lineages, antibiotic resistance, antibiotic concentrations, and disease severity on the cell size distribution in clinical M. tuberculosis strains. Increased cell size and cell-to-cell variation in cell length were associated with bacteria in sputum and infected macrophages rather than liquid culture. Multidrug-resistant (MDR) strains displayed increased cell length heterogeneity compared to sensitive strains in infected macrophages and also during growth under rifampicin (RIF) treatment. Importantly, increased cell length was also associated with pulmonary TB disease severity. Supporting these findings, individual host stresses, such as oxidative stress and iron deficiency, increased cell-length heterogeneity of M. tuberculosis strains. In addition we also observed synergism between host stress and RIF treatment in increasing cell length in MDR-TB strains. This study has identified some clinical factors contributing to cell-length heterogeneity in clinical M. tuberculosis strains. The role of these cellular adaptations to host and antibiotic tolerance needs further investigation. PMID:29209302

Genotyping and drug resistance patterns of M. tuberculosis strains in Pakistan

PubMed Central

Tanveer, Mahnaz; Hasan, Zahra; Siddiqui, Amna R; Ali, Asho; Kanji, Akbar; Ghebremicheal, Solomon; Hasan, Rumina

2008-01-01

Background The incidence of tuberculosis in Pakistan is 181/100,000 population. However, information about transmission and geographical prevalence of Mycobacterium tuberculosis strains and their evolutionary genetics as well as drug resistance remains limited. Our objective was to determine the clonal composition, evolutionary genetics and drug resistance of M. tuberculosis isolates from different regions of the country. Methods M. tuberculosis strains isolated (2003–2005) from specimens submitted to the laboratory through collection units nationwide were included. Drug susceptibility was performed and strains were spoligotyped. Results Of 926 M. tuberculosis strains studied, 721(78%) were grouped into 59 "shared types", while 205 (22%) were identified as "Orphan" spoligotypes. Amongst the predominant genotypes 61% were Central Asian strains (CAS ; including CAS1, CAS sub-families and Orphan Pak clusters), 4% East African-Indian (EAI), 3% Beijing, 2% poorly defined TB strains (T), 2% Haarlem and LAM (0.2). Also TbD1 analysis (M. tuberculosis specific deletion 1) confirmed that CAS1 was of "modern" origin while EAI isolates belonged to "ancestral" strain types. Prevalence of CAS1 clade was significantly higher in Punjab (P < 0.01, Pearsons Chi-square test) as compared with Sindh, North West Frontier Province and Balochistan provinces. Forty six percent of isolates were sensitive to five first line antibiotics tested, 45% were Rifampicin resistant, 50% isoniazid resistant. MDR was significantly associated with Beijing strains (P = 0.01, Pearsons Chi-square test) and EAI (P = 0.001, Pearsons Chi-square test), but not with CAS family. Conclusion Our results show variation of prevalent M. tuberculosis strain with greater association of CAS1 with the Punjab province. The fact that the prevalent CAS genotype was not associated with drug resistance is encouraging. It further suggests a more effective treatment and control programme should be successful in reducing the tuberculosis burden in Pakistan. PMID:19108722

Characterisation of Mycobacterium tuberculosis isolates lacking IS6110 in Viet Nam.

PubMed

Huyen, M N T; Tiemersma, E W; Kremer, K; de Haas, P; Lan, N T N; Buu, T N; Sola, C; Cobelens, F G J; van Soolingen, D

2013-11-01

The molecular diagnosis of tuberculosis (TB) in Viet Nam is often based on the detection of insertion sequence (IS) 6110 in Mycobacterium tuberculosis. However, 8-11% of M. tuberculosis strains in South-East Asia do not contain this target and this undermines the validity of these molecular tests. We quantified the frequency of M. tuberculosis strains lacking IS6110 in rural Viet Nam and studied their epidemiological and clinical characteristics. Consecutively diagnosed adult TB patients in rural Southern Viet Nam submitted two sputum samples for culture, IS6110 restriction fragment length polymorphism (RFLP) spoligotyping and 15-loci variable number tandem repeat typing. Polymerase chain reaction (PCR) was performed to confirm the absence of IS6110 elements in strains lacking IS6110 hybridisation in RFLP. Among 2664 TB patient isolates examined, 109 (4.1%) had no IS6110 element. Compared to other strains, these no-copy strains were less often resistant to anti-tuberculosis drugs, particularly to streptomycin (adjusted OR 0.2, 95%CI 0.1-0.5), and showed significant geographic variation. No associations with TB history or demographic factors were found. Strains without the IS6110 target pose a problem in Viet Nam as regards false-negative molecular TB diagnosis in PCR. Compared to other strains circulating in Viet Nam, no-copy strains are more susceptible to anti-tuberculosis drugs.

Genetic diversity of drug and multidrug-resistant Mycobacterium tuberculosis circulating in Veracruz, Mexico

PubMed Central

Munro-Rojas, Daniela; Fernandez-Morales, Esdras; Zarrabal-Meza, José; Martínez-Cazares, Ma. Teresa; Parissi-Crivelli, Aurora; Fuentes-Domínguez, Javier; Séraphin, Marie Nancy; Lauzardo, Michael; González-y-Merchand, Jorge Alberto; Rivera-Gutierrez, Sandra

2018-01-01

Background Mexico is one of the most important contributors of drug and multidrug-resistant tuberculosis in Latin America; however, knowledge of the genetic diversity of drug-resistant tuberculosis isolates is limited. Methods In this study, the genetic structure of 112 Mycobacterium tuberculosis strains from the southeastern Mexico was determined by spoligotyping and 24-loci MIRU-VNTRs. Findings The results show eight major lineages, the most of which was T1 (24%), followed by LAM (16%) and H (15%). A total of 29 (25%) isolates were identified as orphan. The most abundant SITs were SIT53/T1 and SIT42/LAM9 with 10 isolates each and SIT50/H3 with eight isolates. Fifty-two spoligotype patterns, twenty-seven clusters and ten clonal complexes were observed, demonstrating an important genetic diversity of drug and multidrug-resistant tuberculosis isolates in circulation and transmission level of these aggravated forms of tuberculosis. Being defined as orphan or as part of an orphan cluster, was a risk factor for multidrug resistant-tuberculosis (OR 2.5, IC 1.05–5.86 and OR 3.3, IC 1–11.03, respectively). Multiple correspondence analyses showed association of some clusters and SITs with specific geographical locations. Conclusions Our study provides one of the most detailed description of the genetic structure of drug and multidrug-resistant tuberculosis strains in southeast Mexico, establishing for the first time a baseline of the genotypes observed in resistant isolates circulating, however further studies are required to better elucidate the genetic structure of tuberculosis in region and the factors that could be participating in their dispersion. PMID:29543819

Proteogenomic Investigation of Strain Variation in Clinical Mycobacterium tuberculosis Isolates.

PubMed

Heunis, Tiaan; Dippenaar, Anzaan; Warren, Robin M; van Helden, Paul D; van der Merwe, Ruben G; Gey van Pittius, Nicolaas C; Pain, Arnab; Sampson, Samantha L; Tabb, David L

2017-10-06

Mycobacterium tuberculosis consists of a large number of different strains that display unique virulence characteristics. Whole-genome sequencing has revealed substantial genetic diversity among clinical M. tuberculosis isolates, and elucidating the phenotypic variation encoded by this genetic diversity will be of the utmost importance to fully understand M. tuberculosis biology and pathogenicity. In this study, we integrated whole-genome sequencing and mass spectrometry (GeLC-MS/MS) to reveal strain-specific characteristics in the proteomes of two clinical M. tuberculosis Latin American-Mediterranean isolates. Using this approach, we identified 59 peptides containing single amino acid variants, which covered ∼9% of all coding nonsynonymous single nucleotide variants detected by whole-genome sequencing. Furthermore, we identified 29 distinct peptides that mapped to a hypothetical protein not present in the M. tuberculosis H37Rv reference proteome. Here, we provide evidence for the expression of this protein in the clinical M. tuberculosis SAWC3651 isolate. The strain-specific databases enabled confirmation of genomic differences (i.e., large genomic regions of difference and nonsynonymous single nucleotide variants) in these two clinical M. tuberculosis isolates and allowed strain differentiation at the proteome level. Our results contribute to the growing field of clinical microbial proteogenomics and can improve our understanding of phenotypic variation in clinical M. tuberculosis isolates.

Unique Regulation of the DosR Regulon in the Beijing Lineage of Mycobacterium tuberculosis.

PubMed

Domenech, Pilar; Zou, Jason; Averback, Alexandra; Syed, Nishath; Curtis, Daniele; Donato, Samuel; Reed, Michael B

2017-01-15

The DosR regulon, a set of 48 genes normally expressed in Mycobacterium tuberculosis under conditions that inhibit aerobic respiration, is controlled via the DosR-DosS/DosT two-component system. While the regulon requires induction in most M. tuberculosis isolates, for members of the Beijing lineage, its expression is uncoupled from the need for signaling. In our attempts to understand the mechanistic basis for this uncoupling in the Beijing background, we previously reported the identification of two synonymous single-nucleotide polymorphisms (SNPs) within the adjacent Rv3134c gene. In the present study, we have interrogated the impact of these SNPs on dosR expression in wild-type strains, as well as a range of dosR-dosS-dosT mutants, for both Beijing and non-Beijing M. tuberculosis backgrounds. In this manner, we have unequivocally determined that the C601T dosR promoter SNP is the sole requirement for the dramatic shift in the pattern of DosR regulon expression seen in this globally important lineage. Interestingly, we also show that DosT is completely nonfunctional within these strains. Thus, a complex series of evolutionary steps has led to the present-day Beijing DosR phenotype that, in turn, potentially confers a fitness advantage in the face of some form of host-associated selective pressure. Mycobacterium tuberculosis strains of the Beijing lineage have been described as being of enhanced virulence compared to other lineages, and in certain regions, they are associated with the dramatic spread of multidrug-resistant tuberculosis (TB). In terms of trying to understand the functional basis for these broad epidemiological phenomena, it is interesting that, in contrast to the other major lineages, the Beijing strains all constitutively overexpress members of the DosR regulon. Here, we identify the mutational events that led to the evolution of this unique phenotype. In addition, our work highlights the fact that important phenotypic differences exist between distinct M. tuberculosis lineages, with the potential to impact the efficacy of diagnosis, vaccination, and treatment programs. Copyright © 2016 American Society for Microbiology.

Genetic diversity, transmission dynamics, and drug resistance of Mycobacterium tuberculosis in Luanda, Angola.

PubMed

Perdigão, João; Clemente, Sofia; Ramos, Jorge; Masakidi, Pedro; Machado, Diana; Silva, Carla; Couto, Isabel; Viveiros, Miguel; Taveira, Nuno; Portugal, Isabel

2016-12-01

Despite the important role that the African region plays in a global tuberculosis (TB) epidemiological context, many countries in the region still lack data on the prevalence of specific Mycobacterium tuberculosis strains and drug resistance. This is the case for Angola, which presently lacks any data concerning drug-resistance rates and prevalence of specific M. tuberculosis genotypes and respective population structure. In this study, we made the first characterization of the genetic diversity and drug resistance of M. tuberculosis complex strains circulating in Luanda, Angola's most important setting concerning TB epidemiology. We have analyzed 89 M. tuberculosis isolates recovered from the same number of patients. All clinical isolates were genotyped by spoligotyping and 24-loci mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTRs). First-line drug-susceptibility testing was performed by the standard BACTEC 960 Mycobacteria Growth Indicator Tube (MGIT) procedure. We have detected 33 different spoligotype profiles corresponding to 24 different shared international types (SITs) and nine orphan profiles. SIT 20 (LAM1) was the most prevalent (n=16, 18.2%) followed by SIT 42 (LAM9; n=15, 17.1%). Overall, the M. tuberculosis population structure in this sample was dominated by LAM (64.8%) and T (33.0%) strains. Twenty-four-loci MIRU-VNTR analysis revealed that a total of 13 isolates were grouped into five distinct clusters. Drug-susceptibility testing revealed a worrying situation concerning resistance rates. Drug-susceptibility data showed that 22 (24.7%) of the 89 clinical isolates were resistant to one or more antibacillary drugs of which four (4.5%) were multidrug resistant (MDR). Drug-resistant isolates were found across distinct clades and MIRU-VNTR clusters. This first cross-sectional study conducted in Luanda, Angola, provides a framework for future studies and programmatic management of TB in Angola. We provide sufficient evidence for cluster-based transmission with a high predominance of LAM strains, with differential geographic dispersion. The moderate rate of MDR-TB found in this sample has major public health implications and highlights the need for further studies specifically focused on MDR-TB transmission. Copyright © 2016.

[Multidrug-Resistant Tuberculosis by Strains of Beijing Family, in Patients from Lisbon, Portugal: Preliminary Report].

PubMed

Maltez, Fernando; Martins, Teresa; Póvoas, Diana; Cabo, João; Peres, Helena; Antunes, Francisco; Perdigão, João; Portugal, Isabel

2017-03-31

Beijing family strains of Mycobacterium tuberculosis are associated with multidrug-resistance. Although strains of the Lisboa family are the most common among multidrug-resistant and extensively drug-resistant patients in the region, several studies have reported the presence of the Beijing family. However, the features of patients from whom they were isolated, are not yet known. Retrospective study involving 104 multidrug-resistant and extensively drug-resistant strains of Mycobacterium tuberculosis, from the same number of patients, isolated and genotyped between 1993 and 2015 in Lisbon. We assessed the prevalence of strains of both families and the epidemiologic and clinical features of those infected with Beijing family strains. Seventy-four strains (71.2%) belonged to the Lisboa family, 25 (24.0%) showed a unique genotypic pattern and five (4.8%) belonged to the Beijing family, the latter identified after 2009. Those infected with Beijing family strains were angolan (n = 1), ukrainian (n = 2) and portuguese (n = 2), mainly young-aged and, four of five immunocompetent and with no past history of tuberculosis. All had multidrug-resistant tuberculosis. We did not find any distinctive clinical or radiological features, neither a predominant resistance pattern. Cure rate was high (four patients). Although the number of infected patients with Beijing strains was small, it suggests an important proportion of primary tuberculosis, a potential for transmission in the community but also a better clinical outcome when compared to other reported strains, such as W-Beijing and Lisboa. Although Lisboa family strains account for most of the multidrug and extensively drug-resistant tuberculosis cases in Lisbon area, Beijing strains are transmitted in the city and might change the local characteristics of the epidemics.

Antiprotozoal and antimycobacterial activities of Persea americana seeds.

PubMed

Jiménez-Arellanes, Adelina; Luna-Herrera, Julieta; Ruiz-Nicolás, Ricardo; Cornejo-Garrido, Jorge; Tapia, Amparo; Yépez-Mulia, Lilián

2013-05-16

Persea americana seeds are widely used in traditional Mexican medicine to treat rheumatism, asthma, infectious processes as well as diarrhea and dysentery caused by intestinal parasites. The chloroformic and ethanolic extracts of P. americana seeds were prepared by maceration and their amoebicidal, giardicidal and trichomonicidal activity was evaluated. These extracts were also tested against Mycobacterium tuberculosis H37Rv, four mono-resistant and two multidrug resistant strains of M. tuberculosis as well as five non tuberculosis mycobacterium strains by MABA assay. The chloroformic and ethanolic extracts of P. americana seeds showed significant activity against E. histolytica, G. lamblia and T. vaginalis (IC50 <0.634 μg/ml). The chloroformic extract inhibited the growth of M. tuberculosis H37Rv, M. tuberculosis MDR SIN 4 isolate, three M. tuberculosis H37Rv mono-resistant reference strains and four non tuberculosis mycobacteria (M. fortuitum, M. avium, M. smegmatis and M. absessus) showing MIC values ≤50 μg/ml. Contrariwise, the ethanolic extract affected only the growth of two mono-resistant strains of M. tuberculosis H37Rv and M. smegmatis (MIC ≤50 μg/ml). The CHCl3 and EtOH seed extracts from P. americana showed amoebicidal and giardicidal activity. Importantly, the CHCl3 extract inhibited the growth of a MDR M. tuberculosis isolate and three out of four mono-resistant reference strains of M. tuberculosis H37Rv, showing a MIC = 50 μg/ml. This extract was also active against the NTM strains, M. fortuitum, M. avium, M. smegmatis and M. abscessus, with MIC values <50 μg/ml.

Triclosan Derivatives: Towards Potent Inhibitors of Drug-Sensitive and Drug-Resistant Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Freundlich, Joel S.; Wang, Feng; Vilchèze, Catherine

Isoniazid (INH) is a frontline antitubercular drug that inhibits the enoyl acyl carrier protein reductase InhA. Novel inhibitors of InhA that are not cross-resistant to INH represent a significant goal in antitubercular chemotherapy. The design, synthesis, and biological activity of a series of triclosan-based inhibitors is reported, including their promising efficacy against INH-resistant strains of M. tuberculosis. Triclosan has been previously shown to inhibit InhA, an essential enoyl acyl carrier protein reductase involved in mycolic acid biosynthesis, the inhibition of which leads to the lysis of Mycobacterium tuberculosis. Using a structure-based drug design approach, a series of 5-substituted triclosan derivativesmore » was developed. Two groups of derivatives with alkyl and aryl substituents, respectively, were identified with dramatically enhanced potency against purified InhA. The most efficacious inhibitor displayed an IC{sub 50} value of 21 nM, which was 50-fold more potent than triclosan. X-ray crystal structures of InhA in complex with four triclosan derivatives revealed the structural basis for the inhibitory activity. Six selected triclosan derivatives were tested against isoniazid-sensitive and resistant strains of M. tuberculosis. Among those, the best inhibitor had an MIC value of 4.7 {mu}g mL{sup -1} (13 {mu}M), which represents a tenfold improvement over the bacteriocidal activity of triclosan. A subset of these triclosan analogues was more potent than isoniazid against two isoniazid-resistant M. tuberculosis strains, demonstrating the significant potential for structure-based design in the development of next generation antitubercular drugs.« less

Evaluation of a Simple in-House Test to Presumptively Differentiate Mycobacterium tuberculosis Complex from Nontuberculous Mycobacteria by Detection of p-Nitrobenzoic Acid Metabolites

PubMed Central

Wang, Guirong; Yu, Xia; Liang, Qian; Chen, Suting; Wilson, Stuart; Huang, Hairong

2013-01-01

The timely differentiation of Mycobacterium tuberculosis complex (MTC) and non-tubercular mycobacterium (NTM) species is urgently needed in patient care since the routine laboratory method is time consuming and cumbersome. An easy and cheap method which can successfully distinguish MTC from NTM was established and evaluated. 38 mycobacterial type and reference strains and 65 clinical isolates representing 10 species of mycobacterium were included in this study. Metabolites of p-nitrobenzoic acid (PNB) reduction were identified using liquid chromatography and tandem mass spectrometry (LC/MS/MS). A spectrophotometric method was developed to detect these metabolites, which was evaluated on a number of MTC and NTM species. All of the tested NTM species and strains reduced PNB to p-aminobenzoic acid (PABA), while none of the MTC strains showed a similar activity. Spectrophotometric detection of PABA had 100% sensitivity and specificity for MTC and NTM differentiation among the type strains and the clinical isolates tested. PABA was identified as one of the metabolites of PNB reduction. All the tested NTM species metabolized PNB to PABA whereas the MTC members lacked this activity. A simple, specific and cost-effective method based on PABA production was established in order to discriminate MTC from NTM from cultured organisms. PMID:24260497

Evaluation of Immunogenicity and Protective Efficacy Elicited by Mycobacterium bovis BCG Overexpressing Ag85A Protein against Mycobacterium tuberculosis Aerosol Infection.

PubMed

Xu, Zheng Zhong; Chen, Xiang; Hu, Ting; Meng, Chuang; Wang, Xiao Bo; Rao, Yan; Zhang, Xiao Ming; Yin, Yue Lan; Pan, Zhi Ming; Jiao, Xin An

2016-01-01

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is currently the only vaccine available for preventing tuberculosis (TB), however, BCG has varying success in preventing pulmonary TB. In this study, a recombinant BCG (rBCG::Ag85A) strain overexpressing the immunodominant Ag85A antigen was constructed, and its immunogenicity and protective efficacy were evaluated. Our results indicated that the Ag85A protein was successfully overexpressed in rBCG::Ag85A, and the Ag85A peptide-MHC complexes on draining lymph node dendritic cells of C57BL/6 mice infected with rBCG::Ag85A were detectable 4 h post-infection. The C57BL/6 mice infected with this strain had stronger antigen-specific interferon-gamma (IFN-γ) responses and higher antibody titers than those immunized with BCG, and the protective experiments showed that rBCG::Ag85A can enhance protection against Mycobacterium tuberculosis (M. tuberculosis) H37Rv infection compared to the BCG vaccine alone. Our results demonstrate the potential of rBCG::Ag85A as a candidate vaccine against TB.

Multiprimer PCR system for differential identification of mycobacteria in clinical samples.

PubMed Central

Del Portillo, P; Thomas, M C; Martínez, E; Marañón, C; Valladares, B; Patarroyo, M E; Carlos López, M

1996-01-01

A novel multiprimer PCR method with the potential to identify mycobacteria in clinical samples is presented. The assay relies on the simultaneous amplification of three bacterial DNA genomic fragments by using different sets of oligonucleotide primers. The first set of primers amplifies a 506-bp fragment from the gene for the 32-kDa antigen of Mycobacterium tuberculosis, which is present in most of the species belonging to the genus Mycobacterium. The second set of primers amplifies a 984-bp fragment from the IS6110 insertion sequence of the bacteria belonging to the M. tuberculosis complex. The third set of primers, derived from an M. tuberculosis species-specific sequence named MTP40, amplifies a 396-bp genomic fragment. Thus, while the multiprimer system would render three amplification fragments from the M. tuberculosis genome and two fragments from the Mycobacterium bovis genome, a unique amplification fragment would be obtained from nontuberculous mycobacteria. The results obtained, using reference mycobacterial strains and typed clinical isolates, show that the multiprimer PCR method may be a rapid, sensitive, and specific tool for the differential identification of various mycobacterial strains in a single-step assay. PMID:8789008

The Epidemiological Characteristics of Beijing Lineage Mycobacterium tuberculosis from a National Referral Center in China.

PubMed

Li, Xiao Ying; Li, Ying; Zhang, Yao; Kang, Wan Li; Zhao, Li Ping; Ding, Peng Ju; Dai, Wen Tao; Huang, Hai Rong; Huang, Yan Feng; Li, Wei Min

2015-07-01

Our study was to investigate the epidemiological characteristics of M.tuberculosis from a national tuberculosis referral center in China. All strains isolated from TB patients, were genotyped by the RD105 deletion, 8 and 51 SNP loci and VNTR. The high differentiation SNPs of modern Beijing strains were analyzed for protein function and structure. 413 M. tuberculosis were included. Of 379 Beijing lineage M. tuberculosis, 'modern' and 'ancient' strains respectively represented 85.5% (324/379) and 14.5% (55/379). Rv2494 (V48A) and Rv0245 (S103F) were confirmed as high differentiation SNPs associated with modern strains. In a word, Modern Beijing lineage M.tuberculosis was dominant and the structural models suggested that modern sub-lineage may more easily survive in 'extreme' host condition. Copyright © 2015 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

Antiprotozoal and antimycobacterial activities of Persea americana seeds

PubMed Central

2013-01-01

Background Persea americana seeds are widely used in traditional Mexican medicine to treat rheumatism, asthma, infectious processes as well as diarrhea and dysentery caused by intestinal parasites. Methods The chloroformic and ethanolic extracts of P. americana seeds were prepared by maceration and their amoebicidal, giardicidal and trichomonicidal activity was evaluated. These extracts were also tested against Mycobacterium tuberculosis H37Rv, four mono-resistant and two multidrug resistant strains of M. tuberculosis as well as five non tuberculosis mycobacterium strains by MABA assay. Results The chloroformic and ethanolic extracts of P. americana seeds showed significant activity against E. histolytica, G. lamblia and T. vaginalis (IC50 <0.634 μg/ml). The chloroformic extract inhibited the growth of M. tuberculosis H37Rv, M. tuberculosis MDR SIN 4 isolate, three M. tuberculosis H37Rv mono-resistant reference strains and four non tuberculosis mycobacteria (M. fortuitum, M. avium, M. smegmatis and M. absessus) showing MIC values ≤50 μg/ml. Contrariwise, the ethanolic extract affected only the growth of two mono-resistant strains of M. tuberculosis H37Rv and M. smegmatis (MIC ≤50 μg/ml). Conclusions The CHCl3 and EtOH seed extracts from P. americana showed amoebicidal and giardicidal activity. Importantly, the CHCl3 extract inhibited the growth of a MDR M. tuberculosis isolate and three out of four mono-resistant reference strains of M. tuberculosis H37Rv, showing a MIC = 50 μg/ml. This extract was also active against the NTM strains, M. fortuitum, M. avium, M. smegmatis and M. abscessus, with MIC values <50 μg/ml. PMID:23680126

Clustered regularly interspaced short palindromic repeats (CRISPRs) analysis of members of the Mycobacterium tuberculosis complex.

PubMed

Botelho, Ana; Canto, Ana; Leão, Célia; Cunha, Mónica V

2015-01-01

Typical CRISPR (clustered, regularly interspaced, short palindromic repeat) regions are constituted by short direct repeats (DRs), interspersed with similarly sized non-repetitive spacers, derived from transmissible genetic elements, acquired when the cell is challenged with foreign DNA. The analysis of the structure, in number and nature, of CRISPR spacers is a valuable tool for molecular typing since these loci are polymorphic among strains, originating characteristic signatures. The existence of CRISPR structures in the genome of the members of Mycobacterium tuberculosis complex (MTBC) enabled the development of a genotyping method, based on the analysis of the presence or absence of 43 oligonucleotide spacers separated by conserved DRs. This method, called spoligotyping, consists on PCR amplification of the DR chromosomal region and recognition after hybridization of the spacers that are present. The workflow beneath this methodology implies that the PCR products are brought onto a membrane containing synthetic oligonucleotides that have complementary sequences to the spacer sequences. Lack of hybridization of the PCR products to a specific oligonucleotide sequence indicates absence of the correspondent spacer sequence in the examined strain. Spoligotyping gained great notoriety as a robust identification and typing tool for members of MTBC, enabling multiple epidemiological studies on human and animal tuberculosis.

Importance of confirming data on the in vivo efficacy of novel antibacterial drug regimens against various strains of Mycobacterium tuberculosis.

PubMed

De Groote, Mary A; Gruppo, Veronica; Woolhiser, Lisa K; Orme, Ian M; Gilliland, Janet C; Lenaerts, Anne J

2012-02-01

In preclinical testing of antituberculosis drugs, laboratory-adapted strains of Mycobacterium tuberculosis are usually used both for in vitro and in vivo studies. However, it is unknown whether the heterogeneity of M. tuberculosis stocks used by various laboratories can result in different outcomes in tests of antituberculosis drug regimens in animal infection models. In head-to-head studies, we investigated whether bactericidal efficacy results in BALB/c mice infected by inhalation with the laboratory-adapted strains H37Rv and Erdman differ from each other and from those obtained with clinical tuberculosis strains. Treatment of mice consisted of dual and triple drug combinations of isoniazid (H), rifampin (R), and pyrazinamide (Z). The results showed that not all strains gave the same in vivo efficacy results for the drug combinations tested. Moreover, the ranking of HRZ and RZ efficacy results was not the same for the two H37Rv strains evaluated. The magnitude of this strain difference also varied between experiments, emphasizing the risk of drawing firm conclusions for human trials based on single animal studies. The results also confirmed that the antagonism seen within the standard HRZ regimen by some investigators appears to be an M. tuberculosis strain-specific phenomenon. In conclusion, the specific identity of M. tuberculosis strain used was found to be an important variable that can change the apparent outcome of in vivo efficacy studies in mice. We highly recommend confirmation of efficacy results in late preclinical testing against a different M. tuberculosis strain than the one used in the initial mouse efficacy study, thereby increasing confidence to advance potent drug regimens to clinical trials.

Comparative proteomic analysis of virulent Korean Mycobacterium tuberculosis K-strain with other mycobacteria strain following infection of U-937 macrophage.

PubMed

Ryoo, Sung Weon; Park, Young Kil; Park, Sue-Nie; Shim, Young Soo; Liew, Hyunjeong; Kang, Seongman; Bai, Gill-Han

2007-06-01

In Korea, the Mycobacterium tuberculosis K-strain is the most prevalent clinical isolates and belongs to the Beijing family. In this study, we conducted comparative porteomics of expressed proteins of clinical isolates of the K-strain with H37Rv, H37Ra as well as the vaccine strain of Mycobacterium bovis BCG following phagocytosis by the human monocytic cell line U-937. Proteins were analyzed by 2-D PAGE and MALDITOF-MS. Two proteins, Mb1363 (probable glycogen phosphorylase GlgP) and MT2656 (Haloalkane dehalogenase LinB) were most abundant after phagocytosis of M. tuberculosis K-strain. This approach provides a method to determine specific proteins that may have critical roles in tuberculosis pathogenesis.

Development of a One-Step Multiplex PCR Assay for Differential Detection of Major Mycobacterium Species

PubMed Central

Chae, Hansong; Han, Seung Jung; Kim, Su-Young; Ki, Chang-Seok; Huh, Hee Jae; Yong, Dongeun

2017-01-01

ABSTRACT The prevalence of tuberculosis continues to be high, and nontuberculous mycobacterial (NTM) infection has also emerged worldwide. Moreover, differential and accurate identification of mycobacteria to the species or subspecies level is an unmet clinical need. Here, we developed a one-step multiplex PCR assay using whole-genome analysis and bioinformatics to identify novel molecular targets. The aims of this assay were to (i) discriminate between the Mycobacterium tuberculosis complex (MTBC) and NTM using rv0577 or RD750, (ii) differentiate M. tuberculosis (M. tuberculosis) from MTBC using RD9, (iii) selectively identify the widespread M. tuberculosis Beijing genotype by targeting mtbk_20680, and (iv) simultaneously detect five clinically important NTM (M. avium, M. intracellulare, M. abscessus, M. massiliense, and M. kansasii) by targeting IS1311, DT1, mass_3210, and mkan_rs12360. An initial evaluation of the multiplex PCR assay using reference strains demonstrated 100% specificity for the targeted Mycobacterium species. Analytical sensitivity ranged from 1 to 10 pg for extracted DNA and was 103 and 104 CFU for pure cultures and nonhomogenized artificial sputum cultures, respectively, of the targeted species. The accuracy of the multiplex PCR assay was further evaluated using 55 reference strains and 94 mycobacterial clinical isolates. Spoligotyping, multilocus sequence analysis, and a commercial real-time PCR assay were employed as standard assays to evaluate the multiplex PCR assay with clinical M. tuberculosis and NTM isolates. The PCR assay displayed 100% identification agreement with the standard assays. Our multiplex PCR assay is a simple, convenient, and reliable technique for differential identification of MTBC, M. tuberculosis, M. tuberculosis Beijing genotype, and major NTM species. PMID:28659320

Development of a One-Step Multiplex PCR Assay for Differential Detection of Major Mycobacterium Species.

PubMed

Chae, Hansong; Han, Seung Jung; Kim, Su-Young; Ki, Chang-Seok; Huh, Hee Jae; Yong, Dongeun; Koh, Won-Jung; Shin, Sung Jae

2017-09-01

The prevalence of tuberculosis continues to be high, and nontuberculous mycobacterial (NTM) infection has also emerged worldwide. Moreover, differential and accurate identification of mycobacteria to the species or subspecies level is an unmet clinical need. Here, we developed a one-step multiplex PCR assay using whole-genome analysis and bioinformatics to identify novel molecular targets. The aims of this assay were to (i) discriminate between the Mycobacterium tuberculosis complex (MTBC) and NTM using rv0577 or RD750, (ii) differentiate M. tuberculosis ( M. tuberculosis ) from MTBC using RD9, (iii) selectively identify the widespread M. tuberculosis Beijing genotype by targeting mtbk_20680 , and (iv) simultaneously detect five clinically important NTM ( M. avium , M. intracellulare , M. abscessus , M. massiliense , and M. kansasii ) by targeting IS 1311 , DT1, mass_3210 , and mkan_rs12360 An initial evaluation of the multiplex PCR assay using reference strains demonstrated 100% specificity for the targeted Mycobacterium species. Analytical sensitivity ranged from 1 to 10 pg for extracted DNA and was 10 3 and 10 4 CFU for pure cultures and nonhomogenized artificial sputum cultures, respectively, of the targeted species. The accuracy of the multiplex PCR assay was further evaluated using 55 reference strains and 94 mycobacterial clinical isolates. Spoligotyping, multilocus sequence analysis, and a commercial real-time PCR assay were employed as standard assays to evaluate the multiplex PCR assay with clinical M. tuberculosis and NTM isolates. The PCR assay displayed 100% identification agreement with the standard assays. Our multiplex PCR assay is a simple, convenient, and reliable technique for differential identification of MTBC, M. tuberculosis , M. tuberculosis Beijing genotype, and major NTM species. Copyright © 2017 American Society for Microbiology.

The current status, challenges, and future developments of new tuberculosis vaccines.

PubMed

Gong, Wenping; Liang, Yan; Wu, Xueqiong

2018-03-30

Mycobacterium tuberculosis complex causes tuberculosis (TB), one of the top 10 causes of death worldwide. TB results in more fatalities than multi-drug resistant (MDR) HIV strain related coinfection. Vaccines play a key role in the prevention and control of infectious diseases. Unfortunately, the only licensed preventive vaccine against TB, bacilli Calmette-Guérin (BCG), is ineffective for prevention of pulmonary TB in adults. Therefore, it is very important to develop novel vaccines for TB prevention and control. This literature review provides an overview of the innate and adaptive immune response during M. tuberculosis infection, and presents current developments and challenges to novel TB vaccines. A comprehensive understanding of vaccines in preclinical and clinical studies provides extensive insight for the development of safer and more efficient vaccines, and may inspire new ideas for TB prevention and treatment.

Functional, thermodynamics, structural and biological studies of in silico-identified inhibitors of Mycobacterium tuberculosis enoyl-ACP(CoA) reductase enzyme

NASA Astrophysics Data System (ADS)

Martinelli, Leonardo K. B.; Rotta, Mariane; Villela, Anne D.; Rodrigues-Junior, Valnês S.; Abbadi, Bruno L.; Trindade, Rogério V.; Petersen, Guilherme O.; Danesi, Giuliano M.; Nery, Laura R.; Pauli, Ivani; Campos, Maria M.; Bonan, Carla D.; de Souza, Osmar Norberto; Basso, Luiz A.; Santos, Diogenes S.

2017-04-01

Novel chemotherapeutics agents are needed to kill Mycobacterium tuberculosis, the main causative agent of tuberculosis (TB). The M. tuberculosis 2-trans-enoyl-ACP(CoA) reductase enzyme (MtInhA) is the druggable bona fide target of isoniazid. New chemotypes were previously identified by two in silico approaches as potential ligands to MtInhA. The inhibition mode was determined by steady-state kinetics for seven compounds that inhibited MtInhA activity. Dissociation constant values at different temperatures were determined by protein fluorescence spectroscopy. van’t Hoff analyses of ligand binding to MtInhA:NADH provided the thermodynamic signatures of non-covalent interactions (ΔH°, ΔS°, ΔG°). Phenotypic screening showed that five compounds inhibited in vitro growth of M. tuberculosis H37Rv strain. Labio_16 and Labio_17 compounds also inhibited the in vitro growth of PE-003 multidrug-resistant strain. Cytotoxic effects on Hacat, Vero and RAW 264.7 cell lines were assessed for the latter two compounds. The Labio_16 was bacteriostatic and Labio_17 bactericidal in an M. tuberculosis-infected macrophage model. In Zebrafish model, Labio_16 showed no cardiotoxicity whereas Labio_17 showed dose-dependent cardiotoxicity. Accordingly, a model was built for the MtInhA:NADH:Labio_16 ternary complex. The results show that the Labio_16 compound is a direct inhibitor of MtInhA, and it may represent a hit for the development of chemotherapeutic agents to treat TB.

Spoligotyping based genetic diversity of Mycobacterium tuberculosis in Ethiopia: a systematic review.

PubMed

Tulu, Begna; Ameni, Gobena

2018-03-27

Understanding the types of strains and lineages of Mycobacterium tuberculosis (M. tuberculosis) circulating in a country is of paramount importance for tuberculosis (TB) control program of that country. The main aim of this study was to review and compile the results of studies conducted on strains and lineages of M. tuberculosis in Ethiopia. A systematic search and review of articles published on M. tuberculosis strains and lineages in Ethiopia were made. PubMed and Google Scholar databases were considered for the search while the keywords used were M. tuberculosis, molecular epidemiology, molecular typing spoligotyping and Ethiopia. Twenty-one studies were considered in this review and a total of 3071 M. tuberculosis isolates and 3067 strains were included. These studies used spoligotyping and identified five lineages including Indo-Ocean, East Asian/Beijing, East African-Indian, Euro-American and Ethiopian in a proportion of 7.1%, 0.2%, 23.0%, 64.8%, and 4.1%, respectively. Thus, Euro-American was the most frequently (64.8%) occurring Lineage while East Asian was the least (0.2%) frequently occurring Lineage in the country. Surprisingly, the Ethiopian Lineage seemed to be localized to northeastern Ethiopia. In addition, the top five clades identified by this review were T, CAS, H, Manu and Ethiopian comprising of 48.0%, 23.0%, 11.0%, 6.0% and 4.1% of the strains, respectively. Furthermore, predominant shared types (spoligotype patterns) identified were SIT149, SIT53, SIT25, SIT37, and SIT21, each consisting of 420, 343, 266, 162 and 102 isolates, respectively, while, on the other hand, 15% of the strains were orphan. According to the summary of the results of this review, diversified strains and lineages of M. tuberculosis were found in Ethiopia, and the frequencies of occurrence of these strains and lineages were variable in different regions of the country. This systematic review is registered in the PRISMA with the registration number of 42017059263.

Mixed-Strain Mycobacterium tuberculosis Infections and the Implications for Tuberculosis Treatment and Control

PubMed Central

van Helden, Paul D.; Wilson, Douglas; Colijn, Caroline; McLaughlin, Megan M.; Abubakar, Ibrahim; Warren, Robin M.

2012-01-01

Summary: Numerous studies have reported that individuals can simultaneously harbor multiple distinct strains of Mycobacterium tuberculosis. To date, there has been limited discussion of the consequences for the individual or the epidemiological importance of mixed infections. Here, we review studies that documented mixed infections, highlight challenges associated with the detection of mixed infections, and discuss possible implications of mixed infections for the diagnosis and treatment of patients and for the community impact of tuberculosis control strategies. We conclude by highlighting questions that should be resolved in order to improve our understanding of the importance of mixed-strain M. tuberculosis infections. PMID:23034327

Meta-analysis of human gene expression in response to Mycobacterium tuberculosis infection reveals potential therapeutic targets.

PubMed

Wang, Zhang; Arat, Seda; Magid-Slav, Michal; Brown, James R

2018-01-10

With the global emergence of multi-drug resistant strains of Mycobacterium tuberculosis, new strategies to treat tuberculosis are urgently needed such as therapeutics targeting potential human host factors. Here we performed a statistical meta-analysis of human gene expression in response to both latent and active pulmonary tuberculosis infections from nine published datasets. We found 1655 genes that were significantly differentially expressed during active tuberculosis infection. In contrast, no gene was significant for latent tuberculosis. Pathway enrichment analysis identified 90 significant canonical human pathways, including several pathways more commonly related to non-infectious diseases such as the LRRK2 pathway in Parkinson's disease, and PD-1/PD-L1 signaling pathway important for new immuno-oncology therapies. The analysis of human genome-wide association studies datasets revealed tuberculosis-associated genetic variants proximal to several genes in major histocompatibility complex for antigen presentation. We propose several new targets and drug-repurposing opportunities including intravenous immunoglobulin, ion-channel blockers and cancer immuno-therapeutics for development as combination therapeutics with anti-mycobacterial agents. Our meta-analysis provides novel insights into host genes and pathways important for tuberculosis and brings forth potential drug repurposing opportunities for host-directed therapies.

A marked difference in pathogenesis and immune response induced by different Mycobacterium tuberculosis genotypes

PubMed Central

LÓPEZ, B; AGUILAR, D; OROZCO, H; BURGER, M; ESPITIA, C; RITACCO, V; BARRERA, L; KREMER, K; HERNANDEZ-PANDO, R; HUYGEN, K; VAN SOOLINGEN, D

2003-01-01

In the last decade, an unprecedented genetic diversity has been disclosed among Mycobacterium tuberculosis strains found worldwide. However, well-conserved genotypes seem to prevail in areas with high incidence of tuberculosis. As this may be related to selective advantages, such as advanced mechanisms to circumvent [M. bovis Bacille Calmette–Guerin (BCG)-induced] host defence mechanisms, we investigated the influence of strain diversity on the course of experimental disease. Twelve M. tuberculosis strains, representing four major genotype families found worldwide today, and the laboratory strain H37Rv were each used to infect BALB/c mice by direct intratracheal injection. Compared with H37Rv, infections with Beijng strains were characterized by extensive pneumonia, early but ephemeral tumour necrosis factor-alpha (TNF-α) and inducible isoform of nitric oxide synthetase (iNOS) expression, and significantly higher earlier mortality. Conversely, Canetti strains induced limited pneumonia, sustained TNF-α and iNOS expression in lungs, and almost 100% survival. Strains of the Somali and the Haarlem genotype families displayed less homogeneous, intermediate rates of survival. Previous BCG vaccination protected less effectively against infection with Beijing strains than against the H37Rv strain. In conclusion, genetically different M. tuberculosis strains evoked markedly different immunopathological events. Bacteria with the Beijing genotype, highly prevalent in Asia and the former USSR, elicited a non-protective immune response in mice and were the most virulent. Future immunological research, particularly on candidate vaccines, should include a broad spectrum of M. tuberculosis genotypes rather than a few laboratory strains. PMID:12823275

Express path analysis identifies a tyrosine kinase Src-centric network regulating divergent host responses to Mycobacterium tuberculosis infection.

PubMed

Karim, Ahmad Faisal; Chandra, Pallavi; Chopra, Aanchal; Siddiqui, Zaved; Bhaskar, Ashima; Singh, Amit; Kumar, Dhiraj

2011-11-18

Global gene expression profiling has emerged as a major tool in understanding complex response patterns of biological systems to perturbations. However, a lack of unbiased analytical approaches has restricted the utility of complex microarray data to gain novel system level insights. Here we report a strategy, express path analysis (EPA), that helps to establish various pathways differentially recruited to achieve specific cellular responses under contrasting environmental conditions in an unbiased manner. The analysis superimposes differentially regulated genes between contrasting environments onto the network of functional protein associations followed by a series of iterative enrichments and network analysis. To test the utility of the approach, we infected THP1 macrophage cells with a virulent Mycobacterium tuberculosis strain (H37Rv) or the attenuated non-virulent strain H37Ra as contrasting perturbations and generated the temporal global expression profiles. EPA of the results provided details of response-specific and time-dependent host molecular network perturbations. Further analysis identified tyrosine kinase Src as the major regulatory hub discriminating the responses between wild-type and attenuated Mtb infection. We were then able to verify this novel role of Src experimentally and show that Src executes its role through regulating two vital antimicrobial processes of the host cells (i.e. autophagy and acidification of phagolysosome). These results bear significant potential for developing novel anti-tuberculosis therapy. We propose that EPA could prove extremely useful in understanding complex cellular responses for a variety of perturbations, including pathogenic infections.

Antimycobacterial and cytotoxic activity of selected medicinal plant extracts

PubMed Central

Nguta, Joseph M.; Appiah-Opong, Regina; Nyarko, Alexander K.; Yeboah-Manu, Dorothy; Addo, Phyllis G.A.; Otchere, Isaac; Kissi-Twum, Abena

2016-01-01

Ethnopharmacological relevance Tuberculosis (TB) caused by Mycobacterium tuberculosis remains an ongoing threat to human health. Several medicinal plants are used traditionally to treat tuberculosis in Ghana. The current study was designed to investigate the antimycobacterial activity and cytotoxicity of crude extracts from five selected medicinal plants. Material and methods The microplate alamar blue assay (MABA) was used for antimycobacterial studies while the CellTiter 96® AQueous Assay, which is composed of solutions of a novel tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine methosulfate) PMS, was used for cytotoxic studies. Correlation coefficients were used to compare the activity of crude extracts against nonpathogenic strains and the pathogenic Mycobacterium tuberculosis subsp.tuberculosis. Results Results of the MIC determinations indicated that all the crude extracts were active on all the three tested mycobacterial strains. Minimum inhibitory concentration values as low as 156.3 µg/mL against M. tuberculosis; Strain H37Ra (ATCC® 25,177™) were recorded from the leaves of Solanum torvum Sw. (Solanaceae). Cytotoxicity of the extracts varied, and the leaves from S. torvum had the most promising selectivity index. Activity against M. tuberculosis; Strain H37Ra was the best predictor of activity against pathogenic Mycobacterium tuberculosis subsp.tuberculosis (correlation coefficient=0.8). Conclusion The overall results of the present study provide supportive data on the use of some medicinal plants for tuberculosis treatment. The leaves of Solanum torvum are a potential source of anti-TB natural products and deserve further investigations to develop novel anti-TB agents against sensitive and drug resistant strains of M. tuberculosis. PMID:26875647

Comparison of gene expression profiles between pansensitive and multidrug-resistant strains of Mycobacterium tuberculosis.

PubMed

Peñuelas-Urquides, K; González-Escalante, L; Villarreal-Treviño, L; Silva-Ramírez, B; Gutiérrez-Fuentes, D J; Mojica-Espinosa, R; Rangel-Escareño, C; Uribe-Figueroa, L; Molina-Salinas, G M; Dávila-Velderrain, J; Castorena-Torres, F; Bermúdez de León, M; Said-Fernández, S

2013-09-01

Mycobacterium tuberculosis has developed resistance to anti-tuberculosis first-line drugs. Multidrug-resistant strains complicate the control of tuberculosis and have converted it into a worldwide public health problem. Mutational studies of target genes have tried to envisage the resistance in clinical isolates; however, detection of these mutations in some cases is not sufficient to identify drug resistance, suggesting that other mechanisms are involved. Therefore, the identification of new markers of susceptibility or resistance to first-line drugs could contribute (1) to specifically diagnose the type of M. tuberculosis strain and prescribe an appropriate therapy, and (2) to elucidate the mechanisms of resistance in multidrug-resistant strains. In order to identify specific genes related to resistance in M. tuberculosis, we compared the gene expression profiles between the pansensitive H37Rv strain and a clinical CIBIN:UMF:15:99 multidrug-resistant isolate using microarray analysis. Quantitative real-time PCR confirmed that in the clinical multidrug-resistant isolate, the esxG, esxH, rpsA, esxI, and rpmI genes were upregulated, while the lipF, groES, and narG genes were downregulated. The modified genes could be involved in the mechanisms of resistance to first-line drugs in M. tuberculosis and could contribute to increased efficiency in molecular diagnosis approaches of infections with drug-resistant strains.

Models to understand the population-level impact of mixed strain M. tuberculosis infections.

PubMed

Sergeev, Rinat; Colijn, Caroline; Cohen, Ted

2011-07-07

Over the past decade, numerous studies have identified tuberculosis patients in whom more than one distinct strain of Mycobacterium tuberculosis is present. While it has been shown that these mixed strain infections can reduce the probability of treatment success for individuals simultaneously harboring both drug-sensitive and drug-resistant strains, it is not yet known if and how this phenomenon impacts the long-term dynamics for tuberculosis within communities. Strain-specific differences in immunogenicity and associations with drug resistance suggest that a better understanding of how strains compete within hosts will be necessary to project the effects of mixed strain infections on the future burden of drug-sensitive and drug-resistant tuberculosis. In this paper, we develop a modeling framework that allows us to investigate mechanisms of strain competition within hosts and to assess the long-term effects of such competition on the ecology of strains in a population. These models permit us to systematically evaluate the importance of unknown parameters and to suggest priority areas for future experimental research. Despite the current scarcity of data to inform the values of several model parameters, we are able to draw important qualitative conclusions from this work. We find that mixed strain infections may promote the coexistence of drug-sensitive and drug-resistant strains in two ways. First, mixed strain infections allow a strain with a lower basic reproductive number to persist in a population where it would otherwise be outcompeted if has competitive advantages within a co-infected host. Second, some individuals progressing to phenotypically drug-sensitive tuberculosis from a state of mixed drug-sensitive and drug-resistant infection may retain small subpopulations of drug-resistant bacteria that can flourish once the host is treated with antibiotics. We propose that these types of mixed infections, by increasing the ability of low fitness drug-resistant strains to persist, may provide opportunities for compensatory mutations to accumulate and for relatively fit, highly drug-resistant strains of M. tuberculosis to emerge. Published by Elsevier Ltd.

Identifying Likely Transmission Pathways within a 10-Year Community Outbreak of Tuberculosis by High-Depth Whole Genome Sequencing

PubMed Central

Sadsad, Rosemarie; Martinez, Elena; Jelfs, Peter; Hill-Cawthorne, Grant A.; Gilbert, Gwendolyn L.; Marais, Ben J.; Sintchenko, Vitali

2016-01-01

Background Improved tuberculosis control and the need to contain the spread of drug-resistant strains provide a strong rationale for exploring tuberculosis transmission dynamics at the population level. Whole-genome sequencing provides optimal strain resolution, facilitating detailed mapping of potential transmission pathways. Methods We sequenced 22 isolates from a Mycobacterium tuberculosis cluster in New South Wales, Australia, identified during routine 24-locus mycobacterial interspersed repetitive unit typing. Following high-depth paired-end sequencing using the Illumina HiSeq 2000 platform, two independent pipelines were employed for analysis, both employing read mapping onto reference genomes as well as de novo assembly, to control biases in variant detection. In addition to single-nucleotide polymorphisms, the analyses also sought to identify insertions, deletions and structural variants. Results Isolates were highly similar, with a distance of 13 variants between the most distant members of the cluster. The most sensitive analysis classified the 22 isolates into 18 groups. Four of the isolates did not appear to share a recent common ancestor with the largest clade; another four isolates had an uncertain ancestral relationship with the largest clade. Conclusion Whole genome sequencing, with analysis of single-nucleotide polymorphisms, insertions, deletions, structural variants and subpopulations, enabled the highest possible level of discrimination between cluster members, clarifying likely transmission pathways and exposing the complexity of strain origin. The analysis provides a basis for targeted public health intervention and enhanced classification of future isolates linked to the cluster. PMID:26938641

Definition of the Beijing/W lineage of Mycobacterium tuberculosis on the basis of genetic markers.

PubMed

Kremer, Kristin; Glynn, Judith R; Lillebaek, Troels; Niemann, Stefan; Kurepina, Natalia E; Kreiswirth, Barry N; Bifani, Pablo J; van Soolingen, Dick

2004-09-01

Mycobacterium tuberculosis Beijing genotype strains are highly prevalent in Asian countries and in the territory of the former Soviet Union. They are increasingly reported in other areas of the world and are frequently associated with tuberculosis outbreaks and drug resistance. Beijing genotype strains, including W strains, have been characterized by their highly similar multicopy IS6110 restriction fragment length polymorphism (RFLP) patterns, deletion of spacers 1 to 34 in the direct repeat region (Beijing spoligotype), and insertion of IS6110 in the genomic dnaA-dnaN locus. In this study the suitability and comparability of these three genetic markers to identify members of the Beijing lineage were evaluated. In a well-characterized collection of 1,020 M. tuberculosis isolates representative of the IS6110 RFLP genotypes found in The Netherlands, strains of two clades had spoligotypes characteristic of the Beijing lineage. A set of 19 Beijing reference RFLP patterns was selected to retrieve all Beijing strains from the Dutch database. These reference patterns gave a sensitivity of 98.1% and a specificity of 99.7% for identifying Beijing strains (defined by spoligotyping) in an international database of 1,084 strains. The usefulness of the reference patterns was also assessed with large DNA fingerprint databases in two other European countries and for identification strains from the W lineage found in the United States. A standardized definition for the identification of M. tuberculosis strains belonging to the Beijing/W lineage, as described in this work, will facilitate further studies on the spread and characterization of this widespread genotype family of M. tuberculosis strains.

[Study on molecular characteristics regarding DNA genotype of Mycobacterium tuberculosis clinical strains in Shandong].

PubMed

Deng, Yun-feng; Zhang, Yan-an; Zheng, Jian-li; Jing, Hui; Wang, Yan; Wang, Hai-ying; Ma, Xin; Liu, Zhi-min

2010-03-01

To establish the molecular characteristics of Mycobacterium tuberculosis and on factors influencing the recent transmission of drug resistant isolates in Shandong. Mycobacterium tuberculosis isolated from active pulmonary tuberculosis patients of 13 counties were genotyped by mycobacterial interspersed repetitive units (MIRU) methods. 12 loci of MIRU were detected in 558 isolates and a total of 143 MIRU patterns were confirmed. 66 isolates had distinct patterns, and 481 (86.2%) strains were in clusters. Shandong cluster included 177 strains with 74.6% of the isolates belonged to Beijing family. The recent transmission index of multi-drug resistance strains was in lower level, comparing to the susceptible strains. Our results showed that the Shandong cluster isolates had capacities of facilitating person-to-person transmission and high level of drug resistance.

Targeting Drug-Sensitive and -Resistant Strains of Mycobacterium tuberculosis by Inhibition of Src Family Kinases Lowers Disease Burden and Pathology.

PubMed

Chandra, Pallavi; Rajmani, R S; Verma, Garima; Bhavesh, Neel Sarovar; Kumar, Dhiraj

2016-01-01

In view of emerging drug resistance among bacterial pathogens, including Mycobacterium tuberculosis, the development of novel therapeutic strategies is increasingly being sought. A recent paradigm in antituberculosis (anti-TB) drug development is to target the host molecules that are crucial for intracellular survival of the pathogen. We previously showed the importance of Src tyrosine kinases in mycobacterial pathogenesis. Here, we report that inhibition of Src significantly reduced survival of H37Rv as well as multidrug-resistant (MDR) and extremely drug-resistant (XDR) strains of M. tuberculosis in THP-1 macrophages. Src inhibition was also effective in controlling M. tuberculosis infection in guinea pigs. In guinea pigs, reduced M. tuberculosis burden due to Src inhibition also led to a marked decline in the disease pathology. In agreement with the theoretical framework of host-directed approaches against the pathogen, Src inhibition was equally effective against an XDR strain in controlling infection in guinea pigs. We propose that Src inhibitors could be developed into effective host-directed anti-TB drugs, which could be indiscriminately used against both drug-sensitive and drug-resistant strains of M. tuberculosis. IMPORTANCE The existing treatment regimen for tuberculosis (TB) suffers from deficiencies like high doses of antibiotics, long treatment duration, and inability to kill persistent populations in an efficient manner. Together, these contribute to the emergence of drug-resistant tuberculosis. Recently, several host factors were identified which help intracellular survival of Mycobacterium tuberculosis within the macrophage. These factors serve as attractive targets for developing alternate therapeutic strategies against M. tuberculosis. This strategy promises to be effective against drug-resistant strains. The approach also has potential to considerably lower the risk of emergence of new drug-resistant strains. We explored tyrosine kinase Src as a host factor exploited by virulent M. tuberculosis for intracellular survival. We show that Src inhibition can effectively control tuberculosis in infected guinea pigs. Moreover, Src inhibition ameliorated TB-associated pathology in guinea pigs. Thus, Src inhibitors have strong potential to be developed as possible anti-TB drugs.

Genetic diversity of Mycobacterium tuberculosis strains isolated in Algeria: Results of spoligotyping.

PubMed

Ifticene, Malika; Kaïdi, Saïd; Khechiba, Mesbah-Mounir; Yala, Djamel; Boulahbal, Fadila

2015-12-01

Molecular typing tools, including spoligotyping, are currently widely used in the monitoring and study of the dynamics of tuberculosis epidemics. A study of the molecular profile of a sample of 129 Myobacterium tuberculosis strains isolated during 2011 was carried out in the National Reference Laboratory for Tuberculosis and Mycobacteria at the Pasteur Institute of Algeria. This sample was selected at random from a set of 350 strains isolated from tuberculosis patients from central and eastern areas of the country. Genotypic analysis helped to clarify the frequencies of the different genotypes in the current study population: H family, 29%; LAM family, 26%; T family, 25%; S family, 5%, and other genomic families, including orphan strains, 15%. The study of strains isolated between January and December 2011 has allowed insight into the frequency of different genomic families and the importance of existing clusters in the population of central and eastern Algeria. Copyright © 2015 Asian African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.

An attenuated quadruple gene mutant of Mycobacterium tuberculosis imparts protection against tuberculosis in guinea pigs

PubMed Central

Chauhan, Priyanka

2018-01-01

ABSTRACT Previously we had developed a triple gene mutant of Mycobacterium tuberculosis (MtbΔmms) harboring disruption in three genes, namely mptpA, mptpB and sapM. Though vaccination with MtbΔmms strain induced protection in the lungs of guinea pigs, the mutant strain failed to control the hematogenous spread of the challenge strain to the spleen. Additionally, inoculation with MtbΔmms resulted in some pathological damage to the spleens in the early phase of infection. In order to generate a strain that overcomes the pathology caused by MtbΔmms in spleen of guinea pigs and controls dissemination of the challenge strain, MtbΔmms was genetically modified by disrupting bioA gene to generate MtbΔmmsb strain. Further, in vivo attenuation of MtbΔmmsb was evaluated and its protective efficacy was assessed against virulent M. tuberculosis challenge in guinea pigs. MtbΔmmsb mutant strain was highly attenuated for growth and virulence in guinea pigs. Vaccination with MtbΔmmsb mutant generated significant protection in comparison to sham-immunized animals at 4 and 12 weeks post-infection in lungs and spleen of infected animals. However, the protection imparted by MtbΔmmsb was significantly less in comparison to BCG immunized animals. This study indicates the importance of attenuated multiple gene deletion mutants of M. tuberculosis for generating protection against tuberculosis. PMID:29242198

Molecular characterisation of Mycobacterium tuberculosis isolates in the First National Survey of Anti-tuberculosis Drug Resistance from Venezuela

PubMed Central

Aristimuño, Liselotte; Armengol, Raimond; Cebollada, Alberto; España, Mercedes; Guilarte, Alexis; Lafoz, Carmen; Lezcano, María A; Revillo, María J; Martín, Carlos; Ramírez, Carmen; Rastogi, Nalin; Rojas, Janet; de Salas, Albina Vázques; Sola, Christophe; Samper, Sofía

2006-01-01

Background Molecular typing of Mycobacterium tuberculosis strains has become a valuable tool in the epidemiology of tuberculosis (TB) by allowing detection of outbreaks, tracking of epidemics, identification of genotypes and transmission events among patients who would have remained undetected by conventional contact investigation. This is the first genetic biodiversity study of M. tuberculosis in Venezuela. Thus, we investigated the genetic patterns of strains isolated in the first survey of anti-tuberculosis drug-resistance realised as part of the Global Project of Anti-tuberculosis Drug Resistance Surveillance (WHO/IUATLD). Results Clinical isolates (670/873) were genotyped by spoligotyping. The results were compared with the international spoligotyping database (SpolDB4). Multidrug resistant (MDR) strains (14/18) were also analysed by IS6110-RFLP assays, and resistance to isoniazid and rifampicin was characterised. Spoligotyping grouped 82% (548/670) of the strains into 59 clusters. Twenty new spoligotypes (SITs) specific to Venezuela were identified. Eight new inter-regional clusters were created. The Beijing genotype was not found. The genetic network shows that the Latin American and Mediterranean family constitutes the backbone of the genetic TB population-structure in Venezuela, responsible of >60% of total TB cases studied. MDR was 0.5% in never treated patients and 13.5% in previously treated patients. Mutations in rpoB gene and katG genes were detected in 64% and 43% of the MDR strains, respectively. Two clusters were found to be identical by the four different analysis methods, presumably representing cases of recent transmission of MDR tuberculosis. Conclusion This study gives a first overview of the M. tuberculosis strains circulating in Venezuela during the first survey of anti-tuberculosis drug-resistance. It may aid in the creation of a national database that will be a valuable support for further studies. PMID:17032442

Comparative genomic analysis of Mycobacterium tuberculosis clinical isolates.

PubMed

Liu, Fei; Hu, Yongfei; Wang, Qi; Li, Hong Min; Gao, George F; Liu, Cui Hua; Zhu, Baoli

2014-06-13

Due to excessive antibiotic use, drug-resistant Mycobacterium tuberculosis has become a serious public health threat and a major obstacle to disease control in many countries. To better understand the evolution of drug-resistant M. tuberculosis strains, we performed whole genome sequencing for 7 M. tuberculosis clinical isolates with different antibiotic resistance profiles and conducted comparative genomic analysis of gene variations among them. We observed that all 7 M. tuberculosis clinical isolates with different levels of drug resistance harbored similar numbers of SNPs, ranging from 1409-1464. The numbers of insertion/deletions (Indels) identified in the 7 isolates were also similar, ranging from 56 to 101. A total of 39 types of mutations were identified in drug resistance-associated loci, including 14 previously reported ones and 25 newly identified ones. Sixteen of the identified large Indels spanned PE-PPE-PGRS genes, which represents a major source of antigenic variability. Aside from SNPs and Indels, a CRISPR locus with varied spacers was observed in all 7 clinical isolates, suggesting that they might play an important role in plasticity of the M. tuberculosis genome. The nucleotide diversity (Л value) and selection intensity (dN/dS value) of the whole genome sequences of the 7 isolates were similar. The dN/dS values were less than 1 for all 7 isolates (range from 0.608885 to 0.637365), supporting the notion that M. tuberculosis genomes undergo purifying selection. The Л values and dN/dS values were comparable between drug-susceptible and drug-resistant strains. In this study, we show that clinical M. tuberculosis isolates exhibit distinct variations in terms of the distribution of SNP, Indels, CRISPR-cas locus, as well as the nucleotide diversity and selection intensity, but there are no generalizable differences between drug-susceptible and drug-resistant isolates on the genomic scale. Our study provides evidence strengthening the notion that the evolution of drug resistance among clinical M. tuberculosis isolates is clearly a complex and diversified process.

Relapse, re-infection and mixed infections in tuberculosis disease.

PubMed

McIvor, Amanda; Koornhof, Hendrik; Kana, Bavesh Davandra

2017-04-01

Tuberculosis (TB) disease can be characterized by genotypic and phenotypic complexity in Mycobacterium tuberculosis bacilli within a single patient. This microbiological heterogeneity has become an area of intense study due its perceived importance in drug tolerance, drug resistance and as a surrogate measure of transmission rates. This review presents a descriptive analysis of research describing the prevalence of mixed-strain TB infections in geographically distinct locations. Despite significant variation in disease burden and a rampant human immunodeficiency virus (HIV)-TB co-epidemic, there was no difference in the prevalence range of mixed infections reported in African countries when compared to the rest of the world. The occurrence of recurrent TB was associated with a higher prevalence of mixed-strain infections, but this difference was not reported as statistically significant. These interpretations were limited by differences in the design and overall size of the studies assessed. Factors such as sputum quality, culture media, number of repeated culture steps, molecular typing methods and HIV-infection status can affect the detection of mixed-strain infection. It is recommended that future clinical studies should focus on settings with varying TB burdens, with a common sample processing protocol to gain further insight into these phenomena and develop novel transmission blocking strategies. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Screening mutations in drug-resistant Mycobacterium tuberculosis strains in Yunnan, China.

PubMed

Li, Daoqun; Song, Yuzhu; Zhang, Cheng-Lin; Li, Xiaofei; Xia, Xueshan; Zhang, A-Mei

Drug-resistant tuberculosis (DR-TB), especially multidrug-resistant tuberculosis (MDR-TB), is a serious medical and societal problem in China. The purpose of this study was to evaluate the mutation characteristics of drug-resistant Mycobacterium tuberculosis (M. tuberculosis) isolates in Yunnan, China. Drug susceptibility testing (DST) was performed in 523 clinical M. tuberculosis isolates. Six drug resistance genes (katG, inhA, rpoB, rpsL, embB, and pncA) were selected to screen for mutations. In total, 54 clinical M. tuberculosis strains were identified as drug-resistant by DST, including 18 single drug-resistant (SDR) strains and 36 multidrug-resistant (MDR) strains. Twenty-four types of mutations in five genes (excluding the inhA gene) were screened in forty-one strains. Six novel mutations were identified in this study, including three missense mutations (p.S302R in katG, p.D78G in embB, and p.M1I in pncA), two frameshift mutations (408 ins A and 538-580 del in pncA), and one mutation in a control region (-6 C>T located upstream of rpsL). The mutation frequencies in the hotspot mutation regions in the katG, rpoB, rpsL, embB, and pncA genes were 92.5%, 44.4%, 54.2%, 52.6%, and 37.5%, respectively. The mutation spectra and frequencies seemed somewhat unique in the Yunnan DR-TB strains. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Population Genomics of Mycobacterium tuberculosis in Ethiopia Contradicts the Virgin Soil Hypothesis for Human Tuberculosis in Sub-Saharan Africa

PubMed Central

Comas, Iñaki; Hailu, Elena; Kiros, Teklu; Bekele, Shiferaw; Mekonnen, Wondale; Gumi, Balako; Tschopp, Rea; Ameni, Gobena; Hewinson, R. Glyn; Robertson, Brian D.; Goig, Galo A.; Stucki, David; Gagneux, Sebastien; Aseffa, Abraham; Young, Douglas; Berg, Stefan

2015-01-01

Summary Colonial medical reports claimed that tuberculosis (TB) was largely unknown in Africa prior to European contact, providing a “virgin soil” for spread of TB in highly susceptible populations previously unexposed to the disease [1, 2]. This is in direct contrast to recent phylogenetic models which support an African origin for TB [3, 4, 5, 6]. To address this apparent contradiction, we performed a broad genomic sampling of Mycobacterium tuberculosis in Ethiopia. All members of the M. tuberculosis complex (MTBC) arose from clonal expansion of a single common ancestor [7] with a proposed origin in East Africa [3, 4, 8]. Consistent with this proposal, MTBC lineage 7 is almost exclusively found in that region [9, 10, 11]. Although a detailed medical history of Ethiopia supports the view that TB was rare until the 20th century [12], over the last century Ethiopia has become a high-burden TB country [13]. Our results provide further support for an African origin for TB, with some genotypes already present on the continent well before European contact. Phylogenetic analyses reveal a pattern of serial introductions of multiple genotypes into Ethiopia in association with human migration and trade. In place of a “virgin soil” fostering the spread of TB in a previously naive population, we propose that increased TB mortality in Africa was driven by the introduction of European strains of M. tuberculosis alongside expansion of selected indigenous strains having biological characteristics that carry a fitness benefit in the urbanized settings of post-colonial Africa. PMID:26687624

Population Genomics of Mycobacterium tuberculosis in Ethiopia Contradicts the Virgin Soil Hypothesis for Human Tuberculosis in Sub-Saharan Africa.

PubMed

Comas, Iñaki; Hailu, Elena; Kiros, Teklu; Bekele, Shiferaw; Mekonnen, Wondale; Gumi, Balako; Tschopp, Rea; Ameni, Gobena; Hewinson, R Glyn; Robertson, Brian D; Goig, Galo A; Stucki, David; Gagneux, Sebastien; Aseffa, Abraham; Young, Douglas; Berg, Stefan

2015-12-21

Colonial medical reports claimed that tuberculosis (TB) was largely unknown in Africa prior to European contact, providing a "virgin soil" for spread of TB in highly susceptible populations previously unexposed to the disease [1, 2]. This is in direct contrast to recent phylogenetic models which support an African origin for TB [3-6]. To address this apparent contradiction, we performed a broad genomic sampling of Mycobacterium tuberculosis in Ethiopia. All members of the M. tuberculosis complex (MTBC) arose from clonal expansion of a single common ancestor [7] with a proposed origin in East Africa [3, 4, 8]. Consistent with this proposal, MTBC lineage 7 is almost exclusively found in that region [9-11]. Although a detailed medical history of Ethiopia supports the view that TB was rare until the 20(th) century [12], over the last century Ethiopia has become a high-burden TB country [13]. Our results provide further support for an African origin for TB, with some genotypes already present on the continent well before European contact. Phylogenetic analyses reveal a pattern of serial introductions of multiple genotypes into Ethiopia in association with human migration and trade. In place of a "virgin soil" fostering the spread of TB in a previously naive population, we propose that increased TB mortality in Africa was driven by the introduction of European strains of M. tuberculosis alongside expansion of selected indigenous strains having biological characteristics that carry a fitness benefit in the urbanized settings of post-colonial Africa. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Mycobacterium bovis in Burkina Faso: epidemiologic and genetic links between human and cattle isolates.

PubMed

Sanou, Adama; Tarnagda, Zekiba; Kanyala, Estelle; Zingué, Dezemon; Nouctara, Moumini; Ganamé, Zakaria; Combary, Adjima; Hien, Hervé; Dembele, Mathurin; Kabore, Antoinette; Meda, Nicolas; Van de Perre, Philippe; Neveu, Dorine; Bañuls, Anne Laure; Godreuil, Sylvain

2014-10-01

In sub-Saharan Africa, bovine tuberculosis (bTB) is a potential hazard for animals and humans health. The goal of this study was to improve our understanding of bTB epidemiology in Burkina Faso and especially Mycobacterium bovis transmission within and between the bovine and human populations. Twenty six M. bovis strains were isolated from 101 cattle carcasses with suspected bTB lesions during routine meat inspections at the Bobo Dioulasso and Ouagadougou slaughterhouses. In addition, 7 M. bovis strains were isolated from 576 patients with pulmonary tuberculosis. Spoligotyping, RDAf1 deletion and MIRU-VNTR typing were used for strains genotyping. The isolation of M. bovis strains was confirmed by spoligotyping and 12 spoligotype signatures were detected. Together, the spoligotyping and MIRU-VNTR data allowed grouping the 33 M. bovis isolates in seven clusters including isolates exclusively from cattle (5) or humans (1) or from both (1). Moreover, these data (genetic analyses and phenetic tree) showed that the M. bovis isolates belonged to the African 1 (Af1) clonal complex (81.8%) and the putative African 5 (Af5) clonal complex (18.2%), in agreement with the results of RDAf1 deletion typing. This is the first detailed molecular characterization of M. bovis strains from humans and cattle in Burkina Faso. The distribution of the two Af1 and putative Af5 clonal complexes is comparable to what has been reported in neighbouring countries. Furthermore, the strain genetic profiles suggest that M. bovis circulates across the borders and that the Burkina Faso strains originate from different countries, but have a country-specific evolution. The genetic characterization suggests that, currently, M. bovis transmission occurs mainly between cattle, occasionally between cattle and humans and potentially between humans. This study emphasizes the bTB risk in cattle but also in humans and the difficulty to set up proper disease control strategies in Burkina Faso.

Resistance pattern of multi-drug resistant strains of Mycobacterium tuberculosis and characteristics of patients with multi-drug resistant tuberculosis.

PubMed

Moisoiu, Adriana; Mitran, Cristina Iulia; Mitran, Mãdãlina Irina; Huhu, Mihaela Roxana; Ioghen, Octavian Costin; Gheorghe, Adelina-Silvana; Tampa, Mircea; Georgescu, Simona Roxana; Popa, Mircea Ioan

2016-01-01

Multi-drug resistant tuberculosis (MDR-TB) is a major concern in the medical community. Knowledge about the drug resistance pattern of Mycobacterium tuberculosis strains plays an essential role in the management of the disease. We conducted a retrospective, 3-year study (2009-2011), in an urban area. We collected data on the drug resistance for 497 M. tuberculosis strains, isolated from patients with pulmonary TB. Among the 497 strains, we identified 158 MDR strains. Eighty medical recorders of patients infected with MDR strains were available and we included those patients in the study group. Of the 497 analysed strains, 8% were resistant to a single anti-TB drug. We identified 5.2% polyresistant drug strains, the most frequent combination being INH+EMB (1.4%). Of the 158 MDR strains identified (31.8%), over 60% were resistant to all first line anti-TB drugs tested. Most of them presented resistance to STM (86.1%) and EMB (67.7%). With respect to second line anti-TB drugs resistance to KM (23.4%) was the most common, followed by OFX (8.2%). With respect to the patients with MDR-TB, a percentage of 61.2% of them had a history of anti-TB treatment. Regarding lifestyle habits, 61.2% of the patients were smokers and 18.8% were abusing alcohol. Out of 51 patients, for whom information was available regarding their occupation, only 33.3 % were employees. MDR strains of Mycobacterium tuberculosis display an increased resistance to first line anti-TB drugs. Extension of resistance to second line anti-TB drugs narrows the therapeutic options. Knowledge of MDR-TB risk factors is imperative for the correct and rapid initiation of the treatment.

Primary and acquired drug resistance in Mycobacterium tuberculosis strains in western region of Libyan Arab Jamahiriya.

PubMed

Elghoul, M T; Joshi, R M; Rizghalla, T

1989-10-01

Drug resistance in Mycobacterium tuberculosis strains prevalent in the Western Region of Libyan Arab Jamahiriya was studied for the years 1984, 1985 and 1986 at the regional tuberculosis control centre at Gurgi, Tripoli. Records of resistance to streptomycin, isoniazid, ethambutol and rifampicin were analysed. Whereas primary drug resistance was observed in 5.1%, 19.5% and 3.8%, acquired drug resistance was found in 12.2%, 34.0% and 15.3% of the strains in 1984, 1985 and 1986 respectively. Only 3 out of 598 strains (1.2%) were found to show acquired resistance to rifampicin. No primary resistance to rifampicin was observed. The situation of drug resistance in pulmonary tuberculosis in the Jamahiriya is discussed.

Recent transmission of Mycobacterium tuberculosis in China: the implication of molecular epidemiology for tuberculosis control.

PubMed

Yang, Chongguang; Gao, Qian

2018-02-01

Tuberculosis (TB) has remained an ongoing concern in China. The national scale-up of the Directly Observed Treatment, Short Course (DOTS) program has accelerated the fight against TB in China. Nevertheless, many challenges still remain, including the spread of drug-resistant strains, high disease burden in rural areas, and enormous rural-to-urban migrations. Whether incident active TB represents recent transmission or endogenous reactivation has helped to prioritize the strategies for TB control. Evidence from molecular epidemiology studies has delineated the recent transmission of Mycobacterium tuberculosis (M. tuberculosis) strains in many settings. However, the transmission patterns of TB in most areas of China are still not clear. Studies carried out to date could not capture the real burden of recent transmission of the disease in China because of the retrospective study design, incomplete sampling, and use of low-resolution genotyping methods. We reviewed the implementations of molecular epidemiology of TB in China, the estimated disease burden due to recent transmission of M. tuberculosis strains, the primary transmission of drug-resistant TB, and the evaluation of a feasible genotyping method of M. tuberculosis strains in circulation.

Rv1458c: a new diagnostic marker for identification of Mycobacterium tuberculosis complex in a novel duplex PCR assay.

PubMed

Shrivastava, Kamal; Garima, Kushal; Narang, Anshika; Bhattacharyya, Kausik; Vishnoi, Ekta; Singh, Roshan Kumar; Chaudhry, Anil; Prasad, Rajendra; Bose, Mridula; Varma-Basil, Mandira

2017-03-01

We explored the efficiency of Rv1458c, the gene encoding a putative ABC drug transporter specific for the Mycobacterium tuberculosis complex (MTBC), as a diagnostic marker. A 190 bp region of Rv1458c and a 300 bp region of hsp65 were targeted in a novel duplex PCR assay and the results were compared with those for PCR restriction analysis(PRA) using the restriction enzymes NruI and BamHI. Species identification of a subset of the isolates (n=50) was confirmed by sequencing. Clinical isolates of M. tuberculosis (n=426) obtained from clinically suspected patients of pulmonary tuberculosis and mycobacterial (n=13) and non-mycobacterial (n=8) reference strains were included in the study. The duplex PCR assay correctly identified 320/426 isolates as MTBC and 106/426 isolates as non-tuberculous mycobacteria(NTM). The test was 100 % specific and sensitive when compared with NruI/BamHI PCR restriction analysis and highlighted the use of Rv1458c as a diagnostic marker for MTBC. The duplex PCR assay could be developed for use as a screening test to identify MTBC in clinical specimens in peripheral laboratories with limited resources.

Spatiotemporal Clustering of Mycobacterium tuberculosis Complex Genotypes in Florida: Genetic Diversity Segregated by Country of Birth

PubMed Central

Séraphin, Marie Nancy; Lauzardo, Michael; Morris, J. Glenn; Blackburn, Jason K.

2016-01-01

Background Tuberculosis (TB) is caused by members of the Mycobacterium tuberculosis complex (MTBC). Although the MTBC is highly clonal, between-strain genetic diversity has been observed. In low TB incidence settings, immigration may facilitate the importation of MTBC strains with a potential to complicate TB control efforts. Methods We investigated the genetic diversity and spatiotemporal clustering of 2,510 MTBC strains isolated in Florida, United States, between 2009 and 2013 and genotyped using spoligotyping and 24-locus MIRU-VNTR. We mapped the genetic diversity to the centroid of patient residential zip codes using a geographic information system (GIS). We assessed transmission dynamics and the influence of immigration on genotype clustering using space-time permutation models adjusted for foreign-born population density and county-level HIV risk and multinomial models stratified by country of birth and timing of immigration in SaTScan. Principal Findings Among the 2,510 strains, 1,245 were reported among foreign-born persons; including 408 recent immigrants (<5 years). Strain allelic diversity (h) ranged from low to medium in most locations and was most diverse in urban centers where foreign-born population density was also high. Overall, 21.5% of cases among U.S.-born persons and 4.6% among foreign-born persons clustered genotypically and spatiotemporally and involved strains of the Haarlem family. One Haarlem space-time cluster identified in the mostly rural northern region of Florida included US/Canada-born individuals incarcerated at the time of diagnosis; two clusters in the mostly urban southern region of Florida were composed predominantly of foreign-born persons. Both groups had HIV prevalence above twenty percent. Conclusions/Significance Almost five percent of TB cases reported in Florida during 2009–2013 were potentially due to recent transmission. Improvements to TB screening practices among the prison population and recent immigrants are likely to impact TB control. Due to the monomorphic nature of available markers, whole genome sequencing is needed to conclusively delineate recent transmission events between U.S. and foreign-born persons. PMID:27093156

Utility of Phenotypic and Genotypic Testing in the Study of Mycobacterium tuberculosis Resistance to First-Line Anti-Tuberculosis drugs.

PubMed

Alba Álvarez, Luz María; García García, José María; Pérez Hernández, M Dolores; Martínez González, Susana; Palacios Gutiérrez, Juan José

2017-04-01

To determine the utility of molecular techniques in the diagnosis of resistance and the extent of resistance to first-line drugs in our region. From 2004 to 2013, 1,889 strains of Mycobacterium tuberculosis complex isolated in Asturias, Spain, were studied using phenotypic (Clinical and Laboratory Standards Institute guidelines) and molecular (INNOLiPA RIF-TB © ; GenotypeMDRplus © ; GenotypeMDRsl © ) sensitivity tests. 1,759 strains (94.52%) were sensitive to all first-line drugs, and 102 strains (5.48%) showed some resistance: 81 strains (4.35%) were resistant to 1 single drug, 14 (0.75%) were polyresistant, and 7 (0.37%) were multiresistant (resistant to rifampicin and isoniazid). In total, 137 resistances were identified: 60 to isoniazid (3.22%), 7 to rifampicin (0.37%), 9 to pyrazinamide (0.48%), 11 to ethambutol (0.59%), and 50 to streptomycin (2.68%). Of the mutations detected, 75.9% (63/83) correlated with resistance, while 24.09% of mutations detected (20/83) were not associated with resistance; 16 of these involved a silent mutation at codon 514 of the rpoB gene. Between 0 and 90% of strains, depending on the drug under consideration, were resistant even when no gene mutations were detected using marketed systems. Molecular techniques are very useful, particularly for obtaining rapid results, but these must be confirmed with standard phenotypic sensitivity testing. The rate of resistance in our region is low and multi-drug resistantcases (0.37%) are sporadic. Copyright © 2016 SEPAR. Publicado por Elsevier España, S.L.U. All rights reserved.

Evidence of transmission of Mycobacterium tuberculosis by random amplified polymorphic DNA (RAPD) fingerprinting in Taipei City, Taiwan.

PubMed Central

Harn, H J; Shen, K L; Ho, L I; Yu, K W; Liu, G C; Yueh, K C; Lee, J H

1997-01-01

AIMS: To determine, by strain identification of Mycobacterium tuberculosis, whether transmission has occurred between individuals or whether new strains are present. METHODS: A rapid protocol for random amplified polymorphic DNA (RAPD) analysis was developed. This protocol was applied to 64 strains of M tuberculosis that had been confirmed by culture and microbiological methods. RESULTS: There are five groups of M tuberculosis prevalent in Taipei city, Taiwan. The major types are groups I and III. Groups I and II had been prevalent until the end of last year when, according to our group analysis, they had been eradicated. However, group III was continuously present from the middle of 1995 to the middle of 1996, and group IV was present at the end of both years, which indicated that both groups were transmitted continuously. These clustered strains had demographic characteristics consistent with a finding of transmission tuberculosis. Also, there were 13 of 64 strains with unique RAPD fingerprints that were inferred to be due primarily to the reactivation of infection. In the drug resistance analysis, the major type represented included group III and part of group IV. CONCLUSIONS: Our preliminary data imply, not only that the prevalence of M tuberculosis in Taipei city is due to transmission rather than reactivation, but that drug resistance also may play a role in tuberculosis transmission. Images PMID:9378819

Goats challenged with different members of the Mycobacterium tuberculosis complex display different clinical pictures.

PubMed

Bezos, J; Casal, C; Díez-Delgado, I; Romero, B; Liandris, E; Álvarez, J; Sevilla, I A; Juan, L de; Domínguez, L; Gortázar, C

2015-10-15

Tuberculosis (TB) in goats (Capra hircus) is due to infection with members of the Mycobacterium tuberculosis complex (MTC), mainly Mycobacterium bovis and Mycobacterium caprae. We report a comparative experimental infection of goats with M. bovis, M. caprae and M. tuberculosis strains. We hypothesized that goats experimentally infected with different members of the MTC would display different clinical pictures. Three groups of goats were challenged with either M. bovis SB0134 (group 1, n=5), M. caprae SB0157 (group 2, n=5) and M. tuberculosis SIT58 (group 3, n=4). The highest mean total lesion score was observed in M. bovis challenged goats (mean 15.2, range 9-19), followed by those challenged with M. caprae (10.8, 2-23). The lowest score was recorded in goats challenged with M. tuberculosis (3, 1-6). Culture results coincided with the lesion scores in yielding more positive pools (7/15) in M. bovis challenged goats. By contrast, only three pools were positive from goats challenged M. tuberculosis (3/12) and with M. caprae (3/15), respectively. Differences in the performance of the intradermal and gamma-interferon (IFN-γ) tests depending of the group were observed since all goats from group 1 were diagnosed using intradermal test and these goats reacted earlier to the IFN-γ assay in comparison to the other groups. This study confirmed that goats experimentally infected with different members of the MTC display different clinical pictures and this fact may have implications for MTC maintenance and bacterial shedding. Copyright © 2015 Elsevier B.V. All rights reserved.

Deciphering the recent phylogenetic expansion of the originally deeply rooted Mycobacterium tuberculosis lineage 7.

PubMed

Yimer, Solomon A; Namouchi, Amine; Zegeye, Ephrem Debebe; Holm-Hansen, Carol; Norheim, Gunnstein; Abebe, Markos; Aseffa, Abraham; Tønjum, Tone

2016-06-30

A deeply rooted phylogenetic lineage of Mycobacterium tuberculosis (M. tuberculosis) termed lineage 7 was discovered in Ethiopia. Whole genome sequencing of 30 lineage 7 strains from patients in Ethiopia was performed. Intra-lineage genome variation was defined and unique characteristics identified with a focus on genes involved in DNA repair, recombination and replication (3R genes). More than 800 mutations specific to M. tuberculosis lineage 7 strains were identified. The proportion of non-synonymous single nucleotide polymorphisms (nsSNPs) in 3R genes was higher after the recent expansion of M. tuberculosis lineage 7 strain started. The proportion of nsSNPs in genes involved in inorganic ion transport and metabolism was significantly higher before the expansion began. A total of 22346 bp deletions were observed. Lineage 7 strains also exhibited a high number of mutations in genes involved in carbohydrate transport and metabolism, transcription, energy production and conversion. We have identified unique genomic signatures of the lineage 7 strains. The high frequency of nsSNP in 3R genes after the phylogenetic expansion may have contributed to recent variability and adaptation. The abundance of mutations in genes involved in inorganic ion transport and metabolism before the expansion period may indicate an adaptive response of lineage 7 strains to enable survival, potentially under environmental stress exposure. As lineage 7 strains originally were phylogenetically deeply rooted, this may indicate fundamental adaptive genomic pathways affecting the fitness of M. tuberculosis as a species.

Analysis of uracil phosphoribosyltransferase expression in Mycobacterium tuberculosis and evaluation of upp knockout strain in infected mice.

PubMed

Villela, Anne Drumond; Pham, Ha; Jones, Victoria; Grzegorzewicz, Anna E; Rodrigues-Junior, Valnês da Silva; Campos, Maria Martha; Basso, Luiz Augusto; Jackson, Mary; Santos, Diógenes Santiago

2017-02-01

The upp (Rv3309c)-encoded uracil phosphoribosyltransferase from Mycobacterium tuberculosis (MtUPRT) converts uracil and 5-phosphoribosyl-α-1-pyrophosphate into pyrophosphate and uridine 5΄-monophosphate, the precursor of all pyrimidine nucleotides. A M. tuberculosis knockout strain for upp gene was generated by allelic replacement. Knockout and complemented strains were validated by a functional assay of uracil incorporation. A basal level of MtUPRT expression is shown to be independent of either growth medium used, addition of bases, or oxygen presence/absence. The upp disruption does not affect M. tuberculosis growth in Middlebrook 7H9 medium, and it is not required for M. tuberculosis virulence in a mouse model of infection. Thus, MtUPRT is unlikely to be a good target for drugs against M. tuberculosis. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Rapid and accurate identification of Mycobacterium tuberculosis complex and common non-tuberculous mycobacteria by multiplex real-time PCR targeting different housekeeping genes.

PubMed

Nasr Esfahani, Bahram; Rezaei Yazdi, Hadi; Moghim, Sharareh; Ghasemian Safaei, Hajieh; Zarkesh Esfahani, Hamid

2012-11-01

Rapid and accurate identification of mycobacteria isolates from primary culture is important due to timely and appropriate antibiotic therapy. Conventional methods for identification of Mycobacterium species based on biochemical tests needs several weeks and may remain inconclusive. In this study, a novel multiplex real-time PCR was developed for rapid identification of Mycobacterium genus, Mycobacterium tuberculosis complex (MTC) and the most common non-tuberculosis mycobacteria species including M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and the M. gordonae in three reaction tubes but under same PCR condition. Genetic targets for primer designing included the 16S rDNA gene, the dnaJ gene, the gyrB gene and internal transcribed spacer (ITS). Multiplex real-time PCR was setup with reference Mycobacterium strains and was subsequently tested with 66 clinical isolates. Results of multiplex real-time PCR were analyzed with melting curves and melting temperature (T (m)) of Mycobacterium genus, MTC, and each of non-tuberculosis Mycobacterium species were determined. Multiplex real-time PCR results were compared with amplification and sequencing of 16S-23S rDNA ITS for identification of Mycobacterium species. Sensitivity and specificity of designed primers were each 100 % for MTC, M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and M. gordonae. Sensitivity and specificity of designed primer for genus Mycobacterium was 96 and 100 %, respectively. According to the obtained results, we conclude that this multiplex real-time PCR with melting curve analysis and these novel primers can be used for rapid and accurate identification of genus Mycobacterium, MTC, and the most common non-tuberculosis Mycobacterium species.

[Description of Mycobacterium tuberculosis mutations conferring resistance to rifampicin and isoniazid detected by GenoType® MTBDRplus V.2 in Colombia].

PubMed

Llerena, Claudia; Medina, Raquel

2017-01-24

The GenoType®MTBDRplusV.2 assay is a molecular technique endorsed by the World Health Organization and the Pan American Health Organization that allows for the identification of the Mycobacterium tuberculosis complex and the detection of mutations in the rpoβ gene for rifampicin resistance, and katG and inhA genes for isoniazid resistance. Due to the genetic variability in the circulating strains around the world, the national tuberculosis control programs should assess the performance of these new diagnostic technologies and their use under program conditions as rapid tests. To describe the mutations identified by the GenoType®MTBDRplusV.2 assay in pulmonary samples and Mycobacterium tuberculosis isolates in the Laboratorio Nacional de Referencia of the Instituto Nacional de Salud in 2014. We conducted a retrospective, descriptive study to detect the expression of inhA, KatG and rpoβ genes, responsible for resistence against isoniazid and rifampicin using the GenoType® MTBDRplus V.2 assay in 837 samples and isolates from tuberculosis cases. Several mutations in the rpoβ gene were identified. Ser531Leu was the most frequent (36.6%) followed by Asp516Val (21.6%), while Ser315Thr1 was the most frequent mutation in the katG gene (91.9%). We were able to identify different mutations present in MDR-TB strains in the country, with frequencies similar to those reported in other countries in the South American region.

Mycobacterium tuberculosis strains of the Beijing genotype are rarely observed in tuberculosis patients in South America.

PubMed

Ritacco, Viviana; López, Beatriz; Cafrune, Patricia I; Ferrazoli, Lucilaine; Suffys, Philip N; Candia, Norma; Vásquez, Lucy; Realpe, Teresa; Fernández, Jorge; Lima, Karla V; Zurita, Jeannete; Robledo, Jaime; Rossetti, Maria L; Kritski, Afranio L; Telles, Maria A; Palomino, Juan C; Heersma, Herre; van Soolingen, Dick; Kremer, Kristin; Barrera, Lucía

2008-08-01

The frequency of the Beijing genotype of Mycobacterium tuberculosis as a cause of tuberculosis (TB) in South America was determined by analyzing genotypes of strains isolated from patients that had been diagnosed with the disease between 1997 and 2003 in seven countries of the subcontinent. In total, 19 of the 1,202 (1.6%) TB cases carried Beijing isolates, including 11 of the 185 patients from Peru (5.9%), five of the 512 patients from Argentina (1.0%), two of the 252 Brazilian cases (0.8%), one of the 166 patients from Paraguay (0.6%) and none of the samples obtained from Chile (35), Colombia (36) and Ecuador (16). Except for two patients that were East Asian immigrants, all cases with Beijing strains were native South Americans. No association was found between carrying a strain with the Beijing genotype and having drug or multi-drug resistant disease. Our data show that presently transmission of M. tuberculosis strains of the Beijing genotype is not frequent in Latin America. In addition, the lack of association of drug resistant TB and infection with M. tuberculosis of the Beijing genotype observed presently demands efforts to define better the contribution of the virulence and lack of response to treatment to the growing spread of Beijing strains observed in other parts of the world.

Mycobacterium tuberculosis Whole Genome Sequences From Southern India Suggest Novel Resistance Mechanisms and the Need for Region-Specific Diagnostics.

PubMed

Manson, Abigail L; Abeel, Thomas; Galagan, James E; Sundaramurthi, Jagadish Chandrabose; Salazar, Alex; Gehrmann, Thies; Shanmugam, Siva Kumar; Palaniyandi, Kannan; Narayanan, Sujatha; Swaminathan, Soumya; Earl, Ashlee M

2017-06-01

India is home to 25% of all tuberculosis cases and the second highest number of multidrug resistant cases worldwide. However, little is known about the genetic diversity and resistance determinants of Indian Mycobacterium tuberculosis, particularly for the primary lineages found in India, lineages 1 and 3. We whole genome sequenced 223 randomly selected M. tuberculosis strains from 196 patients within the Tiruvallur and Madurai districts of Tamil Nadu in Southern India. Using comparative genomics, we examined genetic diversity, transmission patterns, and evolution of resistance. Genomic analyses revealed (11) prevalence of strains from lineages 1 and 3, (11) recent transmission of strains among patients from the same treatment centers, (11) emergence of drug resistance within patients over time, (11) resistance gained in an order typical of strains from different lineages and geographies, (11) underperformance of known resistance-conferring mutations to explain phenotypic resistance in Indian strains relative to studies focused on other geographies, and (11) the possibility that resistance arose through mutations not previously implicated in resistance, or through infections with multiple strains that confound genotype-based prediction of resistance. In addition to substantially expanding the genomic perspectives of lineages 1 and 3, sequencing and analysis of M. tuberculosis whole genomes from Southern India highlight challenges of infection control and rapid diagnosis of resistant tuberculosis using current technologies. Further studies are needed to fully explore the complement of diversity and resistance determinants within endemic M. tuberculosis populations. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.

Distinct Clinical and Epidemiological Features of Tuberculosis in New York City Caused by the RDRio Mycobacterium tuberculosis Sublineage

PubMed Central

Weisenberg, Scott A.; Gibson, Andrea L.; Huard, Richard C.; Kurepina, Natalia; Bang, Heejung; Lazzarini, Luiz C O.; Chiu, Yalin; Li, Jiehui; Ahuja, Shama; Driscoll, Jeff; Kreiswirth, Barry N.; Ho, John L.

2011-01-01

Background Genetic tracking of Mycobacterium tuberculosis is a cornerstone of tuberculosis (TB) control programs. The RDRio M. tuberculosis sublineage was previously associated with TB in Brazil. We investigated 3847 M. tuberculosis isolates and registry data from New York City (NYC) (2001–2005) to: 1) affirm the position of RDRio strains within the M. tuberculosis phylogenetic structure, 2) determine its prevalence, and 3) define transmission, demographic, and clinical characteristics associated with RDRio TB. Methods Isolates classified as RDRio or non-RDRio M. tuberculosis by multiplex PCR were further classified as clustered (≥2 isolates) or unique based primarily upon IS6110-RFLP patterns and lineage-specific cluster proportions were calculated. The secondary case rate of RDRio was compared with other prevalent M. tuberculosis lineages. Genotype data were merged with the data from the NYC TB Registry to assess demographic and clinical characteristics. Results RDRio strains were found to: 1) be restricted to the Latin American-Mediterranean family, 2) cause approximately 8% of TB cases in NYC, and 3) be associated with heightened transmission as shown by: i) a higher cluster proportion compared to other prevalent lineages, ii) a higher secondary case rate, and iii) cases in children. Furthermore, RDRio strains were significantly associated with US-born Black or Hispanic race, birth in Latin American and Caribbean countries, and isoniazid resistance. Conclusions The RDRio genotype is a single M. tuberculosis strain population that is emerging in NYC. The findings suggest that expanded RDRio case and exposure identification could be of benefit due to its association with heightened transmission. PMID:21835266

Conspicuous multidrug-resistant Mycobacterium tuberculosis cluster strains do not trespass country borders in Latin America and Spain.

PubMed

Ritacco, Viviana; Iglesias, María-José; Ferrazoli, Lucilaine; Monteserin, Johana; Dalla Costa, Elis R; Cebollada, Alberto; Morcillo, Nora; Robledo, Jaime; de Waard, Jacobus H; Araya, Pamela; Aristimuño, Liselotte; Díaz, Raúl; Gavin, Patricia; Imperiale, Belen; Simonsen, Vera; Zapata, Elsa M; Jiménez, María S; Rossetti, Maria L; Martin, Carlos; Barrera, Lucía; Samper, Sofia

2012-06-01

Multidrug-resistant Mycobacterium tuberculosis strain diversity in Ibero-America was examined by comparing extant genotype collections in national or state tuberculosis networks. To this end, genotypes from over 1000 patients with multidrug-resistant tuberculosis diagnosed from 2004 through 2008 in Argentina, Brazil, Chile, Colombia, Venezuela and Spain were compared in a database constructed ad hoc. Most of the 116 clusters identified by IS6110 restriction fragment length polymorphism were small and restricted to individual countries. The three largest clusters, of 116, 49 and 25 patients, were found in Argentina and corresponded to previously documented locally-epidemic strains. Only 13 small clusters involved more than one country, altogether accounting for 41 patients, of whom 13 were, in turn, immigrants from Latin American countries different from those participating in the study (Peru, Ecuador and Bolivia). Most of these international clusters belonged either to the emerging RD(Rio) LAM lineage or to the Haarlem family of M. tuberculosis and four were further split by country when analyzed with spoligotyping and rifampin resistance-conferring mutations, suggesting that they did not represent ongoing transnational transmission events. The Beijing genotype accounted for 1.3% and 10.2% of patients with multidrug-resistant tuberculosis in Latin America and Spain, respectively, including one international cluster of two cases. In brief, Euro-American genotypes were widely predominant among multidrug-resistant M. tuberculosis strains in Ibero-America, reflecting closely their predominance in the general M. tuberculosis population in the region, and no evidence was found of acknowledged outbreak strains trespassing country borders. Copyright © 2011 Elsevier B.V. All rights reserved.

Molecular characterization of bovine tuberculosis strains in two slaughterhouses in Morocco.

PubMed

Yahyaoui-Azami, Hind; Aboukhassib, Hamid; Bouslikhane, Mohammed; Berrada, Jaouad; Rami, Soukaina; Reinhard, Miriam; Gagneux, Sebastien; Feldmann, Julia; Borrell, Sonia; Zinsstag, Jakob

2017-08-25

Bovine tuberculosis (BTB) is caused by Mycobacterium bovis, which belongs to the Mycobacterium tuberculosis complex. Mycobacterium bovis have been described to be responsible of most cases of bovine tuberculosis. Although M. tuberculosis, M. africanum and non-complex mycobacteria were isolated from cattle. In Morocco, so far, no molecular studies were conducted to characterize the strains responsible of BTB. The present study aims to characterize M. bovis in Morocco. The present study was conducted in slaughterhouses in Rabat and El Jadida. Samples were collected from 327 slaughtered animals with visible lesions suggesting BTB. A total of 225 isolates yielded cultures, 95% (n = 215) of them were acid-fast (AF). Sixty eight per cent of the AF positive samples were confirmed as tuberculous mycobacteria (n = 147), 99% of these (n = 146) having RD9 and among the latter, 98% (n = 143) positive while 2% (n = 3) negative for RD4 A total of 134 samples were analyzed by spoligotyping of which 14 were in cluster and with 41 different spoligotypes, ten of them were new patterns (23%). The most prevalent spoligotypes were SB0121, SB0265, and SB0120, and were already identified in many other countries, such as Algeria, Spain, Tunisia, the United States and Argentina. The shared borders between Algeria and Morocco, in addition to the previous importation of cattle from Europe and the US could explain the similarities found in M. bovis spoligotypes. On the other hand, the desert of Morocco could be considered as an efficient barrier preventing the introduction of BTB to Morocco from West Central and East Africa. Our findings suggest a low level endemic transmission of BTB similar to other African countries. However, more research is needed for further knowledge about the transmission patterns of BTB in Morocco.

Mycobacterium tuberculosis strains exhibit differential and strain-specific molecular signatures in pulmonary epithelial cells.

PubMed

Mvubu, Nontobeko Eunice; Pillay, Balakrishna; Gamieldien, Junaid; Bishai, William; Pillay, Manormoney

2016-12-01

Although pulmonary epithelial cells are integral to innate and adaptive immune responses during Mycobacterium tuberculosis infection, global transcriptomic changes in these cells remain largely unknown. Changes in gene expression induced in pulmonary epithelial cells infected with M. tuberculosis F15/LAM4/KZN, F11, F28, Beijing and Unique genotypes were investigated by RNA sequencing (RNA-Seq). The Illumina HiSeq 2000 platform generated 50 bp reads that were mapped to the human genome (Hg19) using Tophat (2.0.10). Differential gene expression induced by the different strains in infected relative to the uninfected cells was quantified and compared using Cufflinks (2.1.0) and MeV (4.0.9), respectively. Gene expression varied among the strains with the total number of genes as follows: F15/LAM4/KZN (1187), Beijing (1252), F11 (1639), F28 (870), Unique (886) and H37Rv (1179). A subset of 292 genes was commonly induced by all strains, where 52 genes were down-regulated while 240 genes were up-regulated. Differentially expressed genes were compared among the strains and the number of induced strain-specific gene signatures were as follows: F15/LAM4/KZN (138), Beijing (52), F11 (255), F28 (55), Unique (186) and H37Rv (125). Strain-specific molecular gene signatures associated with functional pathways were observed only for the Unique and H37Rv strains while certain biological functions may be associated with other strain signatures. This study demonstrated that strains of M. tuberculosis induce differential gene expression and strain-specific molecular signatures in pulmonary epithelial cells. Specific signatures induced by clinical strains of M. tuberculosis can be further explored for novel host-associated biomarkers and adjunctive immunotherapies. Copyright © 2016 Elsevier Ltd. All rights reserved.

In vitro evaluation of the cyto-genotoxic potential of Ruthenium(II) SCAR complexes: a promising class of antituberculosis agents.

PubMed

De Grandis, Rone Aparecido; Resende, Flávia Aparecida; da Silva, Monize Martins; Pavan, Fernando Rogério; Batista, Alzir Azevedo; Varanda, Eliana Aparecida

2016-03-01

Tuberculosis is a top infectious disease killer worldwide, caused by the bacteria Mycobacterium tuberculosis. Increasing incidences of multiple drug-resistance (MDR) strains are emerging as one of the major public health threats. However, the drugs in use are still incapable of controlling the appalling upsurge of MDR. In recent years a marked number of research groups have devoted their attention toward the development of specific and cost-effective antimicrobial agents against targeted MDR-Tuberculosis. In previous studies, ruthenium(II) complexes (SCAR) have shown a promising activity against MDR-Tuberculosis although few studies have indeed considered ruthenium toxicity. Therefore, within the preclinical requirements, we have sought to determine the cyto-genotoxicity of three SCAR complexes in this present study. The treatment with the SCARs induced a concentration-dependent decrease in cell viability in CHO-K1 and HepG2 cells. Based on the clonogenic survival, SCAR 5 was found to be more cytotoxic while SCAR 6 exhibited selectivity action on tumor cells. Although SCAR 4 and 5 did not indicate any mutagenic activity as evidenced by the Ames and Cytokinesis block micronucleus cytome assays, the complex SCAR 6 was found to engender a frameshift mutation detected by Salmonella typhimurium in the presence of S9. Similarly, we observed a chromosomal damage in HepG2 cells with significant increases of micronuclei and nucleoplasmic bridges. These data indicate that SCAR 4 and 5 complexes did not show genotoxicity in our models while SCAR 6 was considered mutagenic. This study presented a comprehensive genotoxic evaluation of SCAR complexes were shown to be genotoxic in vitro. All in all, further studies are required to fully elucidate how the properties can affect human health. Copyright © 2016 Elsevier B.V. All rights reserved.

Drug Resistance in Newly Presenting and Previously Treated Tuberculosis Patients in Guangxi Province, People's Republic of China.

PubMed

Luo, Dan; Zhao, Jinming; Lin, Mei; Liu, Feiying; Huang, Shuhai; Zhang, Yingkun; Huang, Minying; Li, Juan; Zhou, Yang; Lan, Rushu; Zhao, Yanlin

2017-05-01

Drug-resistant Mycobacterium tuberculosis strains are a major threat to the control of tuberculosis (TB), but the prevalence of drug-resistant TB is still unknown in the southern ethnic region of China. A cluster-randomized sampling method was used to include the study population. Isolates were tested for resistance to 6 antituberculosis drugs, and genotyped to identify Beijing strains. Overall, 11.3% (139/1229) of new cases and 33.0% (126/382) of retreated cases had drug-resistant tuberculosis. Multiple previous TB treatment episodes and multiple treatment interruptions were risk factors for both drug-resistant and multidrug-resistant TB among retreated cases. A total of 53.2% of the patients were infected with a Beijing strain of M tuberculosis. Infection with a Beijing strain was significantly associated with drug resistance among new cases (odds ratio, 1.44; 95% CI, 1.01-2.07). Novel strategies to rapid diagnosis and effective treatment are urgently needed to prevent the development of drug resistance.

Genotyping of Mycobacterium tuberculosis with additional markers enhances accuracy in epidemiological studies.

PubMed Central

Warren, R; Richardson, M; Sampson, S; Hauman, J H; Beyers, N; Donald, P R; van Helden, P D

1996-01-01

Two highly polymorphic Mycobacterium tuberculosis genomic domains, characterized by hybridization to the oligonucleotide (GTG)5, were identified as potential DNA fingerprinting probes. These domains were cloned [pMTB484(1) and pMTB484(2K4), respectively] and shown to be useful for genotype analysis by Southern blotting. These probes were used to genotype geographically linked strains of M. tuberculosis previously shown to have identical IS6110 fingerprints. Subsequent DNA fingerprints generated with MTB484(1) and MTB484(2K4) showed a high degree of polymorphism, allowing subclassification of IS6110-defined clusters into composites of smaller clusters and unique strains. Correlation of the molecular data with patient interviews and clinical records confirmed the sensitivity of these probes, as contacts were established only within subclusters. These findings demonstrate the requirement for multiple probes to accurately classify M. tuberculosis strains, even those with high copy numbers of IS6110. The enhanced accuracy of strain typing should, in turn, further our understanding of the epidemiology of tuberculosis. PMID:8862588

[MOLECULAR CHARACTERISTICS OF THE MULTIDRUG-RESISTANT MYCOBACTERIUM TUBERCULOSIS STRAINS IN THE NORTHWEST RUSSIA].

PubMed

Vyazovaya, A A; Mokrousov, I V; Zhuravlev, V Yu; Solovieva, N S; Otten, T F; Manicheva, O A; Vishnevsky, B I; Narvskaya, O V

2016-01-01

The goal of this work was to study the genotypic characteristics of the multidrug-resistant (MDR, i.e., resistant to at least rifampicine and isoniazid) Mycobacterium tuberculosis strains isolated in 2011-2012 from tuberculosis (TB) patients in the Northwest Russia. Spoligotyping of 195 M. tuberculosis isolates identified 14 different spoligotypes and assigned isolates to the genetic families Beijing (n = 162, 83%), LAM (n = 15), H3/URAL (n = 14), as well as T, Haarlem and X. Spoligotypes SIT1 (Beijing), SIT42 (LAM) and SIT262 (H3/URAL) were the most prevalent. Irrespective to the genotype, all the isolates were resistant to streptomycin. The multidrug resistance was accompanied by the resistance to ethionamide (56%), amikacin (31%), kanamycin (40%), and capreomycin (33%). The ethambutol resistance was found in 71% (n = 115) and 42% (n = 14) of the Beijing and non-Beijing strains, respectively (p < 0.05). In conclusion, the multidrug resistant M. tuberculosis population circulating in the Northwest Russia continues to be dominated by the Beijing family strains.

Transmission of Mycobacterium tuberculosis in China: A Population-Based Molecular Epidemiologic Study

PubMed Central

Yang, Chongguang; Shen, Xin; Peng, Ying; Lan, Rushu; Zhao, Yuling; Long, Bo; Luo, Tao; Sun, Guomei; Li, Xia; Qiao, Ke; Gui, Xiaohong; Wu, Jie; Xu, Jiying; Li, Fabin; Li, Dingyue; Liu, Feiying; Shen, Mei; Hong, Jianjun; Mei, Jian; DeRiemer, Kathryn; Gao, Qian

2015-01-01

Background. Understanding the transmission of Mycobacterium tuberculosis is essential for the development of efficient tuberculosis control strategies. China has the second-largest tuberculosis burden in the world. Recent transmission and infection with M. tuberculosis, particularly drug-resistant strains, may account for many new tuberculosis cases. Methods. We performed a population-based molecular epidemiologic study of pulmonary tuberculosis in China during 1 July 2009 to 30 June 2012. We defined clusters as cases with identical variable number tandem repeat genotype patterns and identified the risk factors associated with clustering, by logistic regression. Relative transmission rates were estimated by the sputum smear status and drug susceptibility status of tuberculosis patients. Results. Among 2274 culture-positive tuberculosis patients with genotyped isolates, there were 705 (31.0%) tuberculosis patients in 287 clusters. Multidrug-resistant (MDR) tuberculosis (adjusted odds ratio [aOR], 1.86; 95% confidence interval [CI], 1.25–2.63) and infection with a Beijing family strain (aOR, 1.56; 95% CI, 1.23–2.96) were associated with clustering. Eighty-four of 280 (30.0%) clusters had a putative source case that was sputum smear negative, and 30.6% of their secondary cases were attributed to transmission by sputum smear–negative patients. The relative transmission rate for sputum smear negative compared with sputum smear–positive patients was 0.89 (95% CI, .68–1.10), and was 1.51 (95% CI, 1.00–2.24) for MDR tuberculosis vs drug-susceptible tuberculosis. Conclusions. Recent transmission of M. tuberculosis, including MDR strains, contributes substantially to tuberculosis disease in China. Sputum smear–negative cases were responsible for at least 30% of the secondary cases. Interventions to reduce the transmission of M. tuberculosis should be implemented in China. PMID:25829000

Sequence analysis of the drug‑resistant rpoB gene in the Mycobacterium tuberculosis L‑form among patients with pneumoconiosis complicated by tuberculosis.

PubMed

Lu, Jun; Jiang, Shan; Ye, Song; Deng, Yun; Ma, Shuai; Li, Chao-Pin

2014-04-01

The aim of the present study was to investigate the mutational characteristics of the drug‑resistant Mycobacterium tuberculosis L‑form of the rpoB gene isolated from patients with pneumoconiosis complicated by tuberculosis, in order to reduce the occurrence of the drug resistance of patients and gain a more complete information on the resistance of the Mycobacterium tuberculosis L‑form. A total of 42 clinically isolated strains of Mycobacterium tuberculosis L‑form were collected, including 31 drug‑resistant strains. The genomic DNA was extracted, then the target genes were amplified by polymerase chain reaction and the hot mutational regions of the rpoB gene were analyzed by direct sequencing. The results revealed that no rpoB gene mutation was present in 11 rifampicin (RFP)‑sensitive strains, while conformational changes were identified in 31 RFP‑resistant strains. The mutation rate was 93.55% (29/31) in the resistant strains, and was frequently concentrated in codons 531 (51.61%; 16/31) and 526 (32.26%; 10/31), mainly occurring by case substitutions, including 27 unit point mutations and two two‑point mutations. The novel mutation identified in codon 516 had not been previously reported. The substitution of highly‑conserved amino acids encoded by the rpoB gene resulted in the molecular mechanism responsible for RFP resistance in the Mycobacterium tuberculosis L‑form. This also demonstrated that the rpoB gene is diversiform.

Comparison of MALDI-TOF MS, nucleic acid hybridization and the MPT64 immunochromatographic test for the identification of M. tuberculosis and non-tuberculosis Mycobacterium species.

PubMed

Şamlı, Asuman; İlki, Arzu

2016-10-01

Mycobacteria are an important cause of morbidity in humans. Rapid and accurate mycobacterial identification is important for improving patient outcomes. However, identification of Mycobacterium species is not easy, due to the slow and fastidious growth of mycobacteria. Recently, biochemical, sequencing, and probing methods have come to be used for identification. This study compared the performance of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of M.tuberculosis and non-tuberculosis Mycobacteria (NTM) to those of nucleic acid hybridization (NAH) and the MPT64 immunochromatographic test. A total of 69 isolates from Marmara University Hospital, Microbiology Laboratory obtained between 2012 and 2013 were included in our study. All strains were grown on Lowenstein-Jensen and Middlebrook 7H9 medium. Among the 69 isolates, 56 (81%) were isolated as Mycobacterium tuberculosis complex (MTC), and 13 (19%) were isolated as NTM by the MPT64 ICT. NAH was able to identify all isolates to the species level. The isolated NTM included M. intracellulare (n:5), M. lentiflavum (n:3), M. xenopi (n:2), M. malmoense (n:1), M. abscessus (n:1), and M. avium (n:1). MALDI-TOF MS identified 88% of the mycobacterial isolates. All M. tuberculosis strains were identified correctly, but the ratio was 38.5% for NTM. Mycobacterial identification using MALDI-TOF MS takes 45 minutes and costs 3 Euro/test, whereas mycobacterial identification using NAH takes 6-7 hours and costs 30 Euro/test. In conclusion, MALDI-TOF MS has the potential to identify mycobacteria in the clinical laboratory setting by reducing identification turnaround time and laboratory costs for isolate referral.

In vitro activity of roxithromycin against the Mycobacterium tuberculosis complex.

PubMed Central

Rastogi, N; Goh, K S; Ruiz, P; Casal, M

1995-01-01

Roxithromycin has recently been shown to possess significant in vitro activity against a variety of atypical mycobacteria such as the M. avium complex, M. scrofulaceum, M. szulgai, M. malmoense, M. xenopi, M. marinum, and M. kansasii and rare pathogens like M. chelonei and M. fortuitum. In the present investigation, screening of its in vitro activity was further extended by testing it against 34 strains belonging to the M. tuberculosis complex (including M. tuberculosis, M. africanum, M. bovis, and M. bovis BCG). The MICs were determined by the radiometric BACTEC 460-TB methodology at pHs of both 6.8 and 7.4, as well as with 7H10 agar medium by the 1% proportion method. With the exception of M. bovis BCG (MIC ranges, 0.5 to 4 micrograms/ml at pH 6.8 and 0.25 to 2 micrograms/ml at pH 7.4), MICs for all of the isolates were significantly greater (MIC ranges, 32 to > 64 micrograms/ml at pH 6.8 and 16 to > 32 micrograms/ml at pH 7.4) than those reported previously for atypical mycobacteria. Roxithromycin MICs of 64 or > 64 micrograms/ml for all of the M. tuberculosis isolates screened were found by the 7H10 agar medium method. Roxithromycin, however, showed a pH-dependent bactericidal effect against M. tuberculosis because the drug was relatively more active when it was used at pH 7.4 than when it was used at pH 6.8. We conclude that roxithromycin per se is not a drug of choice for the treatment of M. tuberculosis infection or disease; however, considering its pharmacokinetics, eventual anti-tubercle bacillus activity in an in vivo system cannot yet be excluded. We suggest that the use of roxithromycin in chemoprophylactic regimens for the prevention of opportunistic infections (including M. avium complex infections) in patients with AIDS should be carefully monitored, and patients should be enrolled in such a regimen only after it has been excluded that the patient das an underlying infection of disease caused by M. tuberculosis. PMID:7625806

Mycobacterium tuberculosis Complex Genotype Diversity and Drug Resistance Profiles in a Pediatric Population in Mexico

PubMed Central

Macías Parra, Mercedes; Kumate Rodríguez, Jesús; Arredondo García, José Luís; López-Vidal, Yolanda; Castañón-Arreola, Mauricio; Balandrano, Susana; Rastogi, Nalin; Gutiérrez Castrellón, Pedro

2011-01-01

The aim of this study was to determine the frequency of drug resistance and the clonality of genotype patterns in M. tuberculosis clinical isolates from pediatric patients in Mexico (n = 90 patients from 19 states; time period—January 2002 to December 2003). Pulmonary disease was the most frequent clinical manifestation (71%). Children with systemic tuberculosis (TB) were significantly younger compared to patients with localized TB infections (mean 7.7 ± 6.2 years versus 15 ± 3.4 years P = 0.001). Resistance to any anti-TB drug was detected in 24/90 (26.7%) of the isolates; 21/90 (23.3%) and 10/90 (11.1%) were resistant to Isoniazid and Rifampicin, respectively, and 10/90 (11.1%) strains were multidrug-resistant (MDR). Spoligotyping produced a total of 55 different patterns; 12/55 corresponded to clustered isolates (n = 47, clustering rate of 52.2%), and 43/55 to unclustered isolates (19 patterns were designated as orphan by the SITVIT2 database). Database comparison led to designation of 36 shared types (SITs); 32 SITs (n = 65 isolates) matched a preexisting shared type in SITVIT2, whereas 4 SITs (n = 6 isolates) were newly created. Lineage classification based on principal genetic groups (PGG) revealed that 10% of the strains belonged to PGG1 (Bovis and Manu lineages). Among PGG2/3 group, the most predominant clade was the Latin-American and Mediterranean (LAM) in 27.8% of isolates, followed by Haarlem and T lineages. The number of single drug-resistant (DR) and multidrug-resistant (MDR-TB) isolates in this study was similar to previously reported in studies from adult population with risk factors. No association between the spoligotype, age, region, or resistance pattern was observed. However, contrary to a study on M. tuberculosis spoligotyping in Acapulco city that characterized a single cluster of SIT19 corresponding to the EAI2-Manila lineage in 70 (26%) of patients, not a single SIT19 isolate was found in our pediatric patient population. Neither did we find any shared type belonging to the EAI family which represents ancestral PGG1 strains within the M. tuberculosis complex. We conclude that the population structure of pediatric TB in our setting is different from the one prevailing in adult TB patient population of Guerrero. PMID:22567263

Molecular characterization of isoniazid-resistant Mycobacterium tuberculosis clinical strains isolated in the Philippines.

PubMed

Herrera, Laura; Valverde, Azucena; Saiz, Pilar; Sáez-Nieto, Juan A; Portero, José L; Jiménez, M Soledad

2004-06-01

The prevalence of mutations in the katG, inhA and oxyR-ahpC genes of isoniazid (INH)-resistant Mycobacterium tuberculosis isolates in the Philippines were determined. Of 306 M. tuberculosis isolates studied, 81 (26.5%) exhibited INH-resistance. Forty-four strains (54.3%) had mutations in the katG gene, eighteen strains (22.2%) had mutations in the putative inhA locus region, seven had mutations in both regions and five strains had mutations in the oxyR-ahpC operon. Only seven strains had no mutations. A total of 71 of the 81 (87.6%) resistant strains and 65 of the 72 (90.3%) INH sensitive randomly selected strains showed amino acid substitution in codon 463 (Arg to Leu) (88.9%). This fact supports the hypothesis that mutations at codon 463 are independent of INH-resistance and are linked to the geographical origins of the strains. Copyright 2004 Elsevier B.V.

Molecular Characteristics and Drug Susceptibility of Mycobacterium tuberculosis Isolates from Patients Co-infected with Human Immunodeficiency Virus in Beijing, China.

PubMed

Liu, Jie; Wang, Hui Zhu; Lian, Lu Lu; Yu, Yan Hua; Zhao, Xiu Qin; Guo, Cai Ping; Liu, Hai Can; Liu, Shu Mei; Zhao, Hui; Zeng, Zhao Ying; Zhao, Xiu Ying; Wan, Kang Lin

2015-03-01

70 clinical Mycobacterium tuberculosis strains isolated from AIDS patients in two HIV/AIDS referral hospitals in Beijing were used in this study. M. tuberculosis and non-tuberculosis mycobacterium (NTM) were identified by using multi-locus PCR. M. tuberculosis was genotyped by using 15-locus MIRU-VNTR technique and spoligotyping afterwards. Meanwhile, the drug susceptibilities of the strains to the four first-line anti TB drugs (rifampin, isoniazid, streptomycin, and ethambutol) and the four second-line anti-TB drugs (capreomycin, kanamycin, ofloxacin, and ethionanide) were tested with proportional method. In this study, M. tuberculosis and NTM strains isolated from AIDS patients with TB-like symptoms were identified and genotyping analysis indicated that Beijing genotype was the predominant genotype. In addition, the prevalence of drug-resistant TB, especially the prevalence of XDR-TB, was higher than that in TB patients without HIV infection. Copyright © 2015 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

PolyTB: A genomic variation map for Mycobacterium tuberculosis

PubMed Central

Coll, Francesc; Preston, Mark; Guerra-Assunção, José Afonso; Hill-Cawthorn, Grant; Harris, David; Perdigão, João; Viveiros, Miguel; Portugal, Isabel; Drobniewski, Francis; Gagneux, Sebastien; Glynn, Judith R.; Pain, Arnab; Parkhill, Julian; McNerney, Ruth; Martin, Nigel; Clark, Taane G.

2014-01-01

Summary Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is the second major cause of death from an infectious disease worldwide. Recent advances in DNA sequencing are leading to the ability to generate whole genome information in clinical isolates of M. tuberculosis complex (MTBC). The identification of informative genetic variants such as phylogenetic markers and those associated with drug resistance or virulence will help barcode Mtb in the context of epidemiological, diagnostic and clinical studies. Mtb genomic datasets are increasingly available as raw sequences, which are potentially difficult and computer intensive to process, and compare across studies. Here we have processed the raw sequence data (>1500 isolates, eight studies) to compile a catalogue of SNPs (n = 74,039, 63% non-synonymous, 51.1% in more than one isolate, i.e. non-private), small indels (n = 4810) and larger structural variants (n = 800). We have developed the PolyTB web-based tool (http://pathogenseq.lshtm.ac.uk/polytb) to visualise the resulting variation and important meta-data (e.g. in silico inferred strain-types, location) within geographical map and phylogenetic views. This resource will allow researchers to identify polymorphisms within candidate genes of interest, as well as examine the genomic diversity and distribution of strains. PolyTB source code is freely available to researchers wishing to develop similar tools for their pathogen of interest. PMID:24637013

Strain-specific estimation of epidemic success provides insights into the transmission dynamics of tuberculosis

PubMed Central

Rasigade, Jean-Philippe; Barbier, Maxime; Dumitrescu, Oana; Pichat, Catherine; Carret, Gérard; Ronnaux-Baron, Anne-Sophie; Blasquez, Ghislaine; Godin-Benhaim, Christine; Boisset, Sandrine; Carricajo, Anne; Jacomo, Véronique; Fredenucci, Isabelle; Pérouse de Montclos, Michèle; Flandrois, Jean-Pierre; Ader, Florence; Supply, Philip; Lina, Gérard; Wirth, Thierry

2017-01-01

The transmission dynamics of tuberculosis involves complex interactions of socio-economic and, possibly, microbiological factors. We describe an analytical framework to infer factors of epidemic success based on the joint analysis of epidemiological, clinical and pathogen genetic data. We derive isolate-specific, genetic distance-based estimates of epidemic success, and we represent success-related time-dependent concepts, namely epidemicity and endemicity, by restricting analysis to specific time scales. The method is applied to analyze a surveillance-based cohort of 1,641 tuberculosis patients with minisatellite-based isolate genotypes. Known predictors of isolate endemicity (older age, native status) and epidemicity (younger age, sputum smear positivity) were identified with high confidence (P < 0.001). Long-term epidemic success also correlated with the ability of Euro-American and Beijing MTBC lineages to cause active pulmonary infection, independent of patient age and country of origin. Our results demonstrate how important insights into the transmission dynamics of tuberculosis can be gained from active surveillance data. PMID:28349973

The viability and protein expression of Beijing/W lineage Mycobacterium tuberculosis circulating in Xinjiang, China.

PubMed

Li, Feng; Li, Hua; Zuo, Wei-Ze; Mi, Ligu; Wang, Xian; Wang, Yuanzhi; Wang, Hong; Shen, Aiping; Cao, Shuaili; Yuan, Li

2015-05-01

Beijing/W lineage strains of Mycobacterium tuberculosis spread faster than other strains, tend to be more virulent and frequently associated with drug resistance. In this study, to distinguish the characteristics of Beijing/W lineage and non-Beijing/W lineage M. tuberculosis, we assessed the growth between the two groups under conditions of hypoxia, nutrient starvation, and intracellular growth in murine macrophages. We also examined the DNA, RNA, and protein levels of 5 major M. tuberculosis proteins, including HspX, Hsp65, 38 kDa, Ag85B, and MPT64 of the different types of strains by sequencing, quantitative RT-PCR, and Western blotting. The results showed that Beijing/W and non-Beijing/W lineage strains of M. tuberculosis have similar viability in ex vivo culture but differ in their ability to survive within macrophages, and the intracellular viability of the Beijing/W lineage strains was significantly more than the viability of the non-Beijing/W lineage strains at 2, 3, and 5 days after infection (P < 0.05). Psts1 and fbpB were expressed at statistically lower levels in Beijing/W lineage strains in their mRNA expression levels (P < 0.05). The expression of their corresponding 38 kDa and Ag85B was lower in the Beijing/W lineage strains than the non-Beijing/W lineage strains (P < 0.05). The expression of HspX and Hsp65 was higher in the Beijing/W lineage strains in their protein expression levels at 24 h after infection of RAW264.7 macrophages (P < 0.05). In conclusion, the increased viability of the Beijing/W lineage strains might be related to the expression levels of these proteins.

Transmission of tuberculosis in the prison of Antananarivo (Madagascar).

PubMed

Rasolofo-Razanamparany, V; Ménard, D; Ratsitorahina, M; Aurégan, G; Gicquel, B; Chanteau, S

2000-11-01

The prevalence of tuberculosis in the Antananarivo prison is 16 times higher than that in the general population of Madagascar. We compared the clustering of Mycobacterium tuberculosis strains within and outside the prison and studied the transmission of strains in the prison. M. tuberculosis strains isolated in 1994 to 1995 from 146 prisoners and from 260 nonprisoner patients from Antananarivo were typed using the genetic markers IS6110 and direct repeat. We compared the strains isolated from prisoners and nonprisoners and found that the clustering rate was higher within (58.9%) than outside the prison (40%) suggesting that the transmission rate was higher in prison. Of the 146 incarcerated patients, 82 were grouped into 22 clusters. We checked for possible tuberculosis transmission between prisoners with identical strains by epidemiological investigation of the various prison clusters. We found that 9.5% of the incarcerated patients could have been sources of infection and that only 15.1% could have been infected in the prison. One hundred and twenty-seven prison patients were new cases. Epidemiological data suggested that 37% of them resulted from a reactivation of an old infection, due to poor living conditions or recent transmission from an index case outside the prison.

[Mycobacterial species repartition: experience of the Antituberculosis Center in Pointe Noire (Republic of Congo)].

PubMed

Ontsira Ngoyi, E N; Obengui; Taty Taty, R; Koumba, E L; Ngala, P; Ossibi Ibara, R B

2014-12-01

The aim of the present work was to describe mycobacteria species isolated in the antituberculosis center of Pointe-Noire city in Congo Brazzaville. It was a descriptive transversal study, conducted between September 2008 and April 2009 (7 months). A simple random sample was established from patients who came to the antituberculosis center of Pointe-Noire City (reference center on diagnosis and treatment of tuberculosis). To those patients consulting with symptoms leading to suspect pulmonary tuberculosis, a sputum sampling in three sessions was conducted. Staining techniques to Ziehl-Neelsen and auramine were performed in Pointe-Noire. Culture, molecular hybridization and antibiotic susceptibility testing to first-line antituberculosis drugs (isoniazid, rifampicin, ethambutol, pyrazinamide or streptomycine) using diffusion method on agar were performed in Cerba Pasteur laboratory in France. In 77 patients, 24 sputum (31.20%) were positive to the microscopic examination and 45 (58.44%) to the culture and identification by molecular hybridization. Mycobacteria species complex isolated were M. tuberculosis with 31 cases (68.9%) and M. africanum with 3 cases (6.67%). Non-tuberculous mycobacteria (NMT) were isolated in association or not with M. tuberculosis in 9 cases (20%) and the most common species were M. intracellulare. In M. tuberculosis species, 7 strains (41.20%) were tested sensitive to the first-line antituberculosis drugs, 8 cases (47%) monoresistance and 2 cases multidrug resistance at both isoniazide and rifampicine (12%) (MDR). This study showed the importance of Mycobacteria species complex and non-mycobacteria species in pulmonary tuberculosis. The data on resistance can help medical physicians in the treatment of pulmonary tuberculosis. Another study with a large population is required to confirm these data.

[Tuberculosis incidence and primary drug resistance rates in young soldiers: data from 14 military hospitals in Turkey].

PubMed

Taş, Dilaver; Taşçı, Cantürk; Demirer, Ersin; Sezer, Ogün; Okutan, Oğuzhan; Kartaloğlu, Zafer

2012-01-01

Tuberculosis is an important health care problem worldwide as well as in Turkey and the control programmes are still in progress. Epidemiological data are necessary to conduct control studies related to the disease. Tuberculosis incidence and drug resistance rates are two necessary parameters which should be monitored for the effective establishment of tuberculosis control. In this objective, tuberculosis incidence and drug resistance rates were studied in young subjects performing their compulsory military service in Turkish Armed Forces. The study was performed in 14 military hospitals which served for the country-wide soldier patients. Based on the computerized medical database of these military hospitals, conscripts diagnosed with tuberculosis between January 01, 2009 and December 31, 2009 were retrospectively evaluated. Drug sensitivity tests of the Mycobacterium tuberculosis complex isolates were done prior to the treatment in the two military medical training hospitals of the two big cities of Turkey (Ankara and Istanbul). There were a total of 259 new tuberculosis cases in 2009 and they were all male with a mean age of 22.51 ± 4.63 years. The number of patients with pulmonary, extrapulmonary (pleuresia, lymphadenitis, others) and both pulmonary and extrapulmonary involvements were 175 (67.5%), 72 (27.8%) and 12 (4.6%), respectively. The total rate of pulmonary tuberculosis cases was 72.2% (187/259) and 64.7% (121/187) of them were smear positive. Since the number of soldiers in Turkish army in the midyear was 537.200; total tuberculosis, pulmonary tuberculosis and smear-positive pulmonary tuberculosis incidences were estimated as 48.2/100.000, 34.8/100.000 and 22.5/100.000, respectively. Drug sensitivity tests was performed for the M.tuberculosis complex strains isolated from 104 cases. Primary resistance rate to at least one drug was detected as 16.3% (n= 17), while the rates of resistance for isoniazid, rifampicin, ethambutol and streptomycin were 12.5% (n= 13), 7.7% (n= 8), 5.8% (n= 6) and 0.9% (n= 1), respectively. Multidrug resistant tuberculosis (isoniazid + rifampicin resistance) was detected in 6 (5.8%) patients. Our data indicated that although tuberculosis incidence among young soldiers was moderately high, a decreasing trend was observed when compared to the previous years. However, the rates of primary anti-tuberculosis drug resistance and multi-drug resistance were found to be high in our study. To decrease the incidence of tuberculosis and multidrug resistant tuberculosis, drug sensitivity tests should be performed for each patient and national tuberculosis programme should be established effectively.

Human macrophage gamma interferon decreases gene expression but not replication of Mycobacterium tuberculosis: analysis of the host-pathogen reciprocal influence on transcription in a comparison of strains H37Rv and CMT97.

PubMed

Cappelli, G; Volpe, P; Sanduzzi, A; Sacchi, A; Colizzi, V; Mariani, F

2001-12-01

Mycobacterium tuberculosis is an intracellular pathogen that readily survives and replicates in human macrophages (MPhi). Host cells have developed different mycobactericidal mechanisms, including the production of inflammatory cytokines. The aim of this study was to compare the MPhi response, in terms of cytokine gene expression, to infection with the M. tuberculosis laboratory strain H37Rv and the clinical M. tuberculosis isolate CMT97. Both strains induce the production of interleukin-12 (IL-12) and IL-16 at comparable levels. However, the clinical isolate induces a significantly higher and more prolonged MPhi activation, as shown by reverse transcription-PCR analysis of IL-1beta, IL-6, IL-10, transforming growth factor beta, tumor necrosis factor alpha, and gamma interferon (IFN-gamma) transcripts. Interestingly, when IFN-gamma transcription is high, the number of M. tuberculosis genes expressed decreases and vice versa, whereas no mycobactericidal effect was observed in terms of bacterial growth. Expression of 11 genes was also studied in the two M. tuberculosis strains by infecting resting or activated MPhi and compared to bacterial intracellular survival. In both cases, a peculiar inverse correlation between expression of these genes and multiplication was observed. The number and type of genes expressed by the two strains differed significantly.

Study of Mycobacterium tuberculosis Complex Genotypic Diversity in Malaysia Reveals a Predominance of Ancestral East-African-Indian Lineage with a Malaysia-Specific Signature

PubMed Central

Ismail, Fazli; Couvin, David; Farakhin, Izzah; Abdul Rahman, Zaidah; Rastogi, Nalin; Suraiya, Siti

2014-01-01

Background Tuberculosis (TB) still constitutes a major public health problem in Malaysia. The identification and genotyping based characterization of Mycobacterium tuberculosis complex (MTBC) isolates causing the disease is important to determine the effectiveness of the control and surveillance programs. Objectives This study intended a first assessment of spoligotyping-based MTBC genotypic diversity in Malaysia followed by a comparison of strains with those prevailing in neighboring countries by comparison with an international MTBC genotyping database. Methods Spoligotyping was performed on a total of 220 M. tuberculosis clinical isolates collected in Kelantan and Kuala Lumpur. The results were compared with the SITVIT2 international database of the Pasteur Institute of Guadeloupe. Results Spoligotyping revealed 77 different patterns: 22 corresponded to orphan patterns while 55 patterns containing 198 isolates were assigned a Spoligo International Type (SIT) designation in the database (the latter included 6 newly created SITs). The eight most common SITs grouped 141 isolates (5 to 56 strains per cluster) as follows: SIT1/Beijing, n = 56, 25.5%; SIT745/EAI1-SOM, n = 33, 15.0%; SIT591/EAI6-BGD1, n = 13, 5.9%; SIT256/EAI5, n = 12, 5.5%; SIT236/EAI5, n = 10, 4.6%; SIT19/EAI2-Manila, n = 9, 4.1%; SIT89/EAI2-Nonthaburi, n = 5, 2.3%; and SIT50/H3, n = 3, 1.4%. The association between city of isolation and lineages was statistically significant; Haarlem and T lineages being higher in Kuala Lumpur (p<0.01). However, no statistically significant differences were noted when comparing drug resistance vs. major lineages, nor between gender and clades. Conclusions The ancestral East-African-Indian (EAI) lineage was most predominant followed by the Beijing lineage. A comparison of strains with those prevailing in neighboring countries in South Asia, East Asia and South East Asia underlined the phylogeographical specificity of SIT745 for Malaysia, and its probable ongoing evolution with locally evolved strains sharing a specific signature characterized by absence of spacers 37, 38, and 40. Pending complementary genotyping confirmation, we propose that SIT745/EAI-SOM is tentatively reclassified as SIT745/EAI-MYS. PMID:25502956

New Approaches and Therapeutic Options for Mycobacterium tuberculosis in a Dormant State.

PubMed

Caño-Muñiz, Santiago; Anthony, Richard; Niemann, Stefan; Alffenaar, Jan-Willem C

2018-01-01

We are far away from the days when tuberculosis (TB) accounted for 1 in 4 deaths during the 19th century. However, Mycobacterium tuberculosis complex (MTBC) strains are still the leading cause of morbidity and mortality by a single infectious disease, with 9.6 million cases and 1.5 million deaths reported. One-third of the world's population is estimated by the WHO to be infected with latent TB. During the last decade, several studies have aimed to define the characteristics of dormant bacteria in these latent infections. General features of the shift to a dormant state encompass several phenotypic changes that reduce metabolic activity. This low metabolic state is thought to increase the resistance of MTBC strains to host/environmental stresses, including antibiotic action. Once the stress ceases (e.g., interruption of treatment), dormant cells can reactivate and cause symptomatic disease again. Therefore, a proper understanding of dormancy could guide the rational development of new treatment regimens that target dormant cells, reducing later relapse. Here, we briefly summarize the latest data on the genetics involved in the regulation of dormancy and discuss new approaches to TB treatment. Copyright © 2017 American Society for Microbiology.

Predicting Mycobacterium tuberculosis Complex Clades Using Knowledge-Based Bayesian Networks

PubMed Central

Bennett, Kristin P.

2014-01-01

We develop a novel approach for incorporating expert rules into Bayesian networks for classification of Mycobacterium tuberculosis complex (MTBC) clades. The proposed knowledge-based Bayesian network (KBBN) treats sets of expert rules as prior distributions on the classes. Unlike prior knowledge-based support vector machine approaches which require rules expressed as polyhedral sets, KBBN directly incorporates the rules without any modification. KBBN uses data to refine rule-based classifiers when the rule set is incomplete or ambiguous. We develop a predictive KBBN model for 69 MTBC clades found in the SITVIT international collection. We validate the approach using two testbeds that model knowledge of the MTBC obtained from two different experts and large DNA fingerprint databases to predict MTBC genetic clades and sublineages. These models represent strains of MTBC using high-throughput biomarkers called spacer oligonucleotide types (spoligotypes), since these are routinely gathered from MTBC isolates of tuberculosis (TB) patients. Results show that incorporating rules into problems can drastically increase classification accuracy if data alone are insufficient. The SITVIT KBBN is publicly available for use on the World Wide Web. PMID:24864238

In vitro activities of levofloxacin used alone and in combination with first- and second-line antituberculous drugs against Mycobacterium tuberculosis.

PubMed Central

Rastogi, N; Goh, K S; Bryskier, A; Devallois, A

1996-01-01

By using the radiometric BACTEC 460-TB methodology, the inhibitory and bactericidal activity of the optically active L-isomer of ofloxacin (levofloxacin) was compared with those of the D-isomer and the commercially available mixture containing equal amounts of DL-isomers (ofloxacin) against the Mycobacterium tuberculosis complex (type strain H37Rv, a panel of drug-susceptible and -resistant clinical isolates including multidrug-resistant isolates of M. tuberculosis, as well as M. africanum, M. bovis, and M. bovis BCG). Levofloxacin MICs (range 0.50 to 0.75 microgram/ml) were about 1 dilution lower than those of ofloxacin (MIC range, 0.75 to 1.00 microgram/ml) and 5 to 6 dilutions lower than those of the D-isomer (MIC range, 32 to 60 micrograms/ml). The MICs of levofloxacin, ofloxacin, and D-ofloxacin at which 90% of the strains are inhibited were 0.50, 1.00, and 64 micrograms/ml, respectively. The multidrug-resistant M. tuberculosis strains resistant to first-line drugs were as susceptible to quinolones as the wild-type drug-susceptible isolates. Levofloxacin at 0.5 microgram/ml showed bactericidal activity comparable to the activities of 1.0 microgram of ofloxacin per ml and 64 micrograms of D-ofloxacin per ml, with MBCs within the range of 0.5 to 2.0 micrograms/ml, compared with MBCs of 0.75 to 4.0 micrograms of ofloxacin per ml for M. tuberculosis, M. africanum, M. bovis BCG. Combination testing of sub-MICs of levolofoxacin with other first-line (isoniazid, rifampin, and ethambutol) and second-line (amikacin and clofazimine) antituberculous drugs was evaluated with various two-, three-, and four-drug combinations; enhanced drug activity was observed in 8 of 25, 12 of 20, and 8 of 15 tests, respectively, indicating that levofloxacin acts in synergy with other antituberculous drugs. PMID:8807049

Innate immune response to Mycobacterium tuberculosis Beijing and other genotypes.

PubMed

Wang, Chongzhen; Peyron, Pascale; Mestre, Olga; Kaplan, Gilla; van Soolingen, Dick; Gao, Qian; Gicquel, Brigitte; Neyrolles, Olivier

2010-10-25

As a species, Mycobacterium tuberculosis is more diverse than previously thought. In particular, the Beijing family of M. tuberculosis strains is spreading and evaluating throughout the world and this is giving rise to public health concerns. Genetic diversity within this family has recently been delineated further and a specific genotype, called Bmyc10, has been shown to represent over 60% of all Beijing clinical isolates in several parts of the world. How the host immune system senses and responds to various M. tuberculosis strains may profoundly influence clinical outcome and the relative epidemiological success of the different mycobacterial lineages. We hypothesised that the success of the Bmyc10 group may, at least in part, rely upon its ability to alter innate immune responses and the secretion of cytokines and chemokines by host phagocytes. We infected human macrophages and dendritic cells with a collection of genetically well-defined M. tuberculosis clinical isolates belonging to various mycobacterial families, including Beijing. We analyzed cytokine and chemokine secretion on a semi-global level using antibody arrays allowing the detection of sixty-five immunity-related soluble molecules. Our data indicate that Beijing strains induce significantly less interleukin (IL)-6, tumor necrosis factor (TNF), IL-10 and GRO-α than the H37Rv reference strain, a feature that is variously shared by other modern and ancient M. tuberculosis families and which constitutes a signature of the Beijing family as a whole. However, Beijing strains did not differ relative to each other in their ability to modulate cytokine secretion. Our results confirm and expand upon previous reports showing that M. tuberculosis Beijing strains in general are poor in vitro cytokine inducers in human phagocytes. The results suggest that the epidemiological success of the Beijing Bmyc10 is unlikely to rely upon any specific ability of this group of strains to impair anti-mycobacterial innate immunity.

Structural and Biophysical Characterization of the Mycobacterium tuberculosis Protein Rv0577, a Protein Associated with Neutral Red Staining of Virulent Tuberculosis Strains and Homologue of the Streptomyces coelicolor Protein KbpA [Structural and Biophysical Characterization of Rv0577, a Protein Associated with Neutral Red Staining of Virulent Strains of Mycobacterium tuberculosis and homolog of the Streptomyces coelicolor protein KbpA

DOE PAGES

Buchko, Garry W.; Echols, Nathaniel; Flynn, E. Megan; ...

2017-07-10

Mycobacterium tuberculosis protein Rv0577 is a prominent antigen in tuberculosis patients, the component responsible for neutral red staining of virulent strains of M. tuberculosis, a putative component in a methylglyoxal detoxification pathway, and an agonist of toll-like receptor 2. It also has an amino acid sequence that is 36% identical to that of Streptomyces coelicolor AfsK-binding protein A (KbpA), a component in the complex secondary metabolite pathways in the Streptomyces genus. To gain insight into the biological function of Rv0577 and the family of KpbA kinase regulators, the crystal structure for Rv0577 was determined to a resolution of 1.75 Å,more » binding properties with neutral red and deoxyadenosine were surveyed, backbone dynamics were measured, and thermal stability was assayed by circular dichroism spectroscopy. The protein is composed of four approximate repeats with a βαβββ topology arranged radially in consecutive pairs to form two continuous eight-strand β-sheets capped on both ends with an α-helix. The two β-sheets intersect in the center at roughly a right angle and form two asymmetric deep “saddles” that may serve to bind ligands. Nuclear magnetic resonance chemical shift perturbation experiments show that neutral red and deoxyadenosine bind to Rv0577. Binding to deoxyadenosine is weaker with an estimated dissociation constants of 4.1 ± 0.3 mM for saddle 1. Heteronuclear steady-state { 1H}– 15N nuclear Overhauser effect, T 1, and T 2 values were generally uniform throughout the sequence with only a few modest pockets of differences. As a result, circular dichroism spectroscopy characterization of the thermal stability of Rv0577 indicated irreversible unfolding upon heating with an estimated melting temperature of 56 °C.« less

Structural and Biophysical Characterization of the Mycobacterium tuberculosis Protein Rv0577, a Protein Associated with Neutral Red Staining of Virulent Tuberculosis Strains and Homologue of the Streptomyces coelicolor Protein KbpA [Structural and Biophysical Characterization of Rv0577, a Protein Associated with Neutral Red Staining of Virulent Strains of Mycobacterium tuberculosis and homolog of the Streptomyces coelicolor protein KbpA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Buchko, Garry W.; Echols, Nathaniel; Flynn, E. Megan

Mycobacterium tuberculosis protein Rv0577 is a prominent antigen in tuberculosis patients, the component responsible for neutral red staining of virulent strains of M. tuberculosis, a putative component in a methylglyoxal detoxification pathway, and an agonist of toll-like receptor 2. It also has an amino acid sequence that is 36% identical to that of Streptomyces coelicolor AfsK-binding protein A (KbpA), a component in the complex secondary metabolite pathways in the Streptomyces genus. To gain insight into the biological function of Rv0577 and the family of KpbA kinase regulators, the crystal structure for Rv0577 was determined to a resolution of 1.75 Å,more » binding properties with neutral red and deoxyadenosine were surveyed, backbone dynamics were measured, and thermal stability was assayed by circular dichroism spectroscopy. The protein is composed of four approximate repeats with a βαβββ topology arranged radially in consecutive pairs to form two continuous eight-strand β-sheets capped on both ends with an α-helix. The two β-sheets intersect in the center at roughly a right angle and form two asymmetric deep “saddles” that may serve to bind ligands. Nuclear magnetic resonance chemical shift perturbation experiments show that neutral red and deoxyadenosine bind to Rv0577. Binding to deoxyadenosine is weaker with an estimated dissociation constants of 4.1 ± 0.3 mM for saddle 1. Heteronuclear steady-state { 1H}– 15N nuclear Overhauser effect, T 1, and T 2 values were generally uniform throughout the sequence with only a few modest pockets of differences. As a result, circular dichroism spectroscopy characterization of the thermal stability of Rv0577 indicated irreversible unfolding upon heating with an estimated melting temperature of 56 °C.« less

Estimating Fitness by Competition Assays between Drug Susceptible and Resistant Mycobacterium tuberculosis of Predominant Lineages in Mumbai, India

PubMed Central

Bhatter, Purva; Chatterjee, Anirvan; D'souza, Desiree; Tolani, Monica; Mistry, Nerges

2012-01-01

Background Multi Drug Resistant Tuberculosis (MDR TB) is a threat to global tuberculosis control. A significant fitness cost has been associated with DR strains from specific lineages. Evaluation of the influence of the competing drug susceptible strains on fitness of drug resistant strains may have an important bearing on understanding the spread of MDR TB. The aim of this study was to evaluate the fitness of MDR TB strains, from a TB endemic region of western India: Mumbai, belonging to 3 predominant lineages namely CAS, Beijing and MANU in the presence of drug susceptible strains from the same lineages. Methodology Drug susceptible strains from a single lineage were mixed with drug resistant strain, bearing particular non synonymous mutation (rpoB D516V; inhA, A16G; katG, S315T1/T2) from the same or different lineages. Fitness of M.tuberculosis (M.tb) strains was evaluated using the difference in growth rates obtained by using the CFU assay system. Conclusion/Significance While MANU were most fit amongst the drug susceptible strains of the 3 lineages, only Beijing MDR strains were found to grow in the presence of any of the competing drug susceptible strains. A disproportionate increase in Beijing MDR could be an alarm for an impending epidemic in this locale. In addition to particular non synonymous substitutions, the competing strains in an environment may impact the fitness of circulating drug resistant strains. PMID:22479407

Impact of Genetic Diversity on the Biology of Mycobacterium tuberculosis Complex Strains.

PubMed

Niemann, Stefan; Merker, Matthias; Kohl, Thomas; Supply, Philip

2016-11-01

Tuberculosis (TB) remains the most deadly bacterial infectious disease worldwide. Its treatment and control are threatened by increasing numbers of multidrug-resistant (MDR) or nearly untreatable extensively drug-resistant (XDR) strains. New concepts are therefore urgently needed to understand the factors driving the TB epidemics and the spread of different strain populations, especially in association with drug resistance. Classical genotyping and, more recently, whole-genome sequencing (WGS) revealed that the world population of tubercle bacilli is more diverse than previously thought. Several major phylogenetic lineages can be distinguished, which are associated with their sympatric host population. Distinct clonal (sub)populations can even coexist within infected patients. WGS is now used as the ultimate approach for differentiating clinical isolates and for linking phenotypic to genomic variation from lineage to strain levels. Multiple lines of evidence indicate that the genetic diversity of TB strains translates into pathobiological consequences, and key molecular mechanisms probably involved in differential pathoadaptation of some main lineages have recently been identified. Evidence also accumulates on molecular mechanisms putatively fostering the emergence and rapid expansion of particular MDR and XDR strain groups in some world regions. However, further integrative studies will be needed for complete elucidation of the mechanisms that allow the pathogen to infect its host, acquire multidrug resistance, and transmit so efficiently. Such knowledge will be key for the development of the most effective new diagnostics, drugs, and vaccination strategies.

Genomic Insight into Mechanisms of Reversion of Antibiotic Resistance in Multidrug Resistant Mycobacterium tuberculosis Induced by a Nanomolecular Iodine-Containing Complex FS-1.

PubMed

Ilin, Aleksandr I; Kulmanov, Murat E; Korotetskiy, Ilya S; Islamov, Rinat A; Akhmetova, Gulshara K; Lankina, Marina V; Reva, Oleg N

2017-01-01

Drug induced reversion of antibiotic resistance is a promising way to combat multidrug resistant infections. However, lacking knowledge of mechanisms of drug resistance reversion impedes employing this approach in medicinal therapies. Induction of antibiotic resistance reversion by a new anti-tuberculosis drug FS-1 has been reported. FS-1 was used in this work in combination with standard anti-tuberculosis antibiotics in an experiment on laboratory guinea pigs infected with an extensively drug resistant (XDR) strain Mycobacterium tuberculosis SCAID 187.0. During the experimental trial, genetic changes in the population were analyzed by sequencing of M. tuberculosis isolates followed by variant calling. In total 11 isolates obtained from different groups of infected animals at different stages of disease development and treatment were sequenced. It was found that despite the selective pressure of antibiotics, FS-1 caused a counter-selection of drug resistant variants that speeded up the recovery of the infected animals from XDR tuberculosis. Drug resistance mutations reported in the genome of the initial strain remained intact in more sensitive isolates obtained in this experiment. Variant calling in the sequenced genomes revealed that the drug resistance reversion could be associated with a general increase in genetic heterogeneity of the population of M. tuberculosis . Accumulation of mutations in PpsA and PpsE subunits of phenolpthiocerol polyketide synthase was observed in the isolates treated with FS-1 that may indicate an increase of persisting variants in the population. It was hypothesized that FS-1 caused an active counter-selection of drug resistant variants from the population by aggravating the cumulated fitness cost of the drug resistance mutations. Action of FS-1 on drug resistant bacteria exemplified the theoretically predicted induced synergy mechanism of drug resistance reversion. An experimental model to study the drug resistance reversion phenomenon is hereby introduced.

Investigating the metabolic capabilities of Mycobacterium tuberculosis H37Rv using the in silico strain iNJ661 and proposing alternative drug targets

PubMed Central

Jamshidi, Neema; Palsson, Bernhard Ø

2007-01-01

Background: Mycobacterium tuberculosis continues to be a major pathogen in the third world, killing almost 2 million people a year by the most recent estimates. Even in industrialized countries, the emergence of multi-drug resistant (MDR) strains of tuberculosis hails the need to develop additional medications for treatment. Many of the drugs used for treatment of tuberculosis target metabolic enzymes. Genome-scale models can be used for analysis, discovery, and as hypothesis generating tools, which will hopefully assist the rational drug development process. These models need to be able to assimilate data from large datasets and analyze them. Results: We completed a bottom up reconstruction of the metabolic network of Mycobacterium tuberculosis H37Rv. This functional in silico bacterium, iNJ661, contains 661 genes and 939 reactions and can produce many of the complex compounds characteristic to tuberculosis, such as mycolic acids and mycocerosates. We grew this bacterium in silico on various media, analyzed the model in the context of multiple high-throughput data sets, and finally we analyzed the network in an 'unbiased' manner by calculating the Hard Coupled Reaction (HCR) sets, groups of reactions that are forced to operate in unison due to mass conservation and connectivity constraints. Conclusion: Although we observed growth rates comparable to experimental observations (doubling times ranging from about 12 to 24 hours) in different media, comparisons of gene essentiality with experimental data were less encouraging (generally about 55%). The reasons for the often conflicting results were multi-fold, including gene expression variability under different conditions and lack of complete biological knowledge. Some of the inconsistencies between in vitro and in silico or in vivo and in silico results highlight specific loci that are worth further experimental investigations. Finally, by considering the HCR sets in the context of known drug targets for tuberculosis treatment we proposed new alternative, but equivalent drug targets. PMID:17555602

Molecular Typing of Mycobacterium Tuberculosis Complex by 24-Locus Based MIRU-VNTR Typing in Conjunction with Spoligotyping to Assess Genetic Diversity of Strains Circulating in Morocco

PubMed Central

Bouklata, Nada; Supply, Philip; Jaouhari, Sanae; Charof, Reda; Seghrouchni, Fouad; Sadki, Khalid; El Achhab, Youness; Nejjari, Chakib; Filali-Maltouf, Abdelkarim

2015-01-01

Background Standard 24-locus Mycobacterial Interspersed Repetitive Unit Variable Number Tandem Repeat (MIRU-VNTR) typing allows to get an improved resolution power for tracing TB transmission and predicting different strain (sub) lineages in a community. Methodology During 2010–2012, a total of 168 Mycobacterium tuberculosis Complex (MTBC) isolates were collected by cluster sampling from 10 different Moroccan cities, and centralized by the National Reference Laboratory of Tuberculosis over the study period. All isolates were genotyped using spoligotyping, and a subset of 75 was genotyped using 24-locus based MIRU-VNTR typing, followed by first line drug susceptibility testing. Corresponding strain lineages were predicted using MIRU-VNTRplus database. Principal Findings Spoligotyping resulted in 137 isolates in 18 clusters (2–50 isolates per cluster: clustering rate of 81.54%) corresponding to a SIT number in the SITVIT database, while 31(18.45%) patterns were unique of which 10 were labelled as “unknown” according to the same database. The most prevalent spoligotype family was LAM; (n = 81 or 48.24% of isolates, dominated by SIT42, n = 49), followed by Haarlem (23.80%), T superfamily (15.47%), >Beijing (2.97%), > U clade (2.38%) and S clade (1.19%). Subsequent 24-Locus MIRU-VNTR typing identified 64 unique types and 11 isolates in 5 clusters (2 to 3isolates per cluster), substantially reducing clusters defined by spoligotyping only. The single cluster of three isolates corresponded to two previously treated MDR-TB cases and one new MDR-TB case known to be contact a same index case and belonging to a same family, albeit residing in 3 different administrative regions. MIRU-VNTR loci 4052, 802, 2996, 2163b, 3690, 1955, 424, 2531, 2401 and 960 were highly discriminative in our setting (HGDI >0.6). Conclusions 24-locus MIRU-VNTR typing can substantially improve the resolution of large clusters initially defined by spoligotyping alone and predominating in Morocco, and could therefore be used to better study tuberculosis transmission in a population-based, multi-year sample context. PMID:26285026

Immunogenicity and protection induced by a Mycobacterium tuberculosis sigE mutant in a BALB/c mouse model of progressive pulmonary tuberculosis.

PubMed

Hernandez Pando, Rogelio; Aguilar, Leon Diana; Smith, Issar; Manganelli, Riccardo

2010-07-01

Tuberculosis is still one of the main challenges to human global health, leading to about two million deaths every year. One of the reasons for its success is the lack of efficacy of the widely used vaccine Mycobacterium bovis BCG. In this article, we analyze the potential use of an attenuated mutant of Mycobacterium tuberculosis H37Rv lacking the sigma factor sigma(E) as a live vaccine. We have demonstrated that BALB/c mice infected by the intratracheal route with this mutant strain showed significantly higher survival rates and less tissue damage than animals infected with the parental or complemented mutant strain. Although animals infected with the sigE mutant had low bacillary loads, their lungs showed significantly higher production of the protective factors gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), inducible nitric oxide synthase (iNOS), and beta-defensins than those of animals infected with the parental or complemented mutant strain. Moreover, we demonstrate that the sigE mutant, when inoculated subcutaneously, was more attenuated than BCG in immunodeficient nude mice, thus representing a good candidate for a novel attenuated live vaccine strain. Finally, when we used the sigE mutant as a subcutaneous vaccine, it was able to induce a higher level of protection than did BCG with both H37Rv and a highly virulent strain of M. tuberculosis (Beijing code 9501000). Taken together, our findings suggest that the sigE mutant is a very promising strain for the development of a new vaccine against tuberculosis.

Mycobacterium tuberculosis population structure and molecular epidemiological analysis in Sucre municipality, Miranda state, Venezuela.

PubMed

Patiño, Margareth A; Abadía, Edgar; Solalba Gómez; Maes, Mailis; Muñoz, Mariana; Gómez, Daniela; Guzmán, Patricia; Méndez, María Victoria; Ramirez, Carmen; Mercedes, España; de Waard, Jacobus; Takiff, Howard

2014-12-01

Sucre municipality is a large, densely populated marginal area in the eastern part of Caracas, Venezuela that consistently has more cases of tuberculosis than other municipalities in the country. To identify the neighborhoods in the municipality with the highest prevalence of tuberculosis, and determine whether the Mycobacterium tuberculosis strain distribution in this municipality is different from that previously found in the western part of Caracas and the rest of Venezuela, we collected data on all tuberculosis cases in the municipality diagnosed in 2005-6. We performed two separate molecular epidemiological studies, spoligotyping 44 strains in a first study, and spoligotyping 131 strains, followed by MIRU-VNTR 15 on 21 clustered isolates in the second. With spoligotyping, the most common patterns were Shared International Type SIT17 (21%); SIT42 (15%); SIT93 (11%); SIT20 (7%); SIT53 (6%), a distribution similar to other parts of Venezuela, except that SIT42 and SIT20 were more common. MIRU-VNTR 15 showed that six of seven SIT17 strains examined belonged to a large cluster previously found circulating in Venezuela, but all of the SIT42 strains were related to a cluster centered in the neighborhoods of Unión and Maca, with a MIRU-VNTR pattern not previously seen in Venezuela. It appears that a large percentage of the tuberculosis in the Sucre municipality is caused by the active transmission of two strain families centered within distinct neighborhoods, one reflecting communication with the rest of the country, and the other suggesting the insular, isolated nature of some sectors.

Tuberculous Lymphadenitis in Ethiopia Predominantly Caused by Strains Belonging to the Delhi/CAS Lineage and Newly Identified Ethiopian Clades of the Mycobacterium tuberculosis Complex

PubMed Central

Biadglegne, Fantahun; Merker, Matthias; Sack, Ulrich; Rodloff, Arne C.; Niemann, Stefan

2015-01-01

Background Recently, newly defined clades of Mycobacterium tuberculosis complex (MTBC) strains, namely Ethiopia 1–3 and Ethiopia H37Rv-like strains, and other clades associated with pulmonary TB (PTB) were identified in Ethiopia. In this study, we investigated whether these new strain types exhibit an increased ability to cause TB lymphadenitis (TBLN) and raised the question, if particular MTBC strains derived from TBLN patients in northern Ethiopia are genetically adapted to their local hosts and/or to the TBLN. Methods Genotyping of 196 MTBC strains isolated from TBLN patients was performed by spoligotyping and 24-loci mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTR) typing. A statistical analysis was carried out to see possible associations between patient characteristics and phylogenetic MTBC strain classification. Results Among 196 isolates, the majority of strains belonged to the Delhi/CAS (38.8%) lineage, followed by Ethiopia 1 (9.7%), Ethiopia 3 (8.7%), Ethiopia H37RV-like (8.2%), Ethiopia 2 and Haarlem (7.7% each), URAL (3.6%), Uganda l and LAM (2% each), S-type (1.5%), X-type (1%), and 0.5% isolates of TUR, EAI, and Beijing genotype, respectively. Overall, 15 strains (7.7%) could not be allocated to a previously described phylogenetic lineage. The distribution of MTBC lineages is similar to that found in studies of PTB samples. The cluster rate (35%) in this study is significantly lower (P = 0.035) compared to 45% in the study of PTB in northwestern Ethiopia. Conclusion In the studied area, lymph node samples are dominated by Dehli/CAS genotype strains and strains of largely not yet defined clades based on MIRU-VNTR 24-loci nomenclature. We found no indication that strains of particular genotypes are specifically associated with TBLN. However, a detailed analysis of specific genetic variants of the locally contained Ethiopian clades by whole genome sequencing may reveal new insights into the host-pathogen co-evolution and specific features that are related to the local host immune system. PMID:26376441

Mycobacterium tuberculosis strains induce strain-specific cytokine and chemokine response in pulmonary epithelial cells.

PubMed

Mvubu, Nontobeko E; Pillay, Balakrishna; McKinnon, Lyle R; Pillay, Manormoney

2018-04-01

M. tuberculosis F15/LAM4/KZN has been associated with high transmission rates of drug resistant tuberculosis in the KwaZulu-Natal province of South Africa. The current study elucidated the cytokine/chemokine responses induced by representatives of the F15/LAM4/KZN and other dominant strain families in pulmonary epithelial cells. Multiplex cytokine analyses were performed at 24, 48 and 72h post infection of the A549 pulmonary epithelial cell line with the F15/LAM4/KZN, F28, F11, Beijing, Unique and H37Rv strains at an MOI of ∼10:1. Twenty-three anti- and pro-inflammatory cytokines/chemokines were detected at all-time intervals. Significantly high concentrations of IL-6, IFN-γ, TNF-α and G-CSF at 48h, and IL-8, IFN-γ, TNF-α, G-CSF and GM-CSF at 72h, were induced by the F28 and F15/LAM4/KZN strains, respectively. Lower levels of cytokines/chemokines were induced by either the Beijing or Unique strains at all three time intervals. All strains induced up-regulation of pathogen recognition receptors (PRRs) (TLR3 and TLR5) while only the F15/LAM4/KZN, F11 and F28 strains induced significant differential expression of TLR2 compared to the Beijing, Unique and H37Rv strains. The low induction of cytokines in epithelial cells by the Beijing strain correlates with its previously reported hypervirulent properties. High concentrations of cytokines and chemokines required for early protection against M. tuberculosis infections induced by the F15/LAM4/KZN and F28 strains suggests a lower virulence of these genotypes compared to the Beijing strain. These findings demonstrate the high diversity in host cytokine/chemokine response to early infection of pulmonary epithelial cells by different strains of M. tuberculosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Immunogenic Properties of Lactobacillus plantarum Producing Surface-Displayed Mycobacterium tuberculosis Antigens

PubMed Central

Kleiveland, Charlotte R.; Minic, Rajna; Moen, Lars F.; Øverland, Lise; Tjåland, Rannei; Carlsen, Harald; Lea, Tor; Eijsink, Vincent G. H.

2016-01-01

ABSTRACT Tuberculosis (TB) remains among the most deadly diseases in the world. The only available vaccine against tuberculosis is the bacille Calmette-Guérin (BCG) vaccine, which does not ensure full protection in adults. There is a global urgency for the development of an effective vaccine for preventing disease transmission, and it requires novel approaches. We are exploring the use of lactic acid bacteria (LAB) as a vector for antigen delivery to mucosal sites. Here, we demonstrate the successful expression and surface display of a Mycobacterium tuberculosis fusion antigen (comprising Ag85B and ESAT-6, referred to as AgE6) on Lactobacillus plantarum. The AgE6 fusion antigen was targeted to the bacterial surface using two different anchors, a lipoprotein anchor directing the protein to the cell membrane and a covalent cell wall anchor. AgE6-producing L. plantarum strains using each of the two anchors induced antigen-specific proliferative responses in lymphocytes purified from TB-positive donors. Similarly, both strains induced immune responses in mice after nasal or oral immunization. The impact of the anchoring strategies was reflected in dissimilarities in the immune responses generated by the two L. plantarum strains in vivo. The present study comprises an initial step toward the development of L. plantarum as a vector for M. tuberculosis antigen delivery. IMPORTANCE This work presents the development of Lactobacillus plantarum as a candidate mucosal vaccine against tuberculosis. Tuberculosis remains one of the top infectious diseases worldwide, and the only available vaccine, bacille Calmette-Guérin (BCG), fails to protect adults and adolescents. Direct antigen delivery to mucosal sites is a promising strategy in tuberculosis vaccine development, and lactic acid bacteria potentially provide easy, safe, and low-cost delivery vehicles for mucosal immunization. We have engineered L. plantarum strains to produce a Mycobacterium tuberculosis fusion antigen and to anchor this antigen to the bacterial cell wall or to the cell membrane. The recombinant strains elicited proliferative antigen-specific T-cell responses in white blood cells from tuberculosis-positive humans and induced specific immune responses after nasal and oral administrations in mice. PMID:27815271

Immunogenic Properties of Lactobacillus plantarum Producing Surface-Displayed Mycobacterium tuberculosis Antigens.

PubMed

Kuczkowska, Katarzyna; Kleiveland, Charlotte R; Minic, Rajna; Moen, Lars F; Øverland, Lise; Tjåland, Rannei; Carlsen, Harald; Lea, Tor; Mathiesen, Geir; Eijsink, Vincent G H

2017-01-15

Tuberculosis (TB) remains among the most deadly diseases in the world. The only available vaccine against tuberculosis is the bacille Calmette-Guérin (BCG) vaccine, which does not ensure full protection in adults. There is a global urgency for the development of an effective vaccine for preventing disease transmission, and it requires novel approaches. We are exploring the use of lactic acid bacteria (LAB) as a vector for antigen delivery to mucosal sites. Here, we demonstrate the successful expression and surface display of a Mycobacterium tuberculosis fusion antigen (comprising Ag85B and ESAT-6, referred to as AgE6) on Lactobacillus plantarum The AgE6 fusion antigen was targeted to the bacterial surface using two different anchors, a lipoprotein anchor directing the protein to the cell membrane and a covalent cell wall anchor. AgE6-producing L. plantarum strains using each of the two anchors induced antigen-specific proliferative responses in lymphocytes purified from TB-positive donors. Similarly, both strains induced immune responses in mice after nasal or oral immunization. The impact of the anchoring strategies was reflected in dissimilarities in the immune responses generated by the two L. plantarum strains in vivo The present study comprises an initial step toward the development of L. plantarum as a vector for M. tuberculosis antigen delivery. This work presents the development of Lactobacillus plantarum as a candidate mucosal vaccine against tuberculosis. Tuberculosis remains one of the top infectious diseases worldwide, and the only available vaccine, bacille Calmette-Guérin (BCG), fails to protect adults and adolescents. Direct antigen delivery to mucosal sites is a promising strategy in tuberculosis vaccine development, and lactic acid bacteria potentially provide easy, safe, and low-cost delivery vehicles for mucosal immunization. We have engineered L. plantarum strains to produce a Mycobacterium tuberculosis fusion antigen and to anchor this antigen to the bacterial cell wall or to the cell membrane. The recombinant strains elicited proliferative antigen-specific T-cell responses in white blood cells from tuberculosis-positive humans and induced specific immune responses after nasal and oral administrations in mice. Copyright © 2016 American Society for Microbiology.

Virulence, immunopathology and transmissibility of selected strains of Mycobacterium tuberculosis in a murine model

PubMed Central

Marquina-Castillo, Brenda; García-García, Lourdes; Ponce-de-León, Alfredo; Jimenez-Corona, Maria-Eugenia; Bobadilla-del Valle, Miriam; Cano-Arellano, Bulmaro; Canizales-Quintero, Sergio; Martinez-Gamboa, Areli; Kato-Maeda, Midori; Robertson, Brian; Young, Douglas; Small, Peter; Schoolnik, Gary; Sifuentes-Osornio, Jose; Hernandez-Pando, Rogelio

2009-01-01

After encounter with Mycobacterium tuberculosis, a series of non-uniform immune responses are triggered that define the course of the infection. Eight M. tuberculosis strains were selected from a prospective population-based study of pulmonary tuberculosis patients (1995–2003) based on relevant clinical/epidemiological patterns and tested in a well-characterized BALB/c mouse model of progressive pulmonary tuberculosis. In addition, a new mouse model of transmissibility consisting of prolonged cohousing (up to 60 days) of infected and naïve animals was tested. Four phenotypes were defined based on strain virulence (mouse survival, lung bacillary load and tissue damage), immunology response (cytokine expression determined by real-time polymerase chain reaction) and transmissibility (lung bacillary loads and cutaneous delayed-type hypersensitivity in naïve animals).We identified four clearly defined strain phenotypes: (1) hypervirulent strain with non-protective immune response and highly transmissible; (2) virulent strain, associated with high expression of proinflammatory cytokines (tumour necrosis factor and interferon) and very low anti-inflammatory cytokine expression (interleukins 4 and 10), which induced accelerated death by immunopathology; (3) strain inducing efficient protective immunity with lower virulence, and (4) strain demonstrating strong and early macrophage activation (innate immunity) with delayed participation of acquired immunity (interferon expression). We were able to correlate virulent and transmissible phenotypes in the mouse model and markers of community transmission such as tuberculin reactivity among contacts, rapid progression to disease and cluster status. However, we were not able to find correlation with the other two phenotypes. Our new transmission model supported the hypothesis that among these strains increased virulence was linked to increased transmission. PMID:19191912

Antimycobacterial activity of methanolic plant extract of Artemisia capillaris containing ursolic acid and hydroquinone against Mycobacterium tuberculosis.

PubMed

Jyoti, Md Anirban; Nam, Kung-Woo; Jang, Woong Sik; Kim, Young-Hee; Kim, Su-Kyung; Lee, Byung-Eui; Song, Ho-Yeon

2016-04-01

In order to protect against Mycobacterium tuberculosis (MTB) infection, novel drugs and new targets should be screened from the vast source of plants. We investigated the potentiality of the herbal plant of Artemisia capillaris extract (AC) against Mycobacterium tuberculosis. In this study, we isolated ursolic acid and hydroquinone by bio-activity guided fractionation from the methanol extracts of AC, and tested the inhibitory effects against several strains of MTB. Anti-mycobacterial evaluation of these compounds was carried out using the MGIT™ 960 and resazurin assay. Mycobacterial morphological changes due to the treatment of these compounds were further evaluated by Transmission electron microscopy (TEM). Ursolic acid (UA) and hydroquinone (HQ) inhibited the growth of both susceptible and resistant strains of M. tuberculosis. The MIC (minimum inhibitory concentration) values of both UA and HQ were 12.5 μg/ml against the susceptible strains of M. tuberculosis. Also both UA and HQ showed 12.5-25 μg/ml of MIC values against MDR/XDR MTB strains. However, against clinical strains of MTB, UA was found sensitive against those strains that are sensitive against both INH and RFP but resistant against those strains that are resistant to INH. On the other hand HQ was sensitive against all clinical strains. TEM image-analysis of the strain H37Ra after treatment with UA revealed cell wall lysis, whereas HQ-treated cells showed deformed cytoplasmic morphology. All these results indicate that AC extracts containing UA and HQ possess promising chemotherapeutic potency against MTB for future use. Copyright © 2015 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Dataset of potential targets for Mycobacterium tuberculosis H37Rv through comparative genome analysis.

PubMed

Asif, Siddiqui M; Asad, Amir; Faizan, Ahmad; Anjali, Malik S; Arvind, Arya; Neelesh, Kapoor; Hirdesh, Kumar; Sanjay, Kumar

2009-12-31

Mycobacterium tuberculosis is the causative agent of the disease, tuberculosis and H37Rv is the most studied clinical strain. We use comparative genome analysis of Mycobacterium tuberculosis H37Rv and human for the identification of potential targets dataset. We used DEG (Database of Essential Genes) to identify essential genes in the H37Rv strain. The analysis shows that 628 of the 3989 genes in Mycobacterium tuberculosis H37Rv were found to be essential of which 324 genes lack similarity to the human genome. Subsequently hypothetical proteins were removed through manual curation. This further resulted in a dataset of 135 proteins with essential function and no homology to human.

Global control of tuberculosis: from extensively drug-resistant to untreatable tuberculosis

PubMed Central

Dheda, Keertan; Gumbo, Tawanda; Gandhi, Neel R; Murray, Megan; Theron, Grant; Udwadia, Zarir; Migliori, G B; Warren, Robin

2017-01-01

Extensively drug-resistant tuberculosis is a burgeoning global health crisis mainly affecting economically active young adults, and has high mortality irrespective of HIV status. In some countries such as South Africa, drug-resistant tuberculosis represents less than 3% of all cases but consumes more than a third of the total national budget for tuberculosis, which is unsustainable and threatens to destabilise national tuberculosis programmes. However, concern about drug-resistant tuberculosis has been eclipsed by that of totally and extremely drug-resistant tuberculosis—ie, resistance to all or nearly all conventional first-line and second-line antituberculosis drugs. In this Review, we discuss the epidemiology, pathogenesis, diagnosis, management, implications for health-care workers, and ethical and medicolegal aspects of extensively drug-resistant tuberculosis and other resistant strains. Finally, we discuss the emerging problem of functionally untreatable tuberculosis, and the issues and challenges that it poses to public health and clinical practice. The emergence and growth of highly resistant strains of tuberculosis make the development of new drugs and rapid diagnostics for tuberculosis—and increased funding to strengthen global control efforts, research, and advocacy—even more pressing. PMID:24717628

Limitations of the Mycobacterium tuberculosis reference genome H37Rv in the detection of virulence-related loci.

PubMed

O'Toole, Ronan F; Gautam, Sanjay S

2017-10-01

The genome sequence of Mycobacterium tuberculosis strain H37Rv is an important and valuable reference point in the study of M. tuberculosis phylogeny, molecular epidemiology, and drug-resistance mutations. However, it is becoming apparent that use of H37Rv as a sole reference genome in analysing clinical isolates presents some limitations to fully investigating M. tuberculosis virulence. Here, we examine the presence of single locus variants and the absence of entire genes in H37Rv with respect to strains that are responsible for cases and outbreaks of tuberculosis. We discuss how these polymorphisms may affect phenotypic properties of H37Rv including pathogenicity. Based on our observations and those of other researchers, we propose that use of a single reference genome, H37Rv, is not sufficient for the detection and characterisation of M. tuberculosis virulence-related loci. We recommend incorporation of genome sequences of other reference strains, in particular, direct clinical isolates, in such analyses in addition to H37Rv. Copyright © 2017 Elsevier Inc. All rights reserved.

Molecular Characteristics of Rifampin- and Isoniazid-Resistant Mycobacterium tuberculosis Strains Isolated in Vietnam

PubMed Central

Van Bac, Nguyen; Son, Nguyen Thai; Lien, Vu Thi Kim; Ha, Chu Hoang; Cuong, Nguyen Huu; Mai, Cung Thi Ngoc; Le, Thanh Hoa

2012-01-01

Molecular characterization of the drug resistance of Mycobacterium tuberculosis strains with different origins can generate information that is useful for developing molecular methods. These methods are widely applicable for rapid detection of drug resistance. A total of 166 rifampin (RIF)- and/or isoniazid (INH)-resistant strains of M. tuberculosis have been isolated from different parts of Vietnam; they were screened for mutations associated with resistance to these drugs by sequence analysis investigating genetic mutations associated with RIF and INH resistance. Seventeen different mutations were identified in 74 RIF-resistant strains, 56 of which (approximately 76%) had mutations in the so-called 81-bp “hot-spot” region of the rpoB gene. The most common point mutations were in codons 531 (37.8%), 526 (23%), and 516 (9.46%) of the rpoB gene. Mutations were not found in three strains (4.05%). In the case of INH resistance, five different mutations in the katG genes of 82 resistant strains were detected, among which the nucleotide substitution at codon 315 (76.83%) is the most common mutation. This study provided the first molecular characterization of INH and RIF resistance of M. tuberculosis strains from Vietnam, and detection of the katG and rpoB mutations of the INH and RIF-resistant strains should be useful for rapid detection of the INH- and RIF-resistant strains by molecular tests. PMID:22170905

Molecular characteristics of rifampin- and isoniazid-resistant mycobacterium tuberculosis strains isolated in Vietnam.

PubMed

Minh, Nghiem Ngoc; Van Bac, Nguyen; Son, Nguyen Thai; Lien, Vu Thi Kim; Ha, Chu Hoang; Cuong, Nguyen Huu; Mai, Cung Thi Ngoc; Le, Thanh Hoa

2012-03-01

Molecular characterization of the drug resistance of Mycobacterium tuberculosis strains with different origins can generate information that is useful for developing molecular methods. These methods are widely applicable for rapid detection of drug resistance. A total of 166 rifampin (RIF)- and/or isoniazid (INH)-resistant strains of M. tuberculosis have been isolated from different parts of Vietnam; they were screened for mutations associated with resistance to these drugs by sequence analysis investigating genetic mutations associated with RIF and INH resistance. Seventeen different mutations were identified in 74 RIF-resistant strains, 56 of which (approximately 76%) had mutations in the so-called 81-bp "hot-spot" region of the rpoB gene. The most common point mutations were in codons 531 (37.8%), 526 (23%), and 516 (9.46%) of the rpoB gene. Mutations were not found in three strains (4.05%). In the case of INH resistance, five different mutations in the katG genes of 82 resistant strains were detected, among which the nucleotide substitution at codon 315 (76.83%) is the most common mutation. This study provided the first molecular characterization of INH and RIF resistance of M. tuberculosis strains from Vietnam, and detection of the katG and rpoB mutations of the INH and RIF-resistant strains should be useful for rapid detection of the INH- and RIF-resistant strains by molecular tests.

Long-Chain Fatty Acyl Coenzyme A Ligase FadD2 Mediates Intrinsic Pyrazinamide Resistance in Mycobacterium tuberculosis

PubMed Central

Rosen, Brandon C.; Dillon, Nicholas A.; Peterson, Nicholas D.; Minato, Yusuke

2016-01-01

ABSTRACT Pyrazinamide (PZA) is a first-line tuberculosis (TB) drug that has been in clinical use for 60 years yet still has an unresolved mechanism of action. Based upon the observation that the minimum concentration of PZA required to inhibit the growth of Mycobacterium tuberculosis is approximately 1,000-fold higher than that of other first-line drugs, we hypothesized that M. tuberculosis expresses factors that mediate intrinsic resistance to PZA. To identify genes associated with intrinsic PZA resistance, a library of transposon-mutagenized Mycobacterium bovis BCG strains was screened for strains showing hypersusceptibility to the active form of PZA, pyrazinoic acid (POA). Disruption of the long-chain fatty acyl coenzyme A (CoA) ligase FadD2 enhanced POA susceptibility by 16-fold on agar medium, and the wild-type level of susceptibility was restored upon expression of fadD2 from an integrating mycobacterial vector. Consistent with the recent observation that POA perturbs mycobacterial CoA metabolism, the fadD2 mutant strain was more vulnerable to POA-mediated CoA depletion than the wild-type strain. Ectopic expression of the M. tuberculosis pyrazinamidase PncA, necessary for conversion of PZA to POA, in the fadD2 transposon insertion mutant conferred at least a 16-fold increase in PZA susceptibility under active growth conditions in liquid culture at neutral pH. Importantly, deletion of fadD2 in M. tuberculosis strain H37Rv also resulted in enhanced susceptibility to POA. These results indicate that FadD2 is associated with intrinsic PZA and POA resistance and provide a proof of concept for the target-based potentiation of PZA activity in M. tuberculosis. PMID:27855077

Molecular diversity of Mycobacterium tuberculosis isolates from patients with tuberculosis in Honduras

PubMed Central

2010-01-01

Background Tuberculosis persists as a public health problem in Honduras. A better knowledge of the molecular characteristics of Mycobacterium tuberculosis strains will contribute to understand the transmission dynamics of the disease within the country. The aim of this study was to provide an insight of the genetic biodiversity of M. tuberculosis clinical isolates collected in Honduras between 1994 and 2002. Genotyping was performed using spoligotyping and RFLP. The spoligotypes obtained were compared with the SITVIT2 proprietary database of the Pasteur Institute of Guadeloupe. Results Spoligotyping grouped 84% of the isolates into 27 clusters (2 to 43 strains per cluster). Of the 44 shared international types (SITs) identified among the Honduran stains, 8 SITs were newly identified either within the present study or after match with an orphan type previously identified in the SITVIT2 database. In addition, 16 patterns corresponded to orphan, previously unreported isolates. The Latin American Mediterranean (LAM) lineage was the most common in this study; 55% of the strains belonged to this family. Other genotypes found were Haarlem (16%), T (16%), X-clade (6%), Unknown signature (5%) and S (1%). Only one Beijing strain was identified (0.5%). We observed a high degree of diversity after characterizing the 43 isolates belonging to the main spoligotyping cluster (SIT 33, LAM3) with IS6110-RFLP. A total of 35 different RFLP-fingerprints were detected, of which 6 patterns corresponded to the same number of clusters comprising 14 strains. Conclusions The findings obtained in this study show that tuberculosis transmission in Honduras is due to modern M. tuberculosis lineages with high level of biodiversity. PMID:20678242

Methodology of mycobacteria tuberculosis bacteria detection by Raman spectroscopy

NASA Astrophysics Data System (ADS)

Zyubin, A.; Lavrova, A.; Manicheva, O.; Dogonadze, M.; Tsibulnikova, A.; Samusev, I.

2018-01-01

We have developed a methodology for the study of deactivated strains of Mycobacterium tuberculosis. Strains of the Beijing species obtained from pulmonary patient secrete (XDR strain) and reference strain (H37Rv) were investigated by Raman spectrometry with He-Ne (632,8 nm) laser excitation source. As a result of the research, the optimal experimental parameters have been obtained to get spectra of mycolic acids, which are part of the cell wall of mycobacteria.

Insights from the docking and molecular dynamics simulation of the Phosphopantetheinyl transferase (PptT) structural model from Mycobacterium tuberculosis.

PubMed

Rohini, Karunakaran; Srikumar, Padmalayam Sadanandan

2013-01-01

A great challenge is posed to the treatment of tuberculosis due to the evolution of multidrug-resistant (MDR) and extensively drugresistant (XDR) strains of Mycobacterium tuberculosis in recent times. The complex cell envelope of the bacterium contains unusual structures of lipids which protects the bacterium from host enzymes and escape immune response. To overcome the drug resistance, targeting "drug targets" which have a critical role in growth and virulence factor is a novel approach for better tuberculosis treatment. The enzyme Phosphopantetheinyl transferase (PptT) is an attractive drug target as it is primarily involved in post translational modification of various types-I polyketide synthases and assembly of mycobactin, which is required for lipid virulence factors. Our in silico studies reported that the structural model of M.tuberculosis PptT characterizes the structure-function activity. The refinement of the model was carried out with molecular dynamics simulations and was analyzed with root mean square deviation (RMSD), and radius of gyration (Rg). This confirmed the structural behavior of PptT in dynamic system. Molecular docking with substrate coenzyme A (CoA) identified the binding pocket and key residues His93, Asp114 and Arg169 involved in PptT-CoA binding. In conclusion, our results show that the M.tuberculosis PptT model and critical CoA binding pocket initiate the inhibitor design of PptT towards tuberculosis treatment.

Structural basis of DNA sequence recognition by the response regulator PhoP in Mycobacterium tuberculosis.

PubMed

He, Xiaoyuan; Wang, Liqin; Wang, Shuishu

2016-04-15

The transcriptional regulator PhoP is an essential virulence factor in Mycobacterium tuberculosis, and it presents a target for the development of new anti-tuberculosis drugs and attenuated tuberculosis vaccine strains. PhoP binds to DNA as a highly cooperative dimer by recognizing direct repeats of 7-bp motifs with a 4-bp spacer. To elucidate the PhoP-DNA binding mechanism, we determined the crystal structure of the PhoP-DNA complex. The structure revealed a tandem PhoP dimer that bound to the direct repeat. The surprising tandem arrangement of the receiver domains allowed the four domains of the PhoP dimer to form a compact structure, accounting for the strict requirement of a 4-bp spacer and the highly cooperative binding of the dimer. The PhoP-DNA interactions exclusively involved the effector domain. The sequence-recognition helix made contact with the bases of the 7-bp motif in the major groove, and the wing interacted with the adjacent minor groove. The structure provides a starting point for the elucidation of the mechanism by which PhoP regulates the virulence of M. tuberculosis and guides the design of screening platforms for PhoP inhibitors.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adams, K L; Steele, P T; Bogan, M J

Two similar mycobacteria, Mycobacteria tuberculosis H37Ra and Mycobacteria smegmatis are rapidly detected and identified within samples containing a complex background of respiratory effluents using Single Particle Aerosol Mass Spectrometry (SPAMS). M. tuberculosis H37Ra (TBa), an avirulent strain, is used as a surrogate for virulent tuberculosis (TBv); M. smegmatis (MSm) is utilized as a near neighbor confounder for TBa. Bovine lung surfactant and human exhaled breath condensate are used as first-order surrogates for infected human lung expirations from patients with pulmonary tuberculosis. This simulated background sputum is mixed with TBa or MSm and nebulized to produce conglomerate aerosol particles, single particlesmore » that contain a bacterium embedded within a background respiratory matrix. Mass spectra of single conglomerate particles exhibit ions associated with both respiratory effluents and mycobacteria. Spectral features distinguishing TBa from MSm in pure and conglomerate particles are shown. SPAMS pattern matching alarm algorithms are able to distinguish TBa containing particles from background matrix and MSm for >50% of the test particles, which is sufficient to enable a high probability of detection and a low false alarm rate if an adequate number of such particles are present. These results indicate the potential usefulness of SPAMS for rapid, reagentless tuberculosis screening.« less

Reduced susceptibility of clinical strains of Mycobacterium tuberculosis to reactive nitrogen species promotes survival in activated macrophages

PubMed Central

Idh, Jonna; Andersson, Blanka; Lerm, Maria; Raffetseder, Johanna; Eklund, Daniel; Woksepp, Hanna; Werngren, Jim; Mansjö, Mikael; Sundqvist, Tommy; Stendahl, Olle

2017-01-01

Background Drugs such as isoniazid (INH) and pretomanid (PRT), used against Mycobacterium tuberculosis are active partly through generation of reactive nitrogen species (RNS). The aim of this study was to explore variability in intracellular susceptibility to nitric oxide (NO) in clinical strains of M. tuberculosis. Method Luciferase-expressing clinical M. tuberculosis strains with or without INH resistance were exposed to RNS donors (DETA/NO and SIN-1) in broth cultures and bacterial survival was analysed by luminometry. NO-dependent intracellular killing in a selection of strains was assessed in interferon gamma/lipopolysaccharide-activated murine macrophages using the NO inhibitor L-NMMA. Results When M. tuberculosis H37Rv was compared to six clinical isolates and CDC1551, three isolates with inhA mediated INH resistance showed significantly reduced NO-susceptibility in broth culture. All strains showed a variable but dose-dependent susceptibility to RNS donors. Two clinical isolates with increased susceptibility to NO exposure in broth compared to H37Rv were significantly inhibited by activated macrophages whereas there was no effect on growth inhibition when activated macrophages were infected by clinical strains with higher survival to NO exposure in broth. Furthermore, the most NO-tolerant clinical isolate showed increased resistance to PRT both in broth culture and the macrophage model compared to H37Rv in the absence of mutational resistance in genes associated to reduced susceptibility against PRT or NO. Conclusion In a limited number of clinical M. tuberculosis isolates we found a significant difference in susceptibility to NO between clinical isolates, both in broth cultures and in macrophages. Our results indicate that mycobacterial susceptibility to cellular host defence mechanisms such as NO need to be taken into consideration when designing new therapeutic strategies. PMID:28704501

[Isolation of a strain of M. tuberculosis which is considered to be rifampicin-dependent, from a patient with long-lasted smear positive and culture difficult (SPCD) mycobacteria].

PubMed

Nakamura, M; Harano, Y; Koga, T

1990-09-01

During the course of clinical examination of drug sensitivity tests for M. tuberculosis, a strain of M. tuberculosis which is considered to be rifampicin-dependent was isolated from a patient with persisting smear positive, culture negative (SPCN) or culture difficult (SPCD) mycobacteria status. The strain isolated produced a few tiny colonies on the control Ogawa-egg yolk medium, whereas it showed abundant growth like a bacteria plaque on the medium containing rifampicin 50 micrograms/ml. Furthermore, the growth of the strain on Ogawa medium containing rifampicin 50 micrograms/ml is much better than that on the medium containing rifampicin 10 micrograms/ml.

Novel semisynthetic antibiotics from caprazamycins A-G: caprazene derivatives and their antibacterial activity.

PubMed

Takahashi, Yoshiaki; Igarashi, Masayuki; Miyake, Toshiaki; Soutome, Hiromi; Ishikawa, Kanae; Komatsuki, Yasuhiro; Koyama, Yoshiko; Nakagawa, Naoko; Hattori, Seiko; Inoue, Kunio; Doi, Norio; Akamatsu, Yuzuru

2013-03-01

Acidic treatment of a mixture of caprazamycins (CPZs) A-G isolated from a screen of novel antimycobacterial agents gave caprazene, a core structure of CPZs, in high yield. Chemical modification of the resulting caprazene was performed to give its various derivatives. The structure-activity relationships of the caprazene derivatives against several mycobacterial species and pathogenic Gram-positive and Gram-negative bacteria were studied. Although caprazene showed no antibacterial activity, the antibacterial activity was restored for its 1'''-alkylamide, 1'''-anilide and 1'''-ester derivatives. Compounds 4b (CPZEN-45), 4d (CPZEN-48), 4f and 4g (CPZEN-51) exhibited more potent activities against Mycobacterium tuberculosis and M. avium complex strains than CPZ-B. These results suggest that caprazene would be a good precursor from which novel semisynthetic antibacterial antibiotics can be designed for the treatment of mycobacterial diseases such as tuberculosis and M. avium complex infection.

PolyTB: a genomic variation map for Mycobacterium tuberculosis.

PubMed

Coll, Francesc; Preston, Mark; Guerra-Assunção, José Afonso; Hill-Cawthorn, Grant; Harris, David; Perdigão, João; Viveiros, Miguel; Portugal, Isabel; Drobniewski, Francis; Gagneux, Sebastien; Glynn, Judith R; Pain, Arnab; Parkhill, Julian; McNerney, Ruth; Martin, Nigel; Clark, Taane G

2014-05-01

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is the second major cause of death from an infectious disease worldwide. Recent advances in DNA sequencing are leading to the ability to generate whole genome information in clinical isolates of M. tuberculosis complex (MTBC). The identification of informative genetic variants such as phylogenetic markers and those associated with drug resistance or virulence will help barcode Mtb in the context of epidemiological, diagnostic and clinical studies. Mtb genomic datasets are increasingly available as raw sequences, which are potentially difficult and computer intensive to process, and compare across studies. Here we have processed the raw sequence data (>1500 isolates, eight studies) to compile a catalogue of SNPs (n = 74,039, 63% non-synonymous, 51.1% in more than one isolate, i.e. non-private), small indels (n = 4810) and larger structural variants (n = 800). We have developed the PolyTB web-based tool (http://pathogenseq.lshtm.ac.uk/polytb) to visualise the resulting variation and important meta-data (e.g. in silico inferred strain-types, location) within geographical map and phylogenetic views. This resource will allow researchers to identify polymorphisms within candidate genes of interest, as well as examine the genomic diversity and distribution of strains. PolyTB source code is freely available to researchers wishing to develop similar tools for their pathogen of interest. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Conserved hypothetical protein Rv1977 in Mycobacterium tuberculosis strains contains sequence polymorphisms and might be involved in ongoing immune evasion.

PubMed

Jiang, Yi; Liu, Haican; Wang, Xuezhi; Li, Guilian; Qiu, Yan; Dou, Xiangfeng; Wan, Kanglin

2015-01-01

Host immune pressure and associated parasite immune evasion are key features of host-pathogen co-evolution. A previous study showed that human T cell epitopes of Mycobacterium tuberculosis are evolutionarily hyperconserved and thus it was deduced that M. tuberculosis lacks antigenic variation and immune evasion. Here, we selected 151 clinical Mycobacterium tuberculosis isolates from China, amplified gene encoding Rv1977 and compared the sequences. The results showed that Rv1977, a conserved hypothetical protein, is not conserved in M. tuberculosis strains and there are polymorphisms existed in the protein. Some mutations, especially one frameshift mutation, occurred in the antigen Rv1977, which is uncommon in M.tb strains and may lead to the protein function altering. Mutations and deletion in the gene all affect one of three T cell epitopes and the changed T cell epitope contained more than one variable position, which may suggest ongoing immune evasion.

[A preliminary study on the molecular characteristics of D-cycloserine resistance of Mycobacterium tuberculosis].

PubMed

Li, C; Li, G L; Luo, Q; Li, S J; Wang, R B; Lou, Y L; Lyu, J X; Wan, K L

2017-02-10

Objective: To investigate the relationship between D-cycloserine resistance and the gene mutations of alrA , ddlA and cycA of Mycobacterium ( M. ) tuberculosis , as well as the association between D-cycloserine resistance and spoligotyping genotyping. Methods: A total of 145 M. tuberculosis strains were selected from the strain bank. D-cycloserine resistant phenotypes of the strains were determined by the proportion method and the minimal inhibitory concentration was determined by resazurin microtiter assay. PCR amplification and DNA direct sequencing methods were used for the analysis of gene mutations. Relationship between the resistance phenotype and genotype was analyzed by chi -square test. Results: Of the 145 clinically collected strains, 24 (16.6%) of them were D-cycloserine resistant and 121 (83.4%) were sensitive. There were only synonymous mutations noticed on alrA , ddlA and cycA in sensitive strains. Of the 24 D-cycloserine resistant strains, 3 (12.5%) isolates' cycA and 1 (4.2%) isolates' alrA happened to be non-synonymous mutations, in which the codes were 188, 318 and 508 of cycA , and 261 of alrA , respectively. Results on drug sensitivity tests confirmed the minimal inhibitory concentration of the mutant strains were all increased to some degrees. The D-cycloserine resistant rates of 88 Beijing genotype and 57 non-Beijing genotype strains were 20.5% and 10.5% , respectively, but with no statistically significant difference ( χ (2) =2.47, P >0.05). Conclusions: The non-synonymous mutations of alrA and cycA might contribute to one of the mechanisms of M. tuberculosis D-cycloserine resistance. M. tuberculosis Beijing genotype or non-Beijing genotype was not considered to be associated with the D-cycloserine resistance.

Bim is a crucial regulator of apoptosis induced by Mycobacterium tuberculosis

PubMed Central

Aguiló, N; Uranga, S; Marinova, D; Martín, C; Pardo, J

2014-01-01

Mycobacterium tuberculosis, the causative agent of tuberculosis, induces apoptosis in infected macrophages in vitro and in vivo. However, the molecular mechanism controlling this process is not known. In order to study the involvement of the mitochondrial apoptotic pathway in M. tuberculosis-induced apoptosis, we analysed cell death in M. tuberculosis-infected embryonic fibroblasts (MEFs) derived from different knockout mice for genes involved in this route. We found that apoptosis induced by M. tuberculosis is abrogated in the absence of Bak and Bax, caspase 9 or the executioner caspases 3 and 7. Notably, we show that MEF deficient in the BH3-only BCL-2-interacting mediator of cell death (Bim) protein were also resistant to this process. The relevance of these results has been confirmed in the mouse macrophage cell line J774, where cell transfection with siRNA targeting Bim impaired apoptosis induced by virulent mycobacteria. Notably, only infection with a virulent strain, but not with attenuated ESX-1-defective strains, such as Bacillus Calmette-Guerin and live-attenuated M. tuberculosis vaccine strain MTBVAC, induced Bim upregulation and apoptosis, probably implicating virulence factor early secreted antigenic target 6-kDa protein in this process. Our results suggest that Bim upregulation and apoptosis is mediated by the p38MAPK-dependent pathway. Our findings show that Bim is a master regulator of apoptosis induced by M. tuberculosis. PMID:25032866

Isolation of a Mycobacterium microti-like organism from a rock hyrax (Procavia capensis) in a Canadian zoo

PubMed Central

Lutze-Wallace, Cyril; Turcotte, Claude; Glover, Gordon; Cousins, Debby; Bell, John; Berlie-Surujballi, Gloria; Barbeau, Yvon; Randall, Geoff

2006-01-01

A Mycobacterium tuberculosis complex organism was isolated from a zoo resident rock hyrax (Procavia capensis) imported into Canada from South Africa. The strain was identified biochemically as Mycobacterium microti. The spoligotype pattern obtained for this isolate was found to be rare. This represents the first report of isolation and spoligotyping of M. microti in North America. PMID:17078252

Combined inhalation and oral supplementation of Vitamin A and Vitamin D: a possible prevention and therapy for tuberculosis.

PubMed

Syal, Kirtimaan; Chakraborty, Surajit; Bhattacharyya, Rajasri; Banerjee, Dibyajyoti

2015-03-01

Tuberculosis is continuing as a problem of mankind. With evolution, MDR and XDR forms of tuberculosis have emerged from drug sensitive strain. MDR and XDR strains are resistant to most of the antibiotics, making the management more difficult. BCG vaccine is not providing complete protection against tuberculosis. Therefore new infections are spreading at a tremendous rate. At the present moment there is experimental evidence to believe that Vitamin A and Vitamin D has anti-mycobacterial property. It is in this context, we have hypothesized a host based approach using the above vitamins that can cause possible prevention and cure of tuberculosis with minimal chance of resistance or toxicity. Copyright © 2015 Elsevier Ltd. All rights reserved.

Genetic diversity of Mycobacterium tuberculosis from Guadalajara, Mexico and identification of a rare multidrug resistant Beijing genotype.

PubMed

Flores-Treviño, Samantha; Morfín-Otero, Rayo; Rodríguez-Noriega, Eduardo; González-Díaz, Esteban; Pérez-Gómez, Héctor R; Bocanegra-García, Virgilio; Vera-Cabrera, Lucio; Garza-González, Elvira

2015-01-01

Determining the genetic diversity of M. tuberculosis strains allows identification of the distinct Mycobacterium tuberculosis genotypes responsible for tuberculosis in different regions. Several studies have reported the genetic diversity of M. tuberculosis strains in Mexico, but little information is available from the state of Jalisco. Therefore, the aim of this study was to determine the genetic diversity of Mycobacterium tuberculosis clinical isolates from Western Mexico. Sixty-eight M. tuberculosis isolates were tested for susceptibility to first-line drugs using manual Mycobacteria Growth Indicator Tube method and genotyped using spoligotyping and IS6110-restriction fragment length polymorphism (RFLP) pattern analyses. Forty-seven (69.1%) isolates were grouped into 10 clusters and 21 isolates displayed single patterns by spoligotyping. Three of the 21 single patterns corresponded to orphan patterns in the SITVITWEB database, and 1 new type that contained 2 isolates was created. The most prevalent lineages were T (38.2%), Haarlem (17.7%), LAM (17.7%), X (7.4%), S (5.9%), EAI (1.5%) and Beijing (1.5%). Six (12.8%) of the clustered isolates were MDR, and type 406 of the Beijing family was among the MDR isolates. Seventeen (26.2%) isolates were grouped into 8 clusters and 48 isolates displayed single patterns by IS6110-RFLP. Combination of IS6110-RFLP and spoligotyping reduced the clustering rate to 20.0%. The results show that T, Haarlem, and LAM are predominant lineages among clinical isolates of M. tuberculosis in Guadalajara, Mexico. Clustering rates indicated low transmission of MDR strains. We detected a rare Beijing genotype, SIT406, which was a highly resistant strain. This is the first report of this Beijing genotype in Latin America.

Genetic Diversity of Mycobacterium tuberculosis from Guadalajara, Mexico and Identification of a Rare Multidrug Resistant Beijing Genotype

PubMed Central

Flores-Treviño, Samantha; Morfín-Otero, Rayo; Rodríguez-Noriega, Eduardo; González-Díaz, Esteban; Pérez-Gómez, Héctor R.; Bocanegra-García, Virgilio; Vera-Cabrera, Lucio; Garza-González, Elvira

2015-01-01

Determining the genetic diversity of M. tuberculosis strains allows identification of the distinct Mycobacterium tuberculosis genotypes responsible for tuberculosis in different regions. Several studies have reported the genetic diversity of M. tuberculosis strains in Mexico, but little information is available from the state of Jalisco. Therefore, the aim of this study was to determine the genetic diversity of Mycobacterium tuberculosis clinical isolates from Western Mexico. Sixty-eight M. tuberculosis isolates were tested for susceptibility to first-line drugs using manual Mycobacteria Growth Indicator Tube method and genotyped using spoligotyping and IS6110-restriction fragment length polymorphism (RFLP) pattern analyses. Forty-seven (69.1%) isolates were grouped into 10 clusters and 21 isolates displayed single patterns by spoligotyping. Three of the 21 single patterns corresponded to orphan patterns in the SITVITWEB database, and 1 new type that contained 2 isolates was created. The most prevalent lineages were T (38.2%), Haarlem (17.7%), LAM (17.7%), X (7.4%), S (5.9%), EAI (1.5%) and Beijing (1.5%). Six (12.8%) of the clustered isolates were MDR, and type 406 of the Beijing family was among the MDR isolates. Seventeen (26.2%) isolates were grouped into 8 clusters and 48 isolates displayed single patterns by IS6110-RFLP. Combination of IS6110-RFLP and spoligotyping reduced the clustering rate to 20.0%. The results show that T, Haarlem, and LAM are predominant lineages among clinical isolates of M. tuberculosis in Guadalajara, Mexico. Clustering rates indicated low transmission of MDR strains. We detected a rare Beijing genotype, SIT406, which was a highly resistant strain. This is the first report of this Beijing genotype in Latin America. PMID:25695431

Molecular Fingerprinting of Mycobacterium tuberculosis and Risk Factors for Tuberculosis Transmission in Paris, France, and Surrounding Area

PubMed Central

Gutiérrez, M. C.; Vincent, V.; Aubert, D.; Bizet, J.; Gaillot, O.; Lebrun, L.; Le Pendeven, C.; Le Pennec, M. P.; Mathieu, D.; Offredo, C.; Pangon, B.; Pierre-Audigier, C.

1998-01-01

Forty-three percent of the tuberculosis cases reported in France are from the Ile de France region. The incidence of tuberculosis in this region is 33 cases per 100,000 inhabitants, twice the national average. A restriction fragment length polymorphism (RFLP) analysis was performed with clinical isolates of Mycobacterium tuberculosis isolated during 1995 in 10 hospitals in Paris and surrounding areas to detect tuberculosis transmission and define the factors associated with clustering in this population. The molecular markers used were the insertion sequence IS6110 and the direct repeat (DR) sequence. Social, demographic, and clinical data were collected from the patients’ medical files. Ten patients with isolates with a single copy of IS6110 were excluded from further analysis. Twenty-four patients with false-positive cultures due to laboratory contamination (based on RFLP analysis with IS6110 and examination of patient data) were also excluded. The study was then conducted with 272 strains isolated from 272 patients. Further fingerprinting was performed by using the DR element with strains with patterns by RFLP analysis with IS6110 that differed by one band only and strains with identical patterns by RFLP analysis with IS6110 and with low numbers of copies of IS6110. The combined use of both markers identified unique patterns for 177 strains and clustered 95 (35.7%) strains in 26 groups, each containing isolates from 2 to 12 patients. The clustering was strongly associated with homelessness and the male sex. It was not associated with age, birth in a foreign country, human immunodeficiency virus positivity, or residence in hostels or prison. Isolates from homeless people were often included in large clusters, and homeless people could be the source of tuberculosis transmission for more than 50% of the clustered patients. These results suggest that homeless people play a key role in the spread of M. tuberculosis in the community and that poor socioeconomic conditions are the main risk factors associated with active tuberculosis transmission. PMID:9466764

Drug Resistance and Population Structure of Mycobacterium tuberculosis Beijing Strains Isolated in Poland.

PubMed

Kozińska, Monika; Augustynowicz-Kopeć, Ewa

2015-01-01

In total, 1095 Mycobacterium tuberculosis clinical isolates from 282 patients with drug-resistant and 813 with drug-sensitive tuberculosis (TB) in Poland during 2007-2011 were analysed. Seventy-one (6.5%) patients were found to have strains of Beijing genotype as defined by spoligotyping. The majority of patients were Polish-born; among foreign-born a large proportion came from Chechnya and Vietnam. Analysis showed strong associations between Beijing genotype infection and MDR, pre-XDR and XDR resistance, with a considerable relative risk among new patients, suggesting that this is due to increased spread of drug-resistant strains rather than acquisition of resistance during treatment.

Primary drug resistance in a region with high burden of tuberculosis. A critical problem.

PubMed

Villa-Rosas, Cecilia; Laniado-Laborín, Rafael; Oceguera-Palao, Lorena

2015-01-01

To determine rates of drug resistance in new cases of pulmonary tuberculosis in a region with a high burden of the disease. New case suspects were referred for drug susceptibility testing. 28.9% of new cases were resistant to at least one first line drug; 3.9% had a multidrug-resistant strain, 15.6% a monoresistant strain and 9.4% a polyresistant strain. Our rate of drug resistant tuberculosis in new cases is very high; this has important clinical implications, since even monoresistance can have a negative impact on the outcome of new cases treated empirically with a six month regimen.

Identification and application of ssDNA aptamers against H₃₇Rv in the detection of Mycobacterium tuberculosis.

PubMed

Aimaiti, Rusitanmujiang; Qin, Lianhua; Cao, Ting; Yang, Hua; Wang, Jie; Lu, Junmei; Huang, Xiaochen; Hu, Zhongyi

2015-11-01

Microscopy of direct smear with the Ziehl-Neelsen stain is still broadly used in tuberculosis diagnosis. However, this method suffers from low specificity and is difficult to distinguish Mycobacterium tuberculosis (MTB) from nontuberculosis mycobacterial (NTM), since all mycobacterial species are positive in Ziehl-Neelsen stain. In this study, we utilized whole cell SELEX to obtain species-specific aptamers for increasing the specificity of MTB detection. Whole cell SELEX was performed in MTB reference strain H37Rv by two selection processes based on enzyme-linked plate or Eppendorf tube, respectively. To increase success rate of generating aptamers, the selection processes were systematically monitored to understand the dynamic evolution of aptamers against complex structure of target bacteria. Two preponderant groups and ten high-affinity aptamers were obtained by analyzing the dynamic evolution. Preponderant aptamer MA1 from group I showed relatively high binding affinity with apparent dissociation constant (KD value) of 12.02 nM. Sandwich ELISA assay revealed five aptamer combinations effectively bound MTB strains in preliminary evaluation, especially the combination based on aptamer MA2 (another preponderant aptamer from group II) and MA1. Further evaluated in many other strains, MA2/MA1 combination effectively identified MTB from NTM or other pathogenic bacteria, and displayed the high specificity and sensitivity. Binding analysis of aptamer MA1 or MA2 by fluorescence microscopy observation showed high binding reactivity with H37Rv, low apparent cross-reactivity with M. marinum, and no apparent cross-reactivity with Enterobacter cloacae. Taken together, this study provides attractive candidate species-specific aptamers to effectively capture or discriminate MTB strains.

New insights into the transposition mechanisms of IS6110 and its dynamic distribution between Mycobacterium tuberculosis Complex lineages

PubMed Central

Uranga, Santiago; Picó, Ana; Lampreave, Carlos; Cebollada, Alberto; Otal, Isabel

2018-01-01

The insertion Sequence IS6110, only present in the pathogens of the Mycobacterium tuberculosis Complex (MTBC), has been the gold-standard epidemiological marker for TB for more than 25 years, but biological implications of IS6110 transposition during MTBC adaptation to humans remain elusive. By studying 2,236 clinical isolates typed by IS6110-RFLP and covering the MTBC, we remarked a lineage-specific content of IS6110 being higher in modern globally distributed strains. Once observed the IS6110 distribution in the MTBC, we selected representative isolates and found a correlation between the normalized expression of IS6110 and its abundance in MTBC chromosomes. We also studied the molecular regulation of IS6110 transposition and we found a synergistic action of two post-transcriptional mechanisms: a -1 ribosomal frameshift and a RNA pseudoknot which interferes translation. The construction of a transcriptionally active transposase resulted in 20-fold increase of the transposition frequency. Finally, we examined transposition in M. bovis and M. tuberculosis during laboratory starvation and in a mouse infection model of TB. Our results shown a higher transposition in M. tuberculosis, that preferably happens during TB infection in mice and after one year of laboratory culture, suggesting that IS6110 transposition is dynamically adapted to the host and to adverse growth conditions. PMID:29649213

Comparative genomic and proteomic analyses of PE/PPE multigene family of Mycobacterium tuberculosis H37Rv and H37Ra reveal novel and interesting differences with implications in virulence

PubMed Central

Kohli, Sakshi; Singh, Yadvir; Sharma, Khushbu; Mittal, Aditya; Ehtesham, Nasreen Z.; Hasnain, Seyed E.

2012-01-01

Tuberculosis, caused by Mycobacterium tuberculosis, remains a leading infectious disease taking one human life every 15 s globally. The two well-characterized strains H37Rv and H37Ra, derived from the same parental strain M. tuberculosis H37, show dramatically different pathogenic phenotypes. PE/PPE gene family, comprising of 176 open reading frames and present exclusively in genus Mycobacterium, accounts for ∼10% of the M. tuberculosis genome. Our comprehensive in silico analyses of PE/PPE family of H37Ra and virulent H37Rv strains revealed genetic differences between these strains in terms of several single nucleotide variations and InDels and these manifested in changes in physico-chemical properties, phosphorylation sites, and protein: protein interacting domains of the corresponding proteomes. Similar comparisons using the 13 sigma factor genes, 36 members of the mammalian cell entry family, 13 mycobacterial membrane protein large family members and 11 two-component signal transduction systems along with 5 orphaned response regulators and 2 orphaned sensor kinases failed to reveal very significant difference between H37Rv and H37Ra, reinforcing the importance of PE/PPE genes. Many of these changes between H37Rv and H37Ra can be correlated to differences in pathogenesis and virulence of the two strains. PMID:22618876

Urgent Implementation in a Hospital Setting of a Strategy To Rule Out Secondary Cases Caused by Imported Extensively Drug-Resistant Mycobacterium tuberculosis Strains at Diagnosis

PubMed Central

Pérez-Lago, Laura; Martínez-Lirola, Miguel; García, Sergio; Herranz, Marta; Mokrousov, Igor; Comas, Iñaki; Martínez-Priego, Llúcia; Bouza, Emilio

2016-01-01

Current migratory movements require new strategies for rapidly tracking the transmission of high-risk imported Mycobacterium tuberculosis strains. Whole-genome sequencing (WGS) enables us to identify single-nucleotide polymorphisms (SNPs) and therefore design PCRs to track specific relevant strains. However, fast implementation of these strategies in the hospital setting is difficult because professionals working in diagnostics, molecular epidemiology, and genomics are generally at separate institutions. In this study, we describe the urgent implementation of a system that integrates genomics and molecular tools in a genuine high-risk epidemiological alert involving 2 independent importations of extensively drug resistant (XDR) and pre-XDR Beijing M. tuberculosis strains from Russia into Spain. Both cases involved commercial sex workers with long-standing tuberculosis (TB). The system was based on strain-specific PCRs tailored from WGS data that were transferred to the local node that was managing the epidemiological alert. The optimized tests were available for prospective implementation in the local node 33 working days after receiving the primary cultures of the XDR strains and were applied to all 42 new incident cases. An interpretable result was obtained in each case (directly from sputum for 27 stain-positive cases) and corresponded to the amplification profiles for strains other than the targeted pre-XDR and XDR strains, which made it possible to prospectively rule out transmission of these high-risk strains at diagnosis. PMID:27682128

Urgent Implementation in a Hospital Setting of a Strategy To Rule Out Secondary Cases Caused by Imported Extensively Drug-Resistant Mycobacterium tuberculosis Strains at Diagnosis.

PubMed

Pérez-Lago, Laura; Martínez-Lirola, Miguel; García, Sergio; Herranz, Marta; Mokrousov, Igor; Comas, Iñaki; Martínez-Priego, Llúcia; Bouza, Emilio; García-de-Viedma, Darío

2016-12-01

Current migratory movements require new strategies for rapidly tracking the transmission of high-risk imported Mycobacterium tuberculosis strains. Whole-genome sequencing (WGS) enables us to identify single-nucleotide polymorphisms (SNPs) and therefore design PCRs to track specific relevant strains. However, fast implementation of these strategies in the hospital setting is difficult because professionals working in diagnostics, molecular epidemiology, and genomics are generally at separate institutions. In this study, we describe the urgent implementation of a system that integrates genomics and molecular tools in a genuine high-risk epidemiological alert involving 2 independent importations of extensively drug resistant (XDR) and pre-XDR Beijing M. tuberculosis strains from Russia into Spain. Both cases involved commercial sex workers with long-standing tuberculosis (TB). The system was based on strain-specific PCRs tailored from WGS data that were transferred to the local node that was managing the epidemiological alert. The optimized tests were available for prospective implementation in the local node 33 working days after receiving the primary cultures of the XDR strains and were applied to all 42 new incident cases. An interpretable result was obtained in each case (directly from sputum for 27 stain-positive cases) and corresponded to the amplification profiles for strains other than the targeted pre-XDR and XDR strains, which made it possible to prospectively rule out transmission of these high-risk strains at diagnosis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Mycobacterium tuberculosis infection causes different levels of apoptosis and necrosis in human macrophages and alveolar epithelial cells.

PubMed

Danelishvili, Lia; McGarvey, Jeffery; Li, Yong-Jun; Bermudez, Luiz E

2003-09-01

Mycobacterium tuberculosis interacts with macrophages and epithelial cells in the alveolar space of the lung, where it is able to invade and replicate in both cell types. M. tuberculosis-associated cytotoxicity to these cells has been well documented, but the mechanisms of host cell death are not well understood. We examined the induction of apoptosis and necrosis of human macrophages (U937) and type II alveolar epithelial cells (A549) by virulent (H37Rv) and attenuated (H37Ra) M. tuberculosis strains. Apoptosis was determined by both enzyme-linked immunosorbent assay (ELISA) and TdT-mediated dUTP nick end labelling (TUNEL) assay, whereas necrosis was evaluated by the release of lactate dehydrogenase (LDH). Both virulent and attenuated M. tuberculosis induced apoptosis in macrophages; however, the attenuated strain resulted in significantly more apoptosis than the virulent strain after 5 days of infection. In contrast, cytotoxicity of alveolar cells was the result of necrosis, but not apoptosis. Although infection with M. tuberculosis strains resulted in apoptosis of 14% of the cells on the monolayer, cell death associated with necrosis was observed in 59% of alveolar epithelial cells after 5 days of infection. Infection with M. tuberculosis suppressed apoptosis of alveolar epithelial cells induced by the kinase inhibitor, staurosporine. Because our findings suggest that M. tuberculosis can modulate the apoptotic response of macrophages and epithelial cells, we carried out an apoptosis pathway-specific cDNA microarray analysis of human macrophages and alveolar epithelial cells. Whereas the inhibitors of apoptosis, bcl-2 and Rb, were upregulated over 2.5-fold in infected (48 h) alveolar epithelial cells, the proapoptotic genes, bad and bax, were downregulated. The opposite was observed when U937 macrophages were infected with M. tuberculosis. Upon infection of alveolar epithelial cells with M. tuberculosis, the generation of apoptosis, as determined by the expression of caspase-1, caspase-3 and caspase-10, was inhibited. Inhibition of replication of intracellular bacteria resulted in an increase in apoptosis in both cell types. Our results showed that the differential induction of apoptosis between macrophages and alveolar epithelial cells represents specific strategies of M. tuberculosis for survival in the host.

Revisiting the role of phospholipases C in virulence and the lifecycle of Mycobacterium tuberculosis

PubMed Central

Le Chevalier, Fabien; Cascioferro, Alessandro; Frigui, Wafa; Pawlik, Alexandre; Boritsch, Eva C.; Bottai, Daria; Majlessi, Laleh; Herrmann, Jean Louis; Brosch, Roland

2015-01-01

Mycobacterium tuberculosis, the agent of human tuberculosis has developed different virulence mechanisms and virulence-associated tools during its evolution to survive and multiply inside the host. Based on previous reports and by analogy with other bacteria, phospholipases C (PLC) of M. tuberculosis were thought to be among these tools. To get deeper insights into the function of PLCs, we investigated their putative involvement in the intracellular lifestyle of M. tuberculosis, with emphasis on phagosomal rupture and virulence, thereby re-visiting a research theme of longstanding interest. Through the construction and use of an M. tuberculosis H37Rv PLC-null mutant (ΔPLC) and control strains, we found that PLCs of M. tuberculosis were not required for induction of phagosomal rupture and only showed marginal, if any, impact on virulence of M. tuberculosis in the cellular and mouse infection models used in this study. In contrast, we found that PLC-encoding genes were strongly upregulated under phosphate starvation and that PLC-proficient M. tuberculosis strains survived better than ΔPLC mutants under conditions where phosphatidylcholine served as sole phosphate source, opening new perspectives for studies on the role of PLCs in the lifecycle of M. tuberculosis. PMID:26603639

Broad-range PCR coupled with mass-spectrometry for the detection of Mycobacterium tuberculosis drug resistance

PubMed Central

Florea, Dragoş; Oţelea, Dan; Olaru, Ioana D.; Hristea, Adriana

2016-01-01

Background The need to limit the spread of drug-resistant Mycobacterium tuberculosis requires rapid detection of resistant strains. The present study aimed to evaluate a commercial assay using broad-range PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) for the rapid detection of isoniazid (INH) and rifampin (RIF) resistance in M. tuberculosis strains isolated from Romanian patients with pulmonary tuberculosis. Methods PCR/ESI-MS was used to detect genotypic resistance to RIF and INH in a panel of 63 M. tuberculosis isolates phenotypically characterized using the absolute concentration method on Löwenstein-Jensen medium. Results Thirty-eight (60%) strains were susceptible to both drugs, 22 (35%) were RIF and INH resistant, one was INH mono-resistant and two were RIF mono-resistant. The sensitivity for INH and RIF resistance mutations detection were 100% and 92% respectively, with a specificity of more than 95% for each drug. Conclusion PCR/ESI-MS is a good method for the detection of RIF and INH resistance and might represent an alternative to other rapid diagnostic tests for the detection of genetic markers of resistance in M. tuberculosis isolates. PMID:27019827

Molecular Epidemiology of Tuberculosis in Malaysia

PubMed Central

Dale, Jeremy W.; Nor, Rohana Mat; Ramayah, Soshila; Tang, Thean Hock; Zainuddin, Zainul F.

1999-01-01

Molecular typing with IS6110 was applied to Mycobacterium tuberculosis isolates from all parts of Malaysia. The degree of clustering increased with patient age, suggesting that reactivation may contribute to clustering. Identical banding patterns were also obtained for isolates from widely separate regions. Therefore, the use of clustering as a measure of recent transmission must be treated with caution. Strains related to the Beijing family were common in Peninsular Malaysia but were less common in Sabah and Sarawak, while a distinct group of strains comprised nearly 40% of isolates from East Malaysia but such strains were rare in Peninsular Malaysia. Single-copy strains, common in South and Southeastern Asia, constituted nearly 20% of isolates from the peninsula but were virtually absent in East Malaysia. The marked geographical difference in the prevailing strains indicates not only a restricted dissemination of M. tuberculosis but also a considerable degree of stability in the banding patterns. PMID:10203468

Molecular epidemiology of tuberculosis in Malaysia.

PubMed

Dale, J W; Nor, R M; Ramayah, S; Tang, T H; Zainuddin, Z F

1999-05-01

Molecular typing with IS6110 was applied to Mycobacterium tuberculosis isolates from all parts of Malaysia. The degree of clustering increased with patient age, suggesting that reactivation may contribute to clustering. Identical banding patterns were also obtained for isolates from widely separate regions. Therefore, the use of clustering as a measure of recent transmission must be treated with caution. Strains related to the Beijing family were common in Peninsular Malaysia but were less common in Sabah and Sarawak, while a distinct group of strains comprised nearly 40% of isolates from East Malaysia but such strains were rare in Peninsular Malaysia. Single-copy strains, common in South and Southeastern Asia, constituted nearly 20% of isolates from the peninsula but were virtually absent in East Malaysia. The marked geographical difference in the prevailing strains indicates not only a restricted dissemination of M. tuberculosis but also a considerable degree of stability in the banding patterns.

Phenotypic and genomic comparison of Mycobacterium aurum and surrogate model species to Mycobacterium tuberculosis: implications for drug discovery.

PubMed

Namouchi, Amine; Cimino, Mena; Favre-Rochex, Sandrine; Charles, Patricia; Gicquel, Brigitte

2017-07-13

Tuberculosis (TB) is caused by Mycobacterium tuberculosis and represents one of the major challenges facing drug discovery initiatives worldwide. The considerable rise in bacterial drug resistance in recent years has led to the need of new drugs and drug regimens. Model systems are regularly used to speed-up the drug discovery process and circumvent biosafety issues associated with manipulating M. tuberculosis. These include the use of strains such as Mycobacterium smegmatis and Mycobacterium marinum that can be handled in biosafety level 2 facilities, making high-throughput screening feasible. However, each of these model species have their own limitations. We report and describe the first complete genome sequence of Mycobacterium aurum ATCC23366, an environmental mycobacterium that can also grow in the gut of humans and animals as part of the microbiota. This species shows a comparable resistance profile to that of M. tuberculosis for several anti-TB drugs. The aims of this study were to (i) determine the drug resistance profile of a recently proposed model species, Mycobacterium aurum, strain ATCC23366, for anti-TB drug discovery as well as Mycobacterium smegmatis and Mycobacterium marinum (ii) sequence and annotate the complete genome sequence of this species obtained using Pacific Bioscience technology (iii) perform comparative genomics analyses of the various surrogate strains with M. tuberculosis (iv) discuss how the choice of the surrogate model used for drug screening can affect the drug discovery process. We describe the complete genome sequence of M. aurum, a surrogate model for anti-tuberculosis drug discovery. Most of the genes already reported to be associated with drug resistance are shared between all the surrogate strains and M. tuberculosis. We consider that M. aurum might be used in high-throughput screening for tuberculosis drug discovery. We also highly recommend the use of different model species during the drug discovery screening process.

First Worldwide Proficiency Study on Variable-Number Tandem-Repeat Typing of Mycobacterium tuberculosis Complex Strains

PubMed Central

de Beer, Jessica L.; Kremer, Kristin; Ködmön, Csaba; Supply, Philip

2012-01-01

Although variable-number tandem-repeat (VNTR) typing has gained recognition as the new standard for the DNA fingerprinting of Mycobacterium tuberculosis complex (MTBC) isolates, external quality control programs have not yet been developed. Therefore, we organized the first multicenter proficiency study on 24-locus VNTR typing. Sets of 30 DNAs of MTBC strains, including 10 duplicate DNA samples, were distributed among 37 participating laboratories in 30 different countries worldwide. Twenty-four laboratories used an in-house-adapted method with fragment sizing by gel electrophoresis or an automated DNA analyzer, nine laboratories used a commercially available kit, and four laboratories used other methods. The intra- and interlaboratory reproducibilities of VNTR typing varied from 0% to 100%, with averages of 72% and 60%, respectively. Twenty of the 37 laboratories failed to amplify particular VNTR loci; if these missing results were ignored, the number of laboratories with 100% interlaboratory reproducibility increased from 1 to 5. The average interlaboratory reproducibility of VNTR typing using a commercial kit was better (88%) than that of in-house-adapted methods using a DNA analyzer (70%) or gel electrophoresis (50%). Eleven laboratories using in-house-adapted manual typing or automated typing scored inter- and intralaboratory reproducibilities of 80% or higher, which suggests that these approaches can be used in a reliable way. In conclusion, this first multicenter study has documented the worldwide quality of VNTR typing of MTBC strains and highlights the importance of international quality control to improve genotyping in the future. PMID:22170917

Complete Genome Sequence of the Clinical Beijing-Like Strain Mycobacterium tuberculosis 323 Using the PacBio Real-Time Sequencing Platform

PubMed Central

Rodríguez, Juan Germán; Pino, Camilo; Tauch, Andreas

2015-01-01

We report here the whole-genome sequence of the multidrug-resistant Beijing-like strain Mycobacterium tuberculosis 323, isolated from a 15-year-old female patient who died shortly after the initiation of second-line drug treatment. This strain is representative of the Beijing-like isolates from Colombia, where this lineage is becoming a public health concern. PMID:25931600

Beijing Sublineages of Mycobacterium tuberculosis Differ in Pathogenicity in the Guinea Pig

PubMed Central

Shanley, Crystal A.; Ackart, David; Jarlsberg, Leah G.; Shang, Shaobin; Obregon-Henao, Andres; Harton, Marisabel; Basaraba, Randall J.; Henao-Tamayo, Marcela; Barrozo, Joyce C.; Rose, Jordan; Kawamura, L. Masae; Coscolla, Mireia; Fofanov, Viacheslav Y.; Koshinsky, Heather; Gagneux, Sebastien; Hopewell, Philip C.; Ordway, Diane J.; Orme, Ian M.

2012-01-01

The Beijing family of Mycobacterium tuberculosis strains is part of lineage 2 (also known as the East Asian lineage). In clinical studies, we have observed that isolates from the sublineage RD207 of lineage 2 were more readily transmitted among humans. To investigate the basis for this difference, we tested representative strains with the characteristic Beijing spoligotype from four of the five sublineages of lineage 2 in the guinea pig model and subjected these strains to comparative whole-genome sequencing. The results of these studies showed that all of the clinical strains were capable of growing and causing lung pathology in guinea pigs after low-dose aerosol exposure. Differences between the abilities of the four sublineages to grow in the lungs of these animals were not overt, but members of RD207 were significantly more pathogenic, resulting in severe lung damage. The RD207 strains also induced much higher levels of markers associated with regulatory T cells and showed a significant loss of activated T cells in the lungs over the course of the infections. Whole-genome sequencing of the strains revealed mutations specific for RD207 which may explain this difference. Based on these data, we hypothesize that the sublineages of M. tuberculosis are associated with distinct pathological and clinical phenotypes and that these differences influence the transmissibility of particular M. tuberculosis strains in human populations. PMID:22718126

Single nucleotide polymorphisms may explain the contrasting phenotypes of two variants of a multidrug-resistant Mycobacterium tuberculosis strain.

PubMed

Bigi, María Mercedes; Lopez, Beatriz; Blanco, Federico Carlos; Sasiain, María Del Carmen; De la Barrera, Silvia; Marti, Marcelo A; Sosa, Ezequiel Jorge; Fernández Do Porto, Darío Augusto; Ritacco, Viviana; Bigi, Fabiana; Soria, Marcelo Abel

2017-03-01

Globally, about 4.5% of new tuberculosis (TB) cases are multi-drug-resistant (MDR), i.e. resistant to the two most powerful first-line anti-TB drugs. Indeed, 480,000 people developed MDR-TB in 2015 and 190,000 people died because of MDR-TB. The MDR Mycobacterium tuberculosis M family, which belongs to the Haarlem lineage, is highly prosperous in Argentina and capable of building up further drug resistance without impairing its ability to spread. In this study, we sequenced the whole genomes of a highly prosperous M-family strain (Mp) and its contemporary variant, strain 410, which produced only one recorded tuberculosis case in the last two decades. Previous reports have demonstrated that Mp induced dysfunctional CD8 + cytotoxic T cell activity, suggesting that this strain has the ability to evade the immune response against M. tuberculosis. Comparative analysis of Mp and 410 genomes revealed non-synonymous polymorphisms in eleven genes and five intergenic regions with polymorphisms between both strains. Some of these genes and promoter regions are involved in the metabolism of cell wall components, others in drug resistance and a SNP in Rv1861, a gene encoding a putative transglycosylase that produces a truncated protein in Mp. The mutation in Rv3787c, a putative S-adenosyl-l-methionine-dependent methyltransferase, is conserved in all of the other prosperous M strains here analysed and absent in non-prosperous M strains. Remarkably, three polymorphic promoter regions displayed differential transcriptional activity between Mp and 410. We speculate that the observed mutations/polymorphisms are associated with the reported higher capacity of Mp for modulating the host's immune response. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characterization of exochelins of the Mycobacterium bovis type strain and BCG substrains.

PubMed

Gobin, J; Wong, D K; Gibson, B W; Horwitz, M A

1999-04-01

Pathogenic mycobacteria must acquire iron in the host in order to multiply and cause disease. To do so, they release abundant quantities of siderophores called exochelins, which have the capacity to scavenge iron from host iron-binding proteins and deliver it to the mycobacteria. In this study, we have characterized the exochelins of Mycobacterium bovis, the causative agent of bovine and occasionally of human tuberculosis, and the highly attenuated descendant of M. bovis, bacillus Calmette-Guérin (BCG), widely used as a vaccine against human tuberculosis. The M. bovis type strain, five substrains of M. bovis BCG (Copenhagen, Glaxo, Japanese, Pasteur, and Tice), and two strains of virulent Mycobacterium tuberculosis all produce the same set of exochelins, although the relative amounts of individual exochelins may differ. Among these mycobacteria, the total amount of exochelins produced is greatest in M. tuberculosis, intermediate in M. bovis, and smallest in M. bovis BCG.

Genomic analysis of globally diverse Mycobacterium tuberculosis strains provides insights into emergence and spread of multidrug resistance

PubMed Central

Manson, Abigail L.; Cohen, Keira A.; Abeel, Thomas; Desjardins, Christopher A.; Armstrong, Derek T.; Barry, Clifton E.; Brand, Jeannette; Chapman, Sinéad B.; Cho, Sang-Nae; Gabrielian, Andrei; Gomez, James; Jodals, Andreea M.; Joloba, Moses; Jureen, Pontus; Lee, Jong Seok; Malinga, Lesibana; Maiga, Mamoudou; Nordenberg, Dale; Noroc, Ecaterina; Romancenco, Elena; Salazar, Alex; Ssengooba, Willy; Velayati, A. A.; Winglee, Kathryn; Zalutskaya, Aksana; Via, Laura E.; Cassell, Gail H.; Dorman, Susan E.; Ellner, Jerrold; Farnia, Parissa; Galagan, James E.; Rosenthal, Alex; Crudu, Valeriu; Homorodean, Daniela; Hsueh, Po-Ren; Narayanan, Sujatha; Pym, Alexander S.; Skrahina, Alena; Swaminathan, Soumya; Van der Walt, Martie; Alland, David; Bishai, William R.; Cohen, Ted; Hoffner, Sven; Birren, Bruce W.; Earl, Ashlee M.

2017-01-01

Multidrug-resistant tuberculosis (MDR-TB), caused by drug resistant strains of Mycobacterium tuberculosis, is an increasingly serious problem worldwide. In this study, we examined a dataset of 5,310 M. tuberculosis whole genome sequences from five continents. Despite great diversity with respect to geographic point of isolation, genetic background and drug resistance, patterns of drug resistance emergence were conserved globally. We have identified harbinger mutations that often precede MDR. In particular, the katG S315T mutation, conferring resistance to isoniazid, overwhelmingly arose before rifampicin resistance across all lineages, geographic regions, and time periods. Molecular diagnostics that include markers for rifampicin resistance alone will be insufficient to identify pre-MDR strains. Incorporating knowledge of pre-MDR polymorphisms, particularly katG S315, into molecular diagnostics will enable targeted treatment of patients with pre-MDR-TB to prevent further development of MDR-TB. PMID:28092681

Shared characteristics between Mycobacterium tuberculosis and fungi contribute to virulence.

PubMed

Willcocks, Sam; Wren, Brendan W

2014-01-01

Mycobacterium tuberculosis, an etiologic agent of tuberculosis, exacts a heavy toll in terms of human morbidity and mortality. Although an ancient disease, new strains are emerging as human population density increases. The emergent virulent strains appear adept at steering the host immune response from a protective Th1 type response towards a Th2 bias, a feature shared with some pathogenic fungi. Other common characteristics include infection site, metabolic features, the composition and display of cell surface molecules, the range of innate immune receptors engaged during infection, and the ability to form granulomas. Literature from these two distinct fields of research are reviewed to propose that the emergent virulent strains of M. tuberculosis are in the process of convergent evolution with pathogenic fungi, and are increasing the prominence of conserved traits from environmental phylogenetic ancestors that facilitate their evasion of host defenses and dissemination.

Fighting an old disease with modern tools: characteristics and molecular detection methods of drug-resistant Mycobacterium tuberculosis.

PubMed

Engström, Anna

2016-01-01

Tuberculosis (TB) is an ancient disease, but not a disease of the past. The increasing prevalence of drug-resistant strains of Mycobacterium tuberculosis, the causative agent of TB, demands new measures to combat the situation. Rapid and accurate detection of the pathogen, and its drug susceptibility pattern, is essential for timely initiation of treatment, and ultimately, control of the disease. Molecular-based methods offer a great chance to improve detection of drug-resistant TB; however, their development and usage should be accompanied with a profound understanding of drug resistance mechanisms and circulating M. tuberculosis strains in specific settings, as otherwise, the usefulness of such tests may be limited. This review gives an overview of the history of TB treatment and drug resistance, drug resistance mechanisms for the most commonly used drugs and molecular methods designed to detect drug-resistant strains.

Neutral-red reaction is related to virulence and cell wall methyl-branched lipids in Mycobacterium tuberculosis.

PubMed

Cardona, P-J; Soto, C Y; Martín, C; Giquel, B; Agustí, G; Andreu, Núria; Guirado, E; Sirakova, T; Kolattukudy, P; Julián, E; Luquin, M

2006-01-01

Searching for virulence marking tests for Mycobacterium tuberculosis, Dubos and Middlebrook reported in 1948 that in an alkaline aqueous solution of neutral-red, the cells of the virulent H37Rv M. tuberculosis strain fixed the dye and became red in color, whereas the cells of the avirulent H37Ra M. tuberculosis strain remained unstained. In the 1950 and 1960s, fresh isolates of M. tuberculosis were tested for this neutral-red cytochemical reaction and it was reported that they were neutral-red positive, whereas other mycobacteria of diverse environmental origins that were non-pathogenic for guinea pigs were neutral-red negative. However, neutral-red has not really been proven to be a virulence marker. To test if virulence is in fact correlated to neutral-red, we studied a clinical isolate of M. tuberculosis that was originally neutral-red positive but, after more than 1 year passing through culture mediums, turned neutral-red negative. We found that, in comparison to the original neutral-red positive strain, this neutral-red negative variant was attenuated in two murine models of experimental tuberculosis. Lipid analysis showed that this neutral-red negative natural mutant lost the capacity to synthesize pthiocerol dimycocerosates, a cell wall methyl-branched lipid that has been related to virulence in M. tuberculosis. We also studied the neutral-red of different gene-targeted M. tuberculosis mutants unable to produce pthiocerol dimycocerosates or other cell wall methyl-branched lipids such as sulfolipids, and polyacyltrehaloses. We found a negative neutral-red reaction in mutants that were deficient in more than one type of methyl-branched lipids. We conclude that neutral-red is indeed a marker of virulence and it indicates important perturbations in the external surface of M. tuberculosis cells.

Secretome profile analysis of multidrug-resistant, monodrug-resistant and drug-susceptible Mycobacterium tuberculosis.

PubMed

Putim, Chanyanuch; Phaonakrop, Narumon; Jaresitthikunchai, Janthima; Gamngoen, Ratikorn; Tragoolpua, Khajornsak; Intorasoot, Sorasak; Anukool, Usanee; Tharincharoen, Chayada Sitthidet; Phunpae, Ponrut; Tayapiwatana, Chatchai; Kasinrerk, Watchara; Roytrakul, Sittiruk; Butr-Indr, Bordin

2018-03-01

The emergence of drug-resistant tuberculosis has generated great concern in the control of tuberculosis and HIV/TB patients have established severe complications that are difficult to treat. Although, the gold standard of drug-susceptibility testing is highly accurate and efficient, it is time-consuming. Diagnostic biomarkers are, therefore, necessary in discriminating between infection from drug-resistant and drug-susceptible strains. One strategy that aids to effectively control tuberculosis is understanding the function of secreting proteins that mycobacteria use to manipulate the host cellular defenses. In this study, culture filtrate proteins from Mycobacterium tuberculosis H37Rv, isoniazid-resistant, rifampicin-resistant and multidrug-resistant strains were gathered and profiled by shotgun-proteomics technique. Mass spectrometric analysis of the secreted proteome identified several proteins, of which 837, 892, 838 and 850 were found in M. tuberculosis H37Rv, isoniazid-resistant, rifampicin-resistant and multidrug-resistant strains, respectively. These proteins have been implicated in various cellular processes, including biological adhesion, biological regulation, developmental process, immune system process localization, cellular process, cellular component organization or biogenesis, metabolic process, and response to stimulus. Analysis based on STITCH database predicted the interaction of DNA topoisomerase I, 3-oxoacyl-(acyl-carrier protein) reductase, ESAT-6-like protein, putative prophage phiRv2 integrase, and 3-phosphoshikimate 1-carboxyvinyltransferase with isoniazid, rifampicin, pyrazinamide, ethambutol and streptomycin, suggesting putative roles in controlling the anti-tuberculosis ability. However, several proteins with no interaction with all first-line anti-tuberculosis drugs might be used as markers for mycobacterial identification.

Predominance of Central Asian and European families among Mycobacterium tuberculosis isolates in Kashmir Valley, India.

PubMed

Bashir, Gulnaz; Wani, Tehmeena; Sharma, Pragya; Katoch, V M; Lone, Rubina; Shah, Azra; Katoch, Kiran; Kakru, D K; Chauhan, Devendra Singh

2017-10-01

As there are no data available regarding the strains of Mycobacterium tuberculosis circulating in Kashmir Valley, India, the current study aimed at describing the genetic diversity of M. tuberculosis strains in this region, by spoligotyping and 12-locus-based MIRU-VNTR typing (Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat). Sputa from 207 smear positive cases with newly diagnosed pulmonary tuberculosis were subjected to culture for M. tuberculosis. Eighty-five isolates confirmed as M. tuberculosis were subjected to drug susceptibility testing and molecular typing by spoligotyping and MIRU-VNTRs. Drug susceptibility results of 72 isolates revealed 76.3% as fully sensitive while 5.5% as multidrug resistant (MDR). Spoligotyping of 85 isolates detected 42 spoligotypes with 50 isolates (58.8%) clustered into seven spoligotypes. SIT26/CAS1_Del was the major spoligotype (23, 27%) followed by SIT127/H4 (12, 14.1%); CAS lineage (37.6%) was predominant, followed by Haarlem (25.8%) and ill-defined T clade (23.5%). MIRU-VNTR analysis displayed 82 MIRU patterns from 85 strains, including 3 small clusters and 79 unique. MIRU 26 was found to be the most discriminatory locus. Kashmir Valley has CAS as the predominant lineage of M. tuberculosis similar to the rest of the Indian sub-continent, while it is peculiar in having Euro American lineages such as Haarlem and ill-defined T clade. Copyright © 2017 Tuberculosis Association of India. Published by Elsevier B.V. All rights reserved.

Spoligotyping of Mycobacterium tuberculosis isolates at a tertiary care hospital in India.

PubMed

Suzana, Shirly; Shanmugam, Sivakumar; Uma Devi, K R; Swarna Latha, P N; Michael, Joy S

2017-06-01

Spoligotyping is a valuable genotyping tool to study the genetic diversity and molecular epidemiology of Mycobacterium tuberculosis (M. tb). The aim of this study was to analyse different spoligotype patterns of M. tb strains isolated from patients with tuberculosis from different parts of India. A total of 163 M. tb isolates were spoligotyped between January 2014 and January 2015. About 47% (n = 77) were from patients with extrapulmonary tuberculosis; of these, 10 were MDR, and seven were Pre-XDR. Of the 86 M. tb isolates from patients with pulmonary tuberculosis, 25 were MDR, and 25 were Pre-XDR. We found 61 spoligo patterns, 128 clusters in the spoligotype data base (spoldb4 data base) with spoligo international type (SIT) number and 35 true unique isolates. The most pre-dominant spoligotype was EAI lineage (56), followed by Beijing (28), CAS (20), T(9), U(7), X(3), H(3), BOVIS_1 BCG(1) and LAM(1). Although our study identified EAI, CAS and Beijing strain lineages as pre-dominant, we also found a large number of orphan strains (20%) in our study. Beijing strains were more significantly associated with MDR TB than CAS and EAI lineages. Further studies on large sample sizes would help to clearly describe the epidemiology of M. tb in India. © 2017 John Wiley & Sons Ltd.

Intragranulomatous necrosis in pulmonary granulomas is not related to resistance against Mycobacterium tuberculosis infection in experimental murine models induced by aerosol.

PubMed

Guirado, Evelyn; Gordillo, Sergi; Gil, Olga; Díaz, Jorge; Tapia, Gustavo; Vilaplana, Cristina; Ausina, Vicenç; Cardona, Pere-Joan

2006-04-01

Intragranulomatous necrosis is a primary feature in the natural history of human tuberculosis (TB). Unfortunately, this phenomenon is not usually seen in the experimental TB murine model. Artificial induction of this necrosis in pulmonary granulomas (INPG) may be achieved through aerosol inoculation of lipopolysaccharide (LPS) 3 weeks after Mycobacterium tuberculosis infection. At week 9 post-infection, the centre of primary granulomas became larger, showing eosinophilic necrosis. Interestingly, INPG induction was related to mice strains C57BL/6 and 129/Sv, but not to BALB/c and DBA/2. Furthermore, the same pattern was obtained with the induction of infection using a clinical M. tuberculosis strain (UTE 0335R) that naturally induces INPG. In all the mice strains tested, the study of pulmonary mRNA expression revealed a tendency to increase or to maintain the expression of RANTES, interferon-gamma, tumour necrosis factor and iNOS, in both LPS- and UTE 0335R-induced INPG, thus suggesting that this response must be necessary but not sufficient for inducing INPG. Our work supports that INPG induction is a local phenomenon unrelated to the resistant (C57BL/6 and BALB/c) or susceptible (129/Sv and DBA/2) background of mice strains against M. tuberculosis infection.

Impact of pe_pgrs33 Gene Polymorphisms on Mycobacterium tuberculosis Infection and Pathogenesis.

PubMed

Camassa, Serena; Palucci, Ivana; Iantomasi, Raffaella; Cubeddu, Tiziana; Minerva, Mariachiara; De Maio, Flavio; Jouny, Samuel; Petruccioli, Elisa; Goletti, Delia; Ria, Francesco; Sali, Michela; Sanguinetti, Maurizio; Manganelli, Riccardo; Rocca, Stefano; Brodin, Priscille; Delogu, Giovanni

2017-01-01

PE_PGRS33 is a surface-exposed protein of Mycobacterium tuberculosis ( Mtb ) which exerts its role in macrophages entry and immunomodulation. In this study, we aimed to investigate the polymorphisms in the pe_pgrs33 gene of Mtb clinical isolates and evaluate their impact on protein functions. We sequenced pe_pgrs33 in a collection of 135 clinical strains, genotyped by 15-loci MIRU-VNTR and spoligotyping and belonging to the Mtb complex (MTBC). Overall, an association between pe_pgrs33 alleles and MTBC genotypes was observed and a dN/dS ratio of 0.64 was obtained, suggesting that a purifying selective pressure is acting on pe_pgrs33 against deleterious SNPs. Among a total of 19 pe_pgrs33 alleles identified in this study, 5 were cloned and used to complement the pe_pgrs33 knock-out mutant strain of Mtb H37Rv ( Mtb Δ33) to assess the functional impact of the respective polymorphisms in in vitro infections of primary macrophages. In human monocyte-derived macrophages (MDMs) infection, large in-frame and frameshift mutations were unable to restore the phenotype of Mtb H37Rv, impairing the cell entry capacity of Mtb , but neither its intracellular replication rate nor its immunomodulatory properties. In vivo studies performed in the murine model of tuberculosis (TB) demonstrated that the Mtb Δ33 mutant strain was not impaired in the ability to infect and replicate in the lung tissue compared to the parental strain. Interestingly, Mtb Δ33 showed an enhanced virulence during the chronic steps of infection compared to Mtb H37Rv. Similarly, the complementation of Mtb Δ33 with a frameshift allele also resulted in a Mtb strain capable of causing a surprisingly enhanced tissue damage in murine lungs, during the chronic steps of infection. Together, these results further support the role of PE_PGRS33 in the pathogenesis and virulence of Mtb .

Pulmonary Bovine-Type Tuberculosis in Rabbits: Bacillary Virulence, Inhaled Dose Effects, Tuberculin Sensitivity, and Mycobacterium vaccae Immunotherapy

PubMed Central

Converse, P. J.; Dannenberg, A. M.; Shigenaga, T.; McMurray, D. N.; Phalen, S. W.; Stanford, J. L.; Rook, G. A. W.; Koru-Sengul, T.; Abbey, Helen; Estep, J. E.; Pitt, M. L. M.

1998-01-01

This report elucidates four aspects of the immunology of pulmonary tuberculosis produced in rabbits: (i) the virulence of bovine-type tubercle bacilli, strain Ravenel S, (ii) systemic factors influencing the generation of visible primary pulmonary tubercles, (iii) differences in tuberculin sensitivity of rabbits and humans, and (iv) the effect of Mycobacterium vaccae immunotherapy on cavitary tuberculosis. Laboratory strain Ravenel S (ATCC 35720) was not fully virulent. Fully virulent strains produce one visible primary pulmonary tubercle for each three bacillary units inhaled. Strain ATCC 35720 produced one such tubercle for each 18 to 107 bacillary units inhaled, indicating that its virulence was reduced by 6- to 36-fold. When a low dose of this Ravenel S strain was inhaled, the host resistance (measured by the number of inhaled bacilli needed to generate one visible primary pulmonary tubercle) was increased at least 3.5-fold compared to the host resistance when a high dose was inhaled. Rabbits and humans differ in the degree and in the maintenance of their dermal sensitivities to tuberculin. Compared to rabbits, humans are 100 times more sensitive to tuberculin. Also, at 33 weeks rabbits with well-controlled cavitary tuberculosis usually showed a decrease in their tuberculin reactions of about 50% from peak values, whereas humans with such well-controlled tuberculosis are thought to maintain strong reactions for many years. These species differences may be due to desensitization to group II mycobacterial antigens in the rabbits because they have a different diet and a different type of digestive tract. M. vaccae immunotherapy of rabbits with cavitary tuberculosis produced no statistically significant effects. Experiments with many more rabbits would be required to prove whether or not such immunotherapy is beneficial. PMID:9801350

Anti-mycobacterial screening of five Indian medicinal plants and partial purification of active extracts of Cassia sophera and Urtica dioica.

PubMed

Singh, Rambir; Hussain, Shariq; Verma, Rajesh; Sharma, Poonam

2013-05-13

To find out the anti-mycobacterial potential of Cassia sophera (C. sophera), Urtica dioica (U. dioica), Momordica dioica, Tribulus terrestris and Coccinia indica plants against multi-drug resistant (MDR) strain of Mycobacterium tuberculosis (M. tuberculosis). Plant materials were extracted successively with solvents of increasing polarity. Solvent extracts were screened for anti-mycobacterial activity against fast growing, non-pathogenic mycobacterium strain, Mycobacterium semegmatis, by disk diffusion method. The active extracts were tested against MDR and clinical isolates of M. tuberculosis by absolute concentration and proportion methods. The active extracts were subjected to bio-autoassay on TLC followed by silica column chromatography for isolation of potential drug leads. Hexane extract of U. dioica (HEUD) and methanol extract of C. sophera (MECS) produced inhibition zone of 20 mm in disc diffusion assay and MIC of 250 and 125 μ g/mL respectively in broth dilution assay against Mycobacterium semegmatis. Semipurified fraction F2 from MECS produced 86% inhibition against clinical isolate and 60% inhibition against MDR strain of M. tuberculosis. F18 from HEUD produced 81% inhibition against clinical isolate and 60% inhibition against MDR strain of M. tuberculosis. Phytochemical analysis indicated that anti-mycobacterial activity of MECS may be due to presence of alkaloids or flavonoids and that of HEUD due to terpenoids. C. sophera and U. dioica plant extracts exhibited promising anti-mycobacterial activity against MDR strain of M. tuberculosis. This is the first report of anti-mycobacterial activity form C. sophera. This study showed possibility of purifying novel anti-mycobacterial compound(s) from C. sophera and U. dioica. Copyright © 2013 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

Study of a series of cobalt(II) sulfonamide complexes: Synthesis, spectroscopic characterization, and microbiological evaluation against M. tuberculosis. Crystal structure of [Co(sulfamethoxazole)2(H2O)2]·H2O

NASA Astrophysics Data System (ADS)

Mondelli, Melina; Pavan, Fernando; de Souza, Paula C.; Leite, Clarice Q.; Ellena, Javier; Nascimento, Otaciro R.; Facchin, Gianella; Torre, María H.

2013-03-01

Nowadays, the research for new and better antimicrobial compounds is an important field due to the increase of immunocompromised patients, the use of invasive medical procedures and extensive surgeries, among others, that can affect the incidence of infections. Another big problem associated is the occurrence of drug-resistant microbial strains that impels a ceaseless search for new antimicrobial agents. In this context, a series of heterocyclic-sulfonamide complexes with Co(II) was synthesized and characterized with the aim of obtaining new antimicrobial compounds. The structural characterization was performed using different spectroscopic methods (UV-Vis, IR, and EPR). In spite of the fact that the general stoichiometry for all the complexes was Co(sulfonamide)2·nH2O, the coordination atoms were different depending on the coordinated sulfonamide. The crystal structure of [Co(sulfamethoxazole)2(H2O)2]·H2O was obtained by X-ray diffraction showing that Co(II) is in a slightly tetragonal distorted octahedron where sulfamethoxazole molecules act as a head-to-tail bridges between two cobalt atoms, forming polymeric chains. Besides, the activity against Mycobacterium tuberculosis, one of the responsible for tuberculosis, and the cytotoxicity on J774A.1 macrophage cells were evaluated.

Role of P27 -P55 operon from Mycobacterium tuberculosis in the resistance to toxic compounds

PubMed Central

2011-01-01

Background The P27-P55 (lprG-Rv1410c) operon is crucial for the survival of Mycobacterium tuberculosis, the causative agent of human tuberculosis, during infection in mice. P55 encodes an efflux pump that has been shown to provide Mycobacterium smegmatis and Mycobacterium bovis BCG with resistance to several drugs, while P27 encodes a mannosylated glycoprotein previously described as an antigen that modulates the immune response against mycobacteria. The objective of this study was to determine the individual contribution of the proteins encoded in the P27-P55 operon to the resistance to toxic compounds and to the cell wall integrity of M. tuberculosis. Method In order to test the susceptibility of a mutant of M. tuberculosis H37Rv in the P27-P55 operon to malachite green, sodium dodecyl sulfate, ethidium bromide, and first-line antituberculosis drugs, this strain together with the wild type strain and a set of complemented strains were cultivated in the presence and in the absence of these drugs. In addition, the malachite green decolorization rate of each strain was obtained from decolorization curves of malachite green in PBS containing bacterial suspensions. Results The mutant strain decolorized malachite green faster than the wild type strain and was hypersensitive to both malachite green and ethidium bromide, and more susceptible to the first-line antituberculosis drugs: isoniazid and ethambutol. The pump inhibitor reserpine reversed M. tuberculosis resistance to ethidium bromide. These results suggest that P27-P55 functions through an efflux-pump like mechanism. In addition, deletion of the P27-P55 operon made M. tuberculosis susceptible to sodium dodecyl sulfate, suggesting that the lack of both proteins causes alterations in the cell wall permeability of the bacterium. Importantly, both P27 and P55 are required to restore the wild type phenotypes in the mutant. Conclusions The results clearly indicate that P27 and P55 are functionally connected in processes that involve the preservation of the cell wall and the transport of toxic compounds away from the cells. PMID:21762531

First evaluation of drug-resistant Mycobacterium tuberculosis clinical isolates from Congo revealed misdetection of fluoroquinolone resistance by line probe assay due to a double substitution T80A-A90G in GyrA.

PubMed

Aubry, Alexandra; Sougakoff, Wladimir; Bodzongo, Pamela; Delcroix, Guy; Armand, Sylvie; Millot, Gérald; Jarlier, Vincent; Courcol, René; Lemaître, Nadine

2014-01-01

Tuberculosis (TB) is one of the major public health problems in Congo. However, data concerning Mycobacterium tuberculosis drug resistance are lacking because of the insufficient processing capacity. So, the aim of this study was to investigate for the first time the resistance patterns and the strain lineages of a sample of M. tuberculosis complex (MTBC) isolates collected in the two main cities of Congo. Over a 9-day period, 114 smear-positive sputa isolated from 114 patients attending centers for the diagnosis and treatment of TB in Brazzaville and Pointe Noire were collected for culture and drug susceptibility testing (DST). Detection of mutations conferring drug resistance was performed by using line probe assays (GenoType MTBDRplus and MTBDRsl) and DNA sequencing. Strain lineages were determined by MIRU-VNTR genotyping. Of the 114 sputa, 46 were culture positive for MTBC. Twenty-one (46%) were resistant to one or more first-line antiTB drugs. Of these, 15 (71%) were multidrug resistant (MDR). The most prevalent mutations involved in rifampin and isoniazid resistance, D516V (60%) in rpoB and S315T (87%) in katG respectively, were well detected by MTBDRplus assay. All the 15 MDR strains were susceptible to fluoroquinolone and injectable second-line drug. No mutation was detected in the rrs locus involved in resistance to amikacin and capreomycin by both the MTBDRsl assay and DNA sequencing. By contrast, 9 MDR strains belonging to the same cluster related to T-family were identified as being falsely resistant to fluoroquinolone by the MTBDRsl assay due to the presence of a double substitution T80A-A90G in GyrA. Taken together, these data revealed a possible spread of a particular MDR clone in Congo, misidentified as fluoroquinolone resistant by MTBDRsl assay. Thus, this test cannot replace gold-standard culture method and should be interpreted carefully in view of the patient's native land.

[Clinical application of testing methods on acid-fast bacteria].

PubMed

Ichiyama, Satoshi; Suzuki, Katsuhiro

2005-02-01

Clinical bacteriology pertaining to acid-fast bacteria has made marked advances over the past decade, initiated by the development of a DNA probe kit for identification of acid-fast bacteria. Wide-spread use of nucleic acid amplification for rapid detection of tubercle bacillus contributed more greatly than any other factor to such advances in this field. At present, 90% of all kits used for nucleic acid amplification in the world are consumed in Japan. Unfortunately, not a few clinicians in Japan have a false idea that the smear method and nucleic acid amplification are necessary but culture is not. In any event nucleic acid amplification has exerted significant impacts on the routine works at bacteriology laboratories. Among others, collecting bacteria by pretreatment with NALC-NaOH has simplified the introduction of the collective mode smear method and liquid media. Furthermore, as clinicians have become increasingly more experienced with various methods of molecular biology, it now seems possible to apply these techniques for detection of genes encoding drug resistance and for utilization of molecular epidemiology in routine laboratory works. Meanwhile, attempts to diagnose acid-fast bacteriosis by checking blood for antibody have also been made, primarily in Japan. At present, two kits for detecting antibodies to glycolipids (LAM, TDM, etc.) are covered by national health insurance in Japan. We have an impression that in Japan clinicians do not have adequate knowledge and skill to make full use of these new testing methods clinically. We, as the chairmen of this symposium, hope that this symposium will help clinicians increase their skill related to new testing methods, eventually leading to stimulation of advances in clinical practices related to acid-fast bacteria in Japan. 1. Smear microscopy by concentration method and broth culture system: Kazunari TSUYUGUCHI (Clinical Research Center, National Hospital Organization Kinki-chuo Chest Medical Center) Smear microscopy and culture still remain the cornerstone to diagnose tuberculosis. However, the classical methods in Japan using direct microscopy and Ogawa solid media were not sufficient for clinical use. In recent years substantial advance has been made in these fields. Concentration of clinical samples by centrifugation improves the sensitivity of smear microscopy with excellent reproducibility. The Mycobacteria Growth Indicator Tube (MGIT) system using liquid media yields high sensitivity and rapidity. Using these methods, more and more tuberculosis cases would be correctly diagnosed and treated adequately based on drug susceptibility testing. 2. New technologies for anti-tuberculosis drug susceptibility testing: Satoshi MITARAI (Bacteriology Division, Reference Centre for Mycobacterium, Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association) Several new technologies have been developed to obtain anti-tuberculosis drug susceptibility testing (AST) results rapidly, utilising liquid culture and molecular technologies. Mycobacterium Growth Indicator Tube (MGIT), as a popular liquid culturing and AST system, was evaluated for its accuracy and usefulness. As for isoniazid, MGIT showed 12.6% of discordant result comparing with standard method. These MGIT resistant and Ogawa susceptible strains had relatively high MICs ranging 0.13 to 2.0 microg/ml. The molecular detection of resistant gene mutation is also a useful method to estimate drug resistance rapidly. The rpoB mutation detection is reliable with high sensitivity and specificity. 3. Nucleic acid amplification and novel diagnostic methods: Shunji TAKAKURA (Department of Clinical Laboratory Medicine, Kyoto University Graduate School of Medicine) Sensitivities of nucleic acid amplification tests (NAATs) for the diagnosis of tuberculosis meet clinical requirement that patients with high-risk of transmission should be identified within a day. Comparison of the performance of various NAATs is difficult because of the difference in sample processing and in samples tested among methods and reports. Considering the limitations of NAATs (low sensitivity compared with culture, inability to differentiate dead bacilli from the living), further advances would be expected when novel technologies could confer additional information, such as drug susceptibility, quantity, viability, and genotype. 4. Serodiagnosis of Mycobacterium avium complex lung disease: Seigo KITADA (Department of Internal Medicine, National Hospital Organization Toneyama National Hospital) Mycobacterium avium complex (MAC) organisms are ubiquitous in environment and a contamination in respiratory tracts is sometimes observed, and that complex the diagnosis. We developed a serodiagnostic method for MAC disease using an enzyme immunoassay with the MAC-specific glycopeptidolipid (GPL) core as antigen. A significant increase in GPL core antibodies was detected in sera of patients with MAC pulmonary diseases compared to patients who were colonized with MAC, patients with M. kansasii disease and tuberculosis and healthy subjects. The serodiagnosis is useful for diagnosis of MAC lung disease. 5. Molecular epidemiologic tools for tuberculosis: IS6110 RFLP, Spoligotyping, and VNTR: Tomoshige MATSUMOTO, Hiromi ANO, Tetsuya TAKASHIMA, Izuo TSUYUGUCHI (Osaka Prefectural Medical Center for Respiratory and Allergic Diseases) We have performed molecular typing on about 1,300 culture positive clinical isolates that made up the majority of tuberculosis strains in part of southeast Osaka since 2001 until now. By spoligotyping, about 75% of entire strains belonged to the Beijing strain. Particular spoligotyping descriptions, which were not described in SpolDBIII, were found in the strains with lower than 6 copies of IS6110 RFLP. We described them as Osaka type. We could also show that direct typing from Tb PCR positive sputum of patients with tuberculosis was possible by VNTR and that VNTR with 16 loci was useful in tuberculosis typing in Osaka.

Factors associated with recently transmitted Mycobacterium tuberculosis strain MS0006 in Hinds County, Mississippi.

PubMed

Temple, Brian; Kwara, Awewura; Sunesara, Imran; Mena, Leandro; Dobbs, Thomas; Henderson, Harold; Holcomb, Mike; Webb, Risa

2011-12-01

The objective of this study was to investigate risk factors associated with tuberculosis (TB) transmission that was caused by Mycobacterium tuberculosis strain MS0006 from 2004 to 2009 in Hinds County, Mississippi. DNA fingerprinting using spoligotyping, mycobacterial interspersed repetitive unit, and IS6110-based restriction fragment length polymorphism of culture-confirmed cases of TB was performed. Clinical and demographic factors associated with strain MS0006 were analyzed by univariate and multivariate analysis. Of the 144 cases of TB diagnosed during the study period, 117 were culture positive with fingerprints available. There were 48 different strains, of which 6 clustered strains were distributed among 74 patients. The MS0006 strain accounted for 46.2% of all culture-confirmed cases. Risk factors for having the MS0006 strain in a univariate analysis included homelessness, HIV co-infection, sputum smear negativity, tuberculin skin test negativity, and noninjectable drug use. Multivariate analysis identified homelessness (odds ratio 7.88, 95% confidence interval 2.90-21.35) and African American race (odds ratio 5.80, 95% confidence interval 1.37-24.55) as independent predictors of having TB caused by the MS0006 strain of M. tuberculosis. Our findings suggest that a majority of recently transmitted TB in the studied county was caused by the MS0006 strain. African American race and homelessness were significant risk factors for inclusion in the cluster. Molecular epidemiology techniques continue to provide in-depth analysis of disease transmission and play a vital role in effective contact tracing and interruption of ongoing transmission.

A non-sense mutation in the putative anti-mutator gene ada/alkA of Mycobacterium tuberculosis and M. bovis isolates suggests convergent evolution

PubMed Central

Nouvel, Laurent X; Vultos, Tiago Dos; Kassa-Kelembho, Eric; Rauzier, Jean; Gicquel, Brigitte

2007-01-01

Background Previous studies have suggested that variations in DNA repair genes of W-Beijing strains may have led to transient mutator phenotypes which in turn may have contributed to host adaptation of this strain family. Single nucleotide polymorphism (SNP) in the DNA repair gene mutT1 was identified in MDR-prone strains from the Central African Republic. A Mycobacteriumtuberculosis H37Rv mutant inactivated in two DNA repair genes, namely ada/alkA and ogt, was shown to display a hypermutator phenotype. We then looked for polymorphisms in these genes in Central African Republic strains (CAR). Results In this study, 55 MDR and 194 non-MDR strains were analyzed. Variations in DNA repair genes ada/alkA and ogt were identified. Among them, by comparison to M. tuberculosis published sequences, we found a non-sense variation in ada/alkA gene which was also observed in M. bovis AF2122 strain. SNPs that are present in the adjacent regions to the amber variation are different in M. bovis and in M. tuberculosis strain. Conclusion An Amber codon was found in the ada/alkA locus of clustered M. tuberculosis isolates and in M. bovis strain AF2122. This is likely due to convergent evolution because SNP differences between strains are incompatible with horizontal transfer of an entire gene. This suggests that such a variation may confer a selective advantage and be implicated in hypermutator phenotype expression, which in turn contributes to adaptation to environmental changes. PMID:17506895

Combined Genotypic, Phylogenetic, and Epidemiologic Analyses of Mycobacterium tuberculosis Genetic Diversity in the Rhône Alpes Region, France.

PubMed

Pichat, Catherine; Couvin, David; Carret, Gérard; Frédénucci, Isabelle; Jacomo, Véronique; Carricajo, Anne; Boisset, Sandrine; Dumitrescu, Oana; Flandrois, Jean-Pierre; Lina, Gérard; Rastogi, Nalin

2016-01-01

The present work relates to identification and a deep molecular characterization of circulating Mycobacterium tuberculosis complex (MTBC) strains in the Rhône-Alpes region, France from 2000 to 2010. It aimed to provide with a first snapshot of MTBC genetic diversity in conjunction with bacterial drug resistance, type of disease and available demographic and epidemiologic characteristics over an eleven-year period, in the south-east of France. Mycobacterium tuberculosis complex (MTBC) strains isolated in the Rhône-Alpes region, France (n = 2257, 1 isolate per patient) between 2000 and 2010 were analyzed by spoligotyping. MIRU-VNTR typing was applied on n = 1698 strains (with full results available for 974 strains). The data obtained were compared with the SITVIT2 database, followed by detailed genotyping, phylogenetic, and epidemiologic analyses in correlation with anonymized data on available demographic, and epidemiologic characteristics, and location of disease (pulmonary or extrapulmonary TB). The most predominant spoligotyping clusters were SIT53/T1 (n = 346, 15.3%) > SIT50/H3 (n = 166, 7.35%) > SIT42/LAM9 (n = 125, 5.5%) > SIT1/Beijing (n = 72, 3.2%) > SIT47/H1 (n = 71, 3.1%). Evolutionary-recent strains belonging to the Principal Genetic Group (PGG) 2/3, or Euro-American lineages (T, LAM, Haarlem, X, S) were predominant and represented 1768 or 78.33% of all isolates. For strains having drug resistance information (n = 1119), any drug resistance accounted for 14.83% cases vs. 1.52% for multidrug resistance (MDR); and was significantly more associated with age group 21-40 years (p-value<0.001). Extra-pulmonary TB was more common among female patients while pulmonary TB predominated among men (p-value<0.001; OR = 2.16 95%CI [1.69; 2.77]). Also, BOV and CAS lineages were significantly well represented in patients affected by extra-pulmonary TB (p-value<0.001). The origin was known for 927/2257 patients: 376 (40.6%) being French-born vs. 551 (59.4%) Foreign-born. French patients were significantly older (mean age: 58.42 yrs 95%CI [56.04; 60.80]) than Foreign-born patients (mean age: 42.38 yrs. 95%CI [40.75; 44.0]). The study underlined the importance of imported TB cases on the genetic diversity and epidemiologic characteristics of circulating MTBC strains in Rhône-Alpes region, France over a large time-period. It helps better understand intricate relationships between certain lineages and geographic origin of the patients, and pinpoints genotypic and phylogenetic specificities of prevailing MTBC strains. Lastly, it also demonstrated a slow decline in isolation of M. africanum lineage in this region between 2000 and 2010.

Nanostructured lipid carriers for incorporation of copper(II) complexes to be used against Mycobacterium tuberculosis

PubMed Central

Sato, Mariana R; Oshiro Junior, João A; Machado, Rachel TA; de Souza, Paula C; Campos, Débora L; Pavan, Fernando R; da Silva, Patricia B; Chorilli, Marlus

2017-01-01

Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis. Cessation of treatment before the recommended conclusion may lead to the emergence of multidrug-resistant strains. The aim of this study was to develop nanostructured lipid carriers (NLCs) for use in the treatment of M. tuberculosis. The NLCs comprised the following lipid phase: 2.07% polyoxyethylene 40 stearate, 2.05% caprylic/capric triglyceride, and 0.88% polyoxyl 40 hydrogenated castor oil; the following aqueous phase: 3.50% poloxamer 407 (F1–F6), and 0.50% cetyltrimethylammonium bromide (F7–F12); and incorporated the copper(II) complexes [CuCl2(INH)2]·H2O (1), [Cu(NCS)2(INH)2]·5H2O (2), and [Cu(NCO)2(INH)2]·4H2O (3) to form compounds F11.1, F11.2, and F11.3, respectively. The mean diameter of F11, F11.1, F11.2, and F11.3 ranged from 111.27±21.86 to 134.25±22.72 nm, 90.27±12.97 to 116.46±9.17 nm, 112.4±10.22 to 149.3±15.82 nm, and 78.65±6.00 to 122.00±8.70 nm, respectively. The polydispersity index values for the NLCs ranged from 0.13±0.01 to 0.30±0.09. The NLCs showed significant changes in zeta potential, except for F11.2, with F11, F11.1, F11.2, and F11.3 ranging from 18.87±4.04 to 23.25±1.13 mV, 17.03±1.77 to 21.42±1.87 mV, 20.51±1.88 to 22.60±3.44 mV, and 17.80±1.96 to 25.25±7.78 mV, respectively. Atomic force microscopy confirmed the formation of nanoscale spherical particle dispersions by the NLCs. Differential scanning calorimetry determined the melting points of the constituents of the NLCs. The in vitro activity of copper(II) complex-loaded NLCs against M. tuberculosis H37Rv showed an improvement in the anti-TB activity of 55.4, 27.1, and 41.1 times the activity for complexes 1, 2, and 3, respectively. An in vivo acute toxicity study of complex-loaded NLCs demonstrated their reduced toxicity. The results suggest that NLCs may be a powerful tool to optimize the activity of copper(II) complexes against M. tuberculosis. PMID:28356717

Identification of new benzamide inhibitor against α-subunit of tryptophan synthase from Mycobacterium tuberculosis through structure-based virtual screening, anti-tuberculosis activity and molecular dynamics simulations.

PubMed

Naz, Sadia; Farooq, Umar; Ali, Sajid; Sarwar, Rizwana; Khan, Sara; Abagyan, Ruben

2018-03-13

Multi-drug-resistant tuberculosis and extensively drug-resistant tuberculosis has emerged as global health threat, causing millions of deaths worldwide. Identification of new drug candidates for tuberculosis (TB) by targeting novel and less explored protein targets will be invaluable for antituberculosis drug discovery. We performed structure-based virtual screening of eMolecules database against a homology model of relatively unexplored protein target: the α-subunit of tryptophan synthase (α-TRPS) from Mycobacterium tuberculosis essential for bacterial survival. Based on physiochemical properties analysis and molecular docking, the seven candidate compounds were selected and evaluated through whole cell-based activity against the H37Rv strain of M. tuberculosis. A new Benzamide inhibitor against α-subunit of tryptophan synthase (α-TRPS) from M. tuberculosis has been identified causing 100% growth inhibition at 25 μg/ml and visible bactericidal activity at 6 μg/ml. This benzamide inhibitor displayed a good predicted binding score (-48.24 kcal/mol) with the α-TRPS binding pocket and has logP value (2.95) comparable to Rifampicin. Further refinement of docking results and evaluation of inhibitor-protein complex stability were investigated through Molecular dynamic (MD) simulations studies. Following MD simulations, Root mean square deviation, Root mean square fluctuation and secondary structure analysis confirmed that protein did not unfold and ligand stayed inside the active pocket of protein during the explored time scale. This identified benzamide inhibitor against the α-subunit of TRPS from M. tuberculosis could be considered as candidate for drug discovery against TB and will be further evaluated for enzyme-based inhibition in future studies.

Assessment of BCG and inactivated Mycobacterium bovis vaccines in an experimental tuberculosis infection model in sheep.

PubMed

Balseiro, Ana; Altuzarra, Raúl; Vidal, Enric; Moll, Xavier; Espada, Yvonne; Sevilla, Iker A; Domingo, Mariano; Garrido, Joseba M; Juste, Ramón A; Prieto, Miguel; Pérez de Val, Bernat

2017-01-01

Animal tuberculosis (TB) is a complex animal health problem that causes disruption to trade and significant economic losses. TB involves a multi-host system where sheep, traditionally considered a rare host of this infection, have been recently included. The aims of this study were to develop an experimental TB infection model in sheep with a Mycobacterium caprae field strain isolated from a tuberculous diseased ewe, and to use this to evaluate the safety and efficacy of two vaccines against TB in sheep, the live-attenuated M. bovis BCG vaccine (Danish strain) and a heat-inactivated M. bovis (HIMB) vaccine. Eighteen 2 month-old lambs were experimentally challenged with M. caprae by the endotracheal route (1.5 × 103 CFU). They were separated per treatment group into parenterally vaccinated with a live BCG Danish strain vaccine (n = 6), orally vaccinated with a suspension of HIMB (n = 6) and unvaccinated controls (n = 6). Clinical, immunological, pathological and bacteriological parameters of infection were measured. All lambs were successfully infected and developed gross TB lesions in the respiratory system. The BCG vaccine conferred considerable protection against experimental TB in lambs, as measured by a reduction of the gross lesion volumes and bacterial load. However, HIMB vaccinated animals did not show protection. This study proposes a reliable new experimental model for a better understanding of tuberculosis in sheep. BCG vaccination offers an effective prospect for controlling the disease. Moreover alternative doses and/or routes of administration should be considered to evaluate the efficacy of the HIMB vaccine candidate.

Tuberculosis diagnostics: Challenges and opportunities

PubMed Central

Nema, Vijay

2012-01-01

Tuberculosis (TB) has been a disease affecting almost all parts of the world since ages. Lot many efforts came in the past for improving diagnosis and treatment. Also, an effective vaccine has been sought after for long. With the emergence of resistant strains of Mycobacterium tuberculosis, the causal organisms of tuberculosis, and complexities emerging due to other associated infections and disease conditions, there is a desperate need for further research input in the field. Be it the better medication and care or better resistance management, proper diagnostics holds the key to success. It has been observed that a high burden of the disease was accompanied by resource limitations and poor research set-up. The scenario remained like this for several decades. With the refreshed vision of resourceful countries and funding agencies, funding is being provided in many areas of research in tuberculosis diagnosis and treatment. This review has been written with an aim to bring forth the limitations of available methods in the field of diagnostics and making researchers aware about the changing scenario with better funding opportunities and support. The author visualizes an enthusiasm from all over the world for the development of better modalities and urges scientists to join the struggle at this very perfect time to take the challenge and come forward with innovations in this field. PMID:22919166

Tuberculosis diagnostics: Challenges and opportunities.

PubMed

Nema, Vijay

2012-07-01

Tuberculosis (TB) has been a disease affecting almost all parts of the world since ages. Lot many efforts came in the past for improving diagnosis and treatment. Also, an effective vaccine has been sought after for long. With the emergence of resistant strains of Mycobacterium tuberculosis, the causal organisms of tuberculosis, and complexities emerging due to other associated infections and disease conditions, there is a desperate need for further research input in the field. Be it the better medication and care or better resistance management, proper diagnostics holds the key to success. It has been observed that a high burden of the disease was accompanied by resource limitations and poor research set-up. The scenario remained like this for several decades. With the refreshed vision of resourceful countries and funding agencies, funding is being provided in many areas of research in tuberculosis diagnosis and treatment. This review has been written with an aim to bring forth the limitations of available methods in the field of diagnostics and making researchers aware about the changing scenario with better funding opportunities and support. The author visualizes an enthusiasm from all over the world for the development of better modalities and urges scientists to join the struggle at this very perfect time to take the challenge and come forward with innovations in this field.

Comparative genomics of archived pyrazinamide resistant Mycobacterium tuberculosis complex isolates from Uganda

USDA-ARS?s Scientific Manuscript database

Bovine tuberculosis is a ‘neglected zoonosis’ and its contribution to the proportion of Mycobacterium tuberculosis complex infections in humans is unknown. A retrospective study on archived Mycobacterium tuberculosis complex (MTC) isolates from a reference laboratory in Uganda was undertaken to iden...

Structural and Biophysical Characterization of the Mycobacterium tuberculosis Protein Rv0577, a Protein Associated with Neutral Red Staining of Virulent Tuberculosis Strains and Homologue of the Streptomyces coelicolor Protein KbpA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Buchko, Garry W.; Echols, Nathaniel; Flynn, E. Megan

The 261-residue Mycobacterium tuberculosis protein Rv0577 is a prominent antigen in tuberculosis patients, the responsible component for neutral red staining of virulent strains of M. tuberculosis, a putative component in a methylglyoxal detoxification pathway, and an agonist of toll-like receptor 2. It also has 36% sequence identity to AfsK-binding protein A (KbpA), a component in the complex secondary metabolite pathways in the Streptomycetes genus from which many commercial antibiotics are derived. To gain insight into the biological function of Rv0577 and the family of KpbA kinase regulators, the crystal structure for Rv0577 was determined to a resolution of 1.75 Åmore » (3OXH), binding properties with neutral red and deoxyadenosine (Ado) surveyed, backbone dynamics measured, and thermal stability assayed by CD spectroscopy. The protein is composed of four approximate repeats with an topology arranged radially in consecutive pairs to form two continuous eight-strand -sheets capped on both ends with an -helix. The two -sheets intersect in the center at roughly a right angle and form an asymmetric deep “saddle” on both sides of the protein, saddle one (P11 to A129) and saddle two (L143 to A258), that may serve to bind ligands. NMR chemical shift perturbation experiments show that neutral red binds to Rv0577, further cementing the role of Rv0577 in the neutral red staining of virulent strains of M. tuberculosis. Similar experiments show that adenosine also bind to Rv0577, although less tightly, with estimated dissociation constants of 4.1 ± 0.3 mM for saddle one and > 1 M for saddle two. Heteronuclear steady-state {1H}-15N NOE, T1, and T2 values were generally uniform through-out the sequence with only a few modest pockets of differences suggestive of slightly different motion in loops between -strands in saddle 1. Circular dichroism spectroscopy characterization of the thermal stability of Rv0577 indicated irreversible unfolding upon heating with an estimated melting temperature of 56 °C. While it is not known if Rv0577 has a kinase regulatory role similar to its Streptomyces homolog KbpA, protein kinase and phosphatase signaling help M. tuberculosis adapt to the hostile host environment during infections. Consequently, new anti-tuberculosis drugs targeting Rv0577 may act by interfering with multiple mechanisms; a potential signaling machinery as well as toll-like receptor 2 activation and the methylglyoxal detoxification pathway.« less

The Forest behind the Tree: Phylogenetic Exploration of a Dominant Mycobacterium tuberculosis Strain Lineage from a High Tuberculosis Burden Country

PubMed Central

Cardoso Oelemann, Maranibia; Gomes, Harrison M.; Willery, Eve; Possuelo, Lia; Batista Lima, Karla Valéria; Allix-Béguec, Caroline; Locht, Camille; Goguet de la Salmonière, Yves-Olivier L.; Gutierrez, Maria Cristina; Suffys, Philip; Supply, Philip

2011-01-01

Background Genotyping of Mycobacterium tuberculosis isolates is a powerful tool for epidemiological control of tuberculosis (TB) and phylogenetic exploration of the pathogen. Standardized PCR-based typing, based on 15 to 24 mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) loci combined with spoligotyping, has been shown to have adequate resolution power for tracing TB transmission and to be useful for predicting diverse strain lineages in European settings. Its informative value needs to be tested in high TB-burden countries, where the use of genotyping is often complicated by dominance of geographically specific, genetically homogeneous strain lineages. Methodology/Principal Findings We tested this genotyping system for molecular epidemiological analysis of 369 M. tuberculosis isolates from 3 regions of Brazil, a high TB-burden country. Deligotyping, targeting 43 large sequence polymorphisms (LSPs), and the MIRU-VNTRplus identification database were used to assess phylogenetic predictions. High congruence between the different typing results consistently revealed the countrywide supremacy of the Latin-American-Mediterranean (LAM) lineage, comprised of three main branches. In addition to an already known RDRio branch, at least one other branch characterized by a phylogenetically informative LAM3 spoligo-signature seems to be globally distributed beyond Brazil. Nevertheless, by distinguishing 321 genotypes in this strain population, combined MIRU-VNTR typing and spoligotyping demonstrated the presence of multiple distinct clones. The use of 15 to 24 loci discriminated 21 to 25% more strains within the LAM lineage, compared to a restricted lineage-specific locus set suggested to be used after SNP analysis. Noteworthy, 23 of the 28 molecular clusters identified were exclusively composed of patient isolates from a same region, consistent with expected patterns of mostly local TB transmission. Conclusions/Significance Standard MIRU-VNTR typing combined with spoligotyping can reveal epidemiologically meaningful clonal diversity behind a dominant M. tuberculosis strain lineage in a high TB-burden country and is useful to explore international phylogenetical ramifications. PMID:21464915

Prospective evaluation of a complex public health intervention: lessons from an initial and follow-up cross-sectional survey of the tuberculosis strain typing service in England.

PubMed

Mears, Jessica; Abubakar, Ibrahim; Crisp, Debbie; Maguire, Helen; Innes, John A; Lilley, Mike; Lord, Joanne; Cohen, Ted; Borgdorff, Martien W; Vynnycky, Emilia; McHugh, Timothy D; Sonnenberg, Pam

2014-10-02

The national tuberculosis strain typing service (TB-STS) was introduced in England in 2010. The TB-STS involves MIRU-VNTR typing of isolates from all TB patients for the prospective identification, reporting and investigation of TB strain typing clusters. As part of a mixed-method evaluation, we report on a repeated cross-sectional survey to illustrate the challenges surrounding the evaluation of a complex national public health intervention. An online initial and follow-up questionnaire survey assessed the knowledge, attitudes and practices of public health staff, physicians and nurses working in TB control in November 2010 and March 2012. It included questions on the implementation, experience and uptake of the TB-STS. Participants that responded to both surveys were included in the analysis. 248 participants responded to the initial survey and 137 of these responded to the follow-up survey (56% retention). Knowledge: A significant increase in knowledge was observed, including a rise in the proportion of respondents who had received training (28.6% to 67.9%, p = 0.003), and the self-rated knowledge of how to use strain typing had improved ('no knowledge' decreased from 43.2% to 27.4%). Attitudes: The majority of respondents found strain typing useful; the proportion that reported strain typing to be useful was similar across the two surveys (95.7% to 94.7%, p = 0.67). Practices: There were significant increases between the initial and follow-up surveys in the number of respondents who reported using strain typing (57.0% to 80.5%, p < 0.001) and the proportion of time health protection staff spent on investigating TB (2.74% to 7.08%, p = 0.04). Evaluation of a complex public health intervention is challenging. In this example, the immediate national roll-out of the TB-STS meant that a controlled survey design was not possible. This study informs the future development of the TB-STS by identifying the need for training to reach wider professional groups, and argues for its continuation based on service users' perception that it is useful. By highlighting the importance of a well-defined sampling frame, collecting baseline information, and including all stakeholders, it provides lessons for the implementation of similar services in other countries and future evaluations of public health interventions.

Contact Investigation in Households of Patients with Tuberculosis in Hanoi, Vietnam: A Prospective Cohort Study

PubMed Central

Fox, Gregory James; Nhung, Nguyen Viet; Sy, Dinh Ngoc; Lien, Luu Thi; Cuong, Nguyen Kim; Britton, Warwick John; Marks, Guy Barrington

2012-01-01

Setting Existing tuberculosis control strategies in Vietnam are based on symptomatic patients attending health services for investigation. This approach has not resulted in substantial reductions in the prevalence of tuberculosis disease, despite the National Tuberculosis Program achieving high treatment completion rates. Alternative approaches are being considered. Objective To determine the feasibility and yield of contact investigation in households of patients with smear positive pulmonary tuberculosis among household members of tuberculosis patients in Hanoi, Vietnam. Methods Household contacts of patients with smear positive pulmonary tuberculosis were recruited at four urban and rural District Tuberculosis Units in Hanoi. Clinical and radiological screening was conducted at baseline, six months and 12 months. Sputum microscopy and culture was performed in contacts suspected of having tuberculosis. MIRU-VNTR molecular testing was used to compare the strains of patients and their contacts with disease. Results Among 545 household contacts of 212 patients, four were diagnosed with tuberculosis at baseline (prevalence 734 cases per 100,000 persons, 95% CI 17–1451) and one was diagnosed with tuberculosis during the subsequent 12 months after initial screening (incidence 180 cases per 100,000 person-years, 95% CI 44–131). Two of these cases were culture positive for M. tuberculosis and both had identical or near-identical MIRU-VNTR strain types. Conclusion Household contacts of patients with potentially infectious forms of tuberculosis have a high prevalence of disease. Household contact investigation is feasible in Vietnam. Further research is required to investigate its effectiveness. PMID:23166785

Clinico-pathological features of tuberculosis due to Mycobacterium tuberculosis Uganda genotype in patients with tuberculous lymphadenitis: a cross sectional study.

PubMed

Wamala, Dan; Asiimwe, Benon; Kigozi, Edgar; Mboowa, Gerald; Joloba, Moses; Kallenius, Gunilla

2014-04-02

Tuberculous lymphadenitis is next to pulmonary tuberculosis as the most common cause of tuberculosis. Uganda genotype, one of the sub-lineages of Mycobacterium tuberculosis, is the most prevalent cause of pulmonary tuberculosis in Uganda. We here investigate the clinicopathological characteristics of patients with tuberculous lymphadenitis infected with M. tuberculosis Uganda genotype compared with those infected with M. tuberculosis non-Uganda genotype strains. Between 2010 and 2012, we enrolled 121 patients (mean age 28.5 yrs, male 48%; female 52%) with tuberculous lymphadenitis, and categorized them by their M. tuberculosis genotypes. The clinical features and lymph node cytopathological parameters were compared between patients in the Uganda and non-Uganda categories using a crude and multivariable logistic regression model with adjustment for confounding factors. Of the 121participants, 56 (46%) were infected with strains of Uganda genotype. Patients infected with this genotype had significantly lower frequency of abdominal lymphadenopathy (odds ratio 0.4, p = 0.046) after adjusting for sex, age and HIV. Abdominal lymphadenopathy was also significantly associated with abnormal chest X-ray (p = 0.027). Tuberculous lymphadenitis patients infected with M. tuberculosis Uganda genotype were significantly less prone to have abdominal lymphadenopathy indicating potential reduced ability to disseminate and supporting the concept that differences in M. tuberculosis genotype may have clinical implications.

Sources of bovine tuberculosis in the United States.

PubMed

Tsao, Kimberly; Robbe-Austerman, Suelee; Miller, Ryan S; Portacci, Katie; Grear, Daniel A; Webb, Colleen

2014-12-01

Despite control and eradication efforts, bovine tuberculosis continues to be identified at low levels among cattle in the United States. We evaluated possible external sources of infection by characterizing the genetic relatedness of bovine tuberculosis from a national database of reported infections, comparing strains circulating among US cattle with those of imported cattle, and farmed and wild cervids. Farmed cervids maintained a genetically distinct Mycobacterium bovis strain, and cattle occasionally became infected with this strain. In contrast, wild cervids acted as an epidemiologically distinct group, instead hosting many of the same strains found in cattle, and the data did not show a clear transmission direction. Cattle from Mexico hosted a higher overall richness of strains than US cattle, and many of those strains were found in both US and Mexican cattle. However, these two populations appeared to be well-mixed with respect to their M. bovis lineages, and higher resolution data is necessary to infer the direction of recent transmission. Overall patterns of both host and geographic distributions were highly variable among strains, suggesting that different sources or transmission mechanisms are contributing to maintaining different strains. Copyright © 2014 Elsevier B.V. All rights reserved.

Mycolic acids: deciphering and targeting the Achilles' heel of the tubercle bacillus

PubMed Central

Nataraj, Vijayashankar; Varela, Cristian; Javid, Asma; Singh, Albel; Besra, Gurdyal S.

2015-01-01

Summary Mycolic acids are unique long chain fatty acids found in the lipid‐rich cell walls of mycobacteria including the tubercle bacillus M ycobacterium tuberculosis. Essential for viability and virulence, enzymes involved in the biosynthesis of mycolic acids represent novel targets for drug development. This is particularly relevant to the impact on global health given the rise of multidrug resistant and extensively drug resistant strains of M . tuberculosis. In this review, we discuss recent advances in our understanding of how mycolic acid are synthesised, especially the potential role of specialised fatty acid synthase complexes. Also, we examine the role of a recently reported mycolic acid transporter MmpL3 with reference to several reports of the targeting of this transporter by diverse compounds with anti‐M . tuberculosis activity. Additionally, we consider recent findings that place mycolic acid biosynthesis in the context of the cell biology of the bacterium, viz its localisation and co‐ordination with the bacterial cytoskeleton, and its role beyond maintaining cell envelope integrity. PMID:26135034

High-throughput identification and dendritic cell-based functional validation of MHC class I-restricted Mycobacterium tuberculosis epitopes

PubMed Central

Nair, Smita K.; Tomaras, Georgia D.; Sales, Ana Paula; Boczkowski, David; Chan, Cliburn; Plonk, Kelly; Cai, Yongting; Dannull, Jens; Kepler, Thomas B.; Pruitt, Scott K.; Weinhold, Kent J.

2014-01-01

Emergence of drug-resistant strains of the pathogen Mycobacterium tuberculosis (Mtb) and the ineffectiveness of BCG in curtailing Mtb infection makes vaccine development for tuberculosis an important objective. Identifying immunogenic CD8+ T cell peptide epitopes is necessary for peptide-based vaccine strategies. We present a three-tiered strategy for identifying and validating immunogenic peptides: first, identify peptides that form stable complexes with class I MHC molecules; second, determine whether cytotoxic T lymphocytes (CTLs) raised against the whole protein antigen recognize and lyse target cells pulsed with peptides that passed step 1; third, determine whether peptides that passed step 2, when administered in vivo as a vaccine in HLA-A2 transgenic mice, elicit CTLs that lyse target cells expressing the whole protein antigen. Our innovative approach uses dendritic cells transfected with Mtb antigen-encoding mRNA to drive antigen expression. Using this strategy, we have identified five novel peptide epitopes from the Mtb proteins Apa, Mtb8.4 and Mtb19. PMID:24755960

Antimicrobial Susceptibility Testing of Mycobacterium bovis Isolates from Michigan White-Tailed Deer during the 2009 Hunting Season.

PubMed

Fitzgerald, Scott D; Schooley, Angie M; Berry, Dale E; Kaneene, John B

2010-12-02

Michigan has had an ongoing outbreak of endemic Mycobacterium bovis which has been recognized within and sustained by its free-ranging white-tailed deer population since 1994. Worldwide, organisms within the Mycobacterium tuberculosis complex have exhibited the ability to develop resistance to antimicrobial agents, resulting in both the multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of human tuberculosis. Michigan's Bovine Tuberculosis Working Group has conducted active antimicrobial susceptibility testing on wildlife isolates of the endemic M. bovis organism at five-year intervals to detect any emerging drug resistance patterns. The results of 33 white-tailed deer origin isolates collected from the 2009 hunting season are reported here. There continues to be no evidence of any drug resistance except for pyrazinamide resistance. These results are likely due to the lack of antibacterial treatment applied to either wildlife or domestic animals which would provide selection pressure for the development of drug resistance.

Antigen 85C Inhibition Restricts Mycobacterium tuberculosis Growth through Disruption of Cord Factor Biosynthesis

PubMed Central

Warrier, Thulasi; Tropis, Marielle; Werngren, Jim; Diehl, Anne; Gengenbacher, Martin; Schlegel, Brigitte; Schade, Markus; Oschkinat, Hartmut; Daffe, Mamadou; Hoffner, Sven; Eddine, Ali Nasser

2012-01-01

The antigen 85 (Ag85) protein family, consisting of Ag85A, -B, and -C, is vital for Mycobacterium tuberculosis due to its role in cell envelope biogenesis. The mycoloyl transferase activity of these proteins generates trehalose dimycolate (TDM), an envelope lipid essential for M. tuberculosis virulence, and cell wall arabinogalactan-linked mycolic acids. Inhibition of these enzymes through substrate analogs hinders growth of mycobacteria, but a link to mycolic acid synthesis has not been established. In this study, we characterized a novel inhibitor of Ag85C, 2-amino-6-propyl-4,5,6,7-tetrahydro-1-benzothiophene-3-carbonitrile (I3-AG85). I3-AG85 was isolated from a panel of four inhibitors that exhibited structure- and dose-dependent inhibition of M. tuberculosis division in broth culture. I3-AG85 also inhibited M. tuberculosis survival in infected primary macrophages. Importantly, it displayed an identical MIC against the drug-susceptible H37Rv reference strain and a panel of extensively drug-resistant/multidrug-resistant M. tuberculosis strains. Nuclear magnetic resonance analysis indicated binding of I3-AG85 to Ag85C, similar to its binding to the artificial substrate octylthioglucoside. Quantification of mycolic acid-linked lipids of the M. tuberculosis envelope showed a specific blockade of TDM synthesis. This was accompanied by accumulation of trehalose monomycolate, while the overall mycolic acid abundance remained unchanged. Inhibition of Ag85C activity also disrupted the integrity of the M. tuberculosis envelope. I3-AG85 inhibited the division of and reduced TDM synthesis in an M. tuberculosis strain deficient in Ag85C. Our results indicate that Ag85 proteins are promising targets for novel antimycobacterial drug design. PMID:22290959

Epidemiologic Consequences of Microvariation in Mycobacterium tuberculosis

PubMed Central

Mathema, Barun; Kurepina, Natalia; Yang, Guibin; Shashkina, Elena; Manca, Claudia; Mehaffy, Carolina; Bielefeldt-Ohmann, Helle; Ahuja, Shama; Fallows, Dorothy A.; Izzo, Angelo; Bifani, Pablo; Dobos, Karen; Kaplan, Gilla

2012-01-01

Background. Evidence from genotype-phenotype studies suggests that genetic diversity in pathogens have clinically relevant manifestations that can impact outcome of infection and epidemiologic success. We studied 5 closely related Mycobacterium tuberculosis strains that collectively caused extensive disease (n = 862), particularly among US-born tuberculosis patients. Methods. Representative isolates were selected using population-based genotyping data from New York City and New Jersey. Growth and cytokine/chemokine response were measured in infected human monocytes. Survival was determined in aerosol-infected guinea pigs. Results. Multiple genotyping methods and phylogenetically informative synonymous single nucleotide polymorphisms showed that all strains were related by descent. In axenic culture, all strains grew similarly. However, infection of monocytes revealed 2 growth phenotypes, slower (doubling ∼55 hours) and faster (∼25 hours). The faster growing strains elicited more tumor necrosis factor α and interleukin 1β than the slower growing strains, even after heat killing, and caused accelerated death of infected guinea pigs (∼9 weeks vs 24 weeks) associated with increased lung inflammation/pathology. Epidemiologically, the faster growing strains were associated with human immunodeficiency virus and more limited in spread, possibly related to their inherent ability to induce a strong protective innate immune response in immune competent hosts. Conclusions. Natural variation, with detectable phenotypic changes, among closely related clinical isolates of M. tuberculosis may alter epidemiologic patterns in human populations. PMID:22315279

Microevolution of the Direct Repeat Locus of Mycobacterium tuberculosis in a Strain Prevalent in San Francisco

PubMed Central

Aga, Roxanne S.; Fair, Elizabeth; Abernethy, Neil F.; DeRiemer, Kathryn; Paz, E. Antonio; Kawamura, L. Masae; Small, Peter M.; Kato-Maeda, Midori

2006-01-01

We describe a microevolutionary event of a prevalent strain of Mycobacterium tuberculosis that caused two outbreaks in San Francisco. During the second outbreak, a direct variable repeat was lost. We discuss the mechanisms of this change and the implications of analyzing multiple genetic loci in this context. PMID:16597893

Correlates between Models of Virulence for Mycobacterium tuberculosis among Isolates of the Central Asian Lineage: a Case for Lysozyme Resistance Testing?

PubMed Central

Casali, Nicola; Clark, Simon O.; Hooper, Richard; Williams, Ann; Velji, Preya; Gonzalo, Ximena

2015-01-01

Virulence factors (VFs) contribute to the emergence of new human Mycobacterium tuberculosis strains, are lineage dependent, and are relevant to the development of M. tuberculosis drugs/vaccines. VFs were sought within M. tuberculosis lineage 3, which has the Central Asian (CAS) spoligotype. Three isolates were selected from clusters previously identified as dominant in London, United Kingdom. Strain-associated virulence was studied in guinea pig, monocyte-derived macrophage, and lysozyme resistance assays. Whole-genome sequencing, single nucleotide polymorphism (SNP) analysis, and a literature review contributed to the identification of SNPs of interest. The animal model revealed borderline differences in strain-associated pathogenicity. Ex vivo, isolate C72 exhibited statistically significant differences in intracellular growth relative to C6 and C14. SNP candidates inducing lower fitness levels included 123 unique nonsynonymous SNPs, including three located in genes (lysX, caeA, and ponA2) previously identified as VFs in the laboratory-adapted reference strain H37Rv and shown to confer lysozyme resistance. C72 growth was most affected by lysozyme in vitro. A BLAST search revealed that all three SNPs of interest (C35F, P76Q, and P780R) also occurred in Tiruvallur, India, and in Uganda. Unlike C72, however, no single isolate identified through BLAST carried all three SNPs simultaneously. CAS isolates representative of three medium-sized human clusters demonstrated differential outcomes in models commonly used to estimate strain-associated virulence, supporting the idea that virulence varies within, not just across, M. tuberculosis lineages. Three VF SNPs of interest were identified in two additional locations worldwide, which suggested independent selection and supported a role for these SNPs in virulence. The relevance of lysozyme resistance to strain virulence remains to be established. PMID:25776753

Characterization of Mycobacterium tuberculosis isolates from Hebei, China: genotypes and drug susceptibility phenotypes.

PubMed

Li, Yanan; Cao, Xinrui; Li, Shiming; Wang, Hao; Wei, Jianlin; Liu, Peng; Wang, Jing; Zhang, Zhi; Gao, Huixia; Li, Machao; Wan, Kanglin; Dai, Erhei

2016-03-03

Tuberculosis remains a major public health problem in China. The Hebei province is located in the Beijing-Tianjin-Hebei integration region; however little information about the genetic diversity of Mycobacterium tuberculosis was available in this area. This study describes the first attempt to map the molecular epidemiology of MTB strains isolated from Hebei. Spoligotyping and 15-locus MIRU-VNTR were performed in combination to yield specific genetic profiles of 1017 MTB strains isolated from ten cities in the Hebei province in China during 2014. Susceptibility testing to first line anti-TB drugs was also conducted for all strains using the L-J proportion method. Based on the SpolDB4.0 database, the predominant spoligotype belonged to the Beijing family (90.5%), followed by T family (6.3%). Using 15-locus MIRU-VNTR clustering analysis, 846 different patterns were identified, including 84 clusters (2-17 strains per cluster) and 764 individual types. Drug susceptibility pattern showed that 347 strains (34.1%) were resistant to at least one of the first line drugs, including 134 (13.2%) multi-drug resistance strains. Statistical analysis indicated that drug resistance was associated with treatment history. The Beijing family was associated with genetic clustering. However, no significant difference was observed between the Beijing and non-Beijing family in gender, age, treatment history and drug resistance. The Mycobacterium tuberculosis strains in Hebei exhibit high genetic diversity. The Beijing family is the most prevalent lineage in this area. Spoligotyping in combination with 15-locus MIRU-VNTR is a useful tool to study the molecular epidemiology of the MTB strains in Hebei.

The Association between Mycobacterium Tuberculosis Genotype and Drug Resistance in Peru

PubMed Central

Grandjean, Louis; Iwamoto, Tomotada; Lithgow, Anna; Gilman, Robert H; Arikawa, Kentaro; Nakanishi, Noriko; Martin, Laura; Castillo, Edith; Alarcon, Valentina; Coronel, Jorge; Solano, Walter; Aminian, Minoo; Guezala, Claudia; Rastogi, Nalin; Couvin, David; Sheen, Patricia; Zimic, Mirko; Moore, David AJ

2015-01-01

Background The comparison of Mycobacterium tuberculosis bacterial genotypes with phenotypic, demographic, geospatial and clinical data improves our understanding of how strain lineage influences the development of drug-resistance and the spread of tuberculosis. Methods To investigate the association of Mycobacterium tuberculosis bacterial genotype with drug-resistance. Drug susceptibility testing together with genotyping using both 15-loci MIRU-typing and spoligotyping, was performed on 2,139 culture positive isolates, each from a different patient in Lima, Peru. Demographic, geospatial and socio-economic data were collected using questionnaires, global positioning equipment and the latest national census. Results The Latin American Mediterranean (LAM) clade (OR 2.4, p<0.001) was significantly associated with drug-resistance and alone accounted for more than half of all drug resistance in the region. Previously treated patients, prisoners and genetically clustered cases were also significantly associated with drug-resistance (OR's 2.5, 2.4 and 1.8, p<0.001, p<0.05, p<0.001 respectively). Conclusions Tuberculosis disease caused by the LAM clade was more likely to be drug resistant independent of important clinical, genetic and socio-economic confounding factors. Explanations for this include; the preferential co-evolution of LAM strains in a Latin American population, a LAM strain bacterial genetic background that favors drug-resistance or the "founder effect" from pre-existing LAM strains disproportionately exposed to drugs. PMID:25984723

Tuberculosis: An Inorganic Medicinal Chemistry Perspective.

PubMed

Viganor, Livia; Skerry, Ciaran; McCann, Malachy; Devereux, Michael

2015-01-01

Tuberculosis (TB) which is caused by the resilient pathogen Mycobacterium tuberculosis (MTB) has re-emerged to become a leading public health problem in the world. The growing number of multi-drug resistant MTB strains and the more recently emerging problem with the extensively drug resistant strains of the pathogen are greatly undermining conventional anti-TB therapeutic strategies which are lengthy and expose patients to toxicity and other unwanted side effects. The search for new anti-TB drugs essentially involves either the repurposing of existing organic drugs which are now off patent and already FDA approved, the synthesis of modified analogues of existing organic drugs, with the aim of shortening and improving drug treatment for the disease, or the search for novel structures that offer the possibility of new mechanisms of action against the mycobacterium. Inorganic medicinal chemistry offers an alternative to organic drugs through opportunities for the design of therapeutics that target different biochemical pathways. The incorporation of metal ions into the molecular structure of a potential drug offers the medicinal chemist an opportunity to exploit structural diversity, have access to various oxidation states of the metal and also offer the possibility of enhancing the activity of an established organic drug through its coordination to the metal centre. In this review, we summarize what is currently known about the antitubercular capability of metal complexes, their mechanisms of action and speculate on their potential applications in the clinic.

Performance Assessment of the CapitalBio Mycobacterium Identification Array System for Identification of Mycobacteria

PubMed Central

Liu, Jingbo; Yan, Zihe; Han, Min; Han, Zhijun; Jin, Lingjie; Zhao, Yanlin

2012-01-01

The CapitalBio Mycobacterium identification microarray system is a rapid system for the detection of Mycobacterium tuberculosis. The performance of this system was assessed with 24 reference strains, 486 Mycobacterium tuberculosis clinical isolates, and 40 clinical samples and then compared to the “gold standard” of DNA sequencing. The CapitalBio Mycobacterium identification microarray system showed highly concordant identification results of 100% and 98.4% for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM), respectively. The sensitivity and specificity of the CapitalBio Mycobacterium identification array for identification of Mycobacterium tuberculosis isolates were 99.6% and 100%, respectively, for direct detection and identification of clinical samples, and the overall sensitivity was 52.5%. It was 100% for sputum, 16.7% for pleural fluid, and 10% for bronchoalveolar lavage fluid, respectively. The total assay was completed in 6 h, including DNA extraction, PCR, and hybridization. The results of this study confirm the utility of this system for the rapid identification of mycobacteria and suggest that the CapitalBio Mycobacterium identification array is a molecular diagnostic technique with high sensitivity and specificity that has the capacity to quickly identify most mycobacteria. PMID:22090408

The Structure of the Anti-tuberculosis Antibiotics Viomycin and Capreomycin Bound to the 70S Ribosome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stanley, R.; Blaha, G; Grodzicki, R

2010-01-01

Viomycin and capreomycin belong to the tuberactinomycin family of antibiotics, which are among the most effective antibiotics against multidrug-resistant tuberculosis. Here we present two crystal structures of the 70S ribosome in complex with three tRNAs and bound to either viomycin or capreomycin at 3.3- and 3.5-{angstrom} resolution, respectively. Both antibiotics bind to the same site on the ribosome, which lies at the interface between helix 44 of the small ribosomal subunit and helix 69 of the large ribosomal subunit. The structures of these complexes suggest that the tuberactinomycins inhibit translocation by stabilizing the tRNA in the A site in themore » pretranslocation state. In addition, these structures show that the tuberactinomycins bind adjacent to the binding sites for the paromomycin and hygromycin B antibiotics, which may enable the development of new derivatives of tuberactinomycins that are effective against drug-resistant strains.« less

The structures of the anti-tuberculosis antibiotics viomycin and capreomycin bound to the 70S ribosome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stanley, Robin E.; Blaha, Gregor; Grodzicki, Robert L.

2010-05-03

Viomycin and capreomycin belong to the tuberactinomycin family of antibiotics, which are among the most effective antibiotics against multidrug-resistant tuberculosis. Here we present two crystal structures of the 70S ribosome in complex with three tRNAs and bound to either viomycin or capreomycin at 3.3- and 3.5-{angstrom} resolution, respectively. Both antibiotics bind to the same site on the ribosome, which lies at the interface between helix 44 of the small ribosomal subunit and helix 69 of the large ribosomal subunit. The structures of these complexes suggest that the tuberactinomycins inhibit translocation by stabilizing the tRNA in the A site in themore » pretranslocation state. In addition, these structures show that the tuberactinomycins bind adjacent to the binding sites for the paromomycin and hygromycin B antibiotics, which may enable the development of new derivatives of tuberactinomycins that are effective against drug-resistant strains.« less

Progenitor “Mycobacterium canettii” clone responsible for lymph node tuberculosis epidemic, Djibouti.

PubMed

Blouin, Yann; Cazajous, Géraldine; Dehan, Céline; Soler, Charles; Vong, Rithy; Hassan, Mohamed Osman; Hauck, Yolande; Boulais, Christian; Andriamanantena, Dina; Martinaud, Christophe; Martin, Émilie; Pourcel, Christine; Vergnaud, Gilles

2014-01-01

“Mycobacterium canettii,” an opportunistic human pathogen living in an unknown environmental reservoir, is the progenitor species from which Mycobacterium tuberculosis emerged. Since its discovery in 1969, most of the ≈70 known M. canettii strains were isolated in the Republic of Djibouti, frequently from expatriate children and adults. We show here, by whole-genome sequencing, that most strains collected from February 2010 through March 2013, and associated with 2 outbreaks of lymph node tuberculosis in children, belong to a unique epidemic clone within M. canettii. Evolution of this clone, which has been recovered regularly since 1983, may mimic the birth of M. tuberculosis. Thus, recognizing this organism and identifying its reservoir are clinically important.

Progenitor “Mycobacterium canettii” Clone Responsible for Lymph Node Tuberculosis Epidemic, Djibouti

PubMed Central

Blouin, Yann; Cazajous, Géraldine; Dehan, Céline; Soler, Charles; Vong, Rithy; Hassan, Mohamed Osman; Hauck, Yolande; Boulais, Christian; Andriamanantena, Dina; Martinaud, Christophe; Martin, Émilie; Pourcel, Christine

2014-01-01

“Mycobacterium canettii,” an opportunistic human pathogen living in an unknown environmental reservoir, is the progenitor species from which Mycobacterium tuberculosis emerged. Since its discovery in 1969, most of the ≈70 known M. canettii strains were isolated in the Republic of Djibouti, frequently from expatriate children and adults. We show here, by whole-genome sequencing, that most strains collected from February 2010 through March 2013, and associated with 2 outbreaks of lymph node tuberculosis in children, belong to a unique epidemic clone within M. canettii. Evolution of this clone, which has been recovered regularly since 1983, may mimic the birth of M. tuberculosis. Thus, recognizing this organism and identifying its reservoir are clinically important. PMID:24520560

Contribution of spoligotyping and MIRU-VNTRs to characterize prevalent Mycobacterium tuberculosis genotypes infecting tuberculosis patients in Morocco.

PubMed

Chaoui, Imane; Zozio, Thierry; Lahlou, Ouafae; Sabouni, Radia; Abid, Mohammed; El Aouad, Rajae; Akrim, Mohammed; Amzazi, Said; Rastogi, Nalin; El Mzibri, Mohammed

2014-01-01

In the present study, Mycobacterium tuberculosis complex (MTBC) clinical isolates from culture-positive TB patients in Morocco were studied by spoligotyping and 12-loci MIRU-VNTR typing methods to characterize prevalent genotypes (n = 219 isolates from 208 patients). Spoligotyping resulted in 39 unique patterns and 167 strains in 30 clusters (2-50 strains per cluster). Comparison with international database showed that 29 of 39 unique patterns matched existing shared spoligotype international types (SITs). Nine shared types containing 10 strains were newly created (SIT 2891 to SIT 2899); this led to the description of 69 SITs with 206 strains and two orphan patterns. The most prevalent spoligotype was SIT42 (LAM; n = 50 or 24% of isolates). The repartition of strains according to major MTBC clades was as follows LAM (46.1%)> Haarlem (26%) >ill-defined T superfamily (22.6%) and S clade (0.96%). On the other hand, Beijing, CAS (Central Asian) and EAI (East-African Indian) strains were absent in this setting. Subsequent 12-Loci MIRU typing resulted in a total of 25 SIT/MIT clusters (n = 66 isolates, 2-6 isolates per cluster), with a resulting recent transmission rate of 22.3%. The MIRU-VNTR patterns corresponded to 69 MITs for 138 strains and 46 orphan patterns. The most frequent patterns were MIT43 (n = 8), MIT9 (n = 7) and MIT42 (n = 7). HGDI analysis of the 12 MIRU loci showed that loci 10, 23 and 40 were highly discriminative in our setting. The results also underlined the usefulness of spoligotyping and MIRU-VNTR to detect mixed infections among certain of our TB patients. Globally, the results obtained showed that TB is almost exclusively transmitted in Morocco through evolutionary-modern MTBC lineages belonging to principal genetic groups 2/3 strains (Haarlem, LAM, T), with a high level of biodiversity seen by MIRU typing. This study provides with a 1st global snapshot of MTBC population structure in Morocco, and validates the potential use of spoligotyping in conjunction with minisatellites for future investigations in Morocco that should in future ideally include optimized 15- or 24-loci MIRU-VNTRs. Copyright © 2013 Elsevier B.V. All rights reserved.

FIND Tuberculosis Strain Bank: a Resource for Researchers and Developers Working on Tests To Detect Mycobacterium tuberculosis and Related Drug Resistance.

PubMed

Tessema, Belay; Nabeta, Pamela; Valli, Eloise; Albertini, Audrey; Collantes, Jimena; Lan, Nguyen Huu; Romancenco, Elena; Tukavdze, Nestani; Denkinger, Claudia M; Dolinger, David L

2017-04-01

The spread of multidrug-resistant (MDR) tuberculosis (TB) and extensively drug-resistant (XDR) TB hampers global efforts in the fight against tuberculosis. To enhance the development and evaluation of diagnostic tests quickly and efficiently, well-characterized strains and samples from drug-resistant tuberculosis patients are necessary. In this project, the Foundation for Innovative New Diagnostics (FIND) has focused on the collection, characterization, and storage of such well-characterized reference materials and making them available to researchers and developers. The collection is being conducted at multiple centers in Southeast Asia, South America, Eastern Europe, and soon the sub-Saharan Africa regions. Strains are characterized for their phenotypic resistances and MICs to first-line drugs (FLDs) and second-line drugs (SLDs) using the automated MGIT 960 system following validated procedures and WHO criteria. Analysis of resistance-associated mutations is done by whole-genome sequencing (WGS) using the Illumina NextSeq system. Mycobacterial interspersed repetitive-unit-variable-number tandem-repeat analysis and WGS are used to determine strain lineages. All strains are maintained frozen at -80°C ± 10°C as distinct mother and daughter lots. All strains are extensively quality assured. The data presented here represent an analysis of the initial part of the collection. Currently, the bank contains 118 unique strains with extracted genomic DNA and matched sputum, serum, and plasma samples and will be expanded to a minimum of 1,000 unique strains over the next 3 years. Analysis of the current strains by phenotypic resistance testing shows 102 (86.4%), 10 (8.5%), and 6 (5.1%) MDR, XDR, and mono/poly resistant strains, respectively. Two of the strains are resistant to all 11 drugs that were phenotypically tested. WGS mutation analysis revealed FLD resistance-associated mutations in the rpoB , katG , inhA , embB , embA , and pncA genes; SLD resistance in the gyrA , gyrB , rrs , eis , and tlyA genes; and ethionamide resistance in the ethA genes. Most important lineages are represented in the bank, and further collections have been initiated to increase geographic and lineage diversity. The bank provides highly characterized and high-quality strains as a resource for researchers and developers in support of the development and evaluation of new diagnostics and drug resistance detection tools. Copyright © 2017 Tessema et al.

Mycobacterium tuberculosis infection in cattle from the Eastern Cape Province of South Africa.

PubMed

Hlokwe, Tiny Motlatso; Said, Halima; Gcebe, Nomakorinte

2017-10-10

Mycobacterium tuberculosis is the main causative agent of tuberculosis (TB) in human and Mycobacterium bovis commonly causes tuberculosis in animals. Transmission of tuberculosis caused by both pathogens can occur from human to animals and vice versa. In the current study, M. tuberculosis, as confirmed by polymerase chain reaction (PCR) using primers targeting 3 regions of difference (RD4, RD9 and RD12) on the genomes, was isolated from cattle originating from two epidemiologically unrelated farms in the Eastern Cape (E.C) Province of South Africa. Although the isolates were genotyped with variable number of tandem repeat (VNTR) typing, no detailed epidemiological investigation was carried out on the respective farms to unequivocally confirm or link humans as sources of TB transmission to cattle, a move that would have embraced the 'One Health' concept. In addition, strain comparison with human M. tuberculosis in the database from the E.C Province and other provinces in the country did not reveal any match. This is the first report of cases of M. tuberculosis infection in cattle in South Africa. The VNTR profiles of the M. tuberculosis strains identified in the current study will form the basis for creating M. tuberculosis VNTR database for animals including cattle for future epidemiological studies. Our findings however, call for urgent reinforcement of collaborative efforts between the veterinary and the public health services of the country.

Mycobacterium tuberculosis resistance to antituberculosis drugs in Mozambique*, **

PubMed Central

Pires, Germano Manuel; Folgosa, Elena; Nquobile, Ndlovu; Gitta, Sheba; Cadir, Nureisha

2014-01-01

OBJECTIVE: To determine the drug resistance profile of Mycobacterium tuberculosis in Mozambique. METHODS: We analyzed secondary data from the National Tuberculosis Referral Laboratory, in the city of Maputo, Mozambique, and from the Beira Regional Tuberculosis Referral Laboratory, in the city of Beira, Mozambique. The data were based on culture-positive samples submitted to first-line drug susceptibility testing (DST) between January and December of 2011. We attempted to determine whether the frequency of DST positivity was associated with patient type or provenance. RESULTS: During the study period, 641 strains were isolated in culture and submitted to DST. We found that 374 (58.3%) were resistant to at least one antituberculosis drug and 280 (43.7%) were resistant to multiple antituberculosis drugs. Of the 280 multidrug-resistant tuberculosis cases, 184 (65.7%) were in previously treated patients, most of whom were from southern Mozambique. Two (0.71%) of the cases of multidrug-resistant tuberculosis were confirmed to be cases of extensively drug-resistant tuberculosis. Multidrug-resistant tuberculosis was most common in males, particularly those in the 21-40 year age bracket. CONCLUSIONS: M. tuberculosis resistance to antituberculosis drugs is high in Mozambique, especially in previously treated patients. The frequency of M. tuberculosis strains that were resistant to isoniazid, rifampin, and streptomycin in combination was found to be high, particularly in samples from previously treated patients. PMID:24831398

Comparative 'omics analyses differentiate Mycobacterium tuberculosis and Mycobacterium bovis and reveal distinct macrophage responses to infection with the human and bovine tubercle bacilli

PubMed Central

Malone, Kerri M.; Rue-Albrecht, Kévin; Magee, David A.; Conlon, Kevin; Schubert, Olga T.; Nalpas, Nicolas C.; Browne, John A.; Smyth, Alicia; Gormley, Eamonn; Aebersold, Ruedi; MacHugh, David E.; Gordon, Stephen V.

2018-01-01

Members of the Mycobacterium tuberculosis complex (MTBC) are the causative agents of tuberculosis in a range of mammals, including humans. A key feature of MTBC pathogens is their high degree of genetic identity yet distinct host tropism. Notably, while Mycobacterium bovis is highly virulent and pathogenic for cattle, the human pathogen M. tuberculosis is attenuated in cattle. Previous research also suggests that host preference amongst MTBC members has a basis in host innate immune responses. To explore MTBC host tropism, we present in-depth profiling of the MTBC reference strains M. bovis AF2122/97 and M. tuberculosis H37Rv at both the global transcriptional and the translational level via RNA-sequencing and SWATH MS. Furthermore, a bovine alveolar macrophage infection time course model was used to investigate the shared and divergent host transcriptomic response to infection with M. tuberculosis H37Rv or M. bovis AF2122/97. Significant differential expression of virulence-associated pathways between the two bacilli was revealed, including the ESX-1 secretion system. A divergent transcriptional response was observed between M. tuberculosis H37Rv and M. bovis AF2122/97 infection of bovine alveolar macrophages, in particular cytosolic DNA-sensing pathways at 48 h post-infection, and highlights a distinct engagement of M. bovis with the bovine innate immune system. The work presented here therefore provides a basis for the identification of host innate immune mechanisms subverted by virulent host-adapted mycobacteria to promote their survival during the early stages of infection. PMID:29557774

Synergism between ethanolic extract of propolis (EEP) and anti-tuberculosis drugs on growth of mycobacteria.

PubMed

Scheller, S; Dworniczak, S; Waldemar-Klimmek, K; Rajca, M; Tomczyk, A; Shani, J

1999-01-01

Ethanolic extract of propolis exerts a strong anti-bacterial activity, in addition to antifungal, antiviral and antiprotozoal properties. In previous studies from these laboratories we have demonstrated that the intensity of the bactericidal activity of EEP is correlated with the virulence of the mycobacteria tested, and that EEP has a synergistic effect with antibiotics on growth of staphylococcus aureus. In the present study we investigated whether the same synergism and correlation exists between EEP and some anti-tuberculosis drugs on tuberculosis mycobacteria with different degrees of virulence. Six standard strains and 11 wild strains of mycobacteria were exposed for 30 days to EEP, with or without streptomycin, rifamycin, isoniazid or ethambutol. Out of the 17 strains, 8 were resistant to at least two standard antibiotics, and were considered "multi-resistant strains". The rest were either susceptible or resistant to only one of the antimycobacterial drugs. Antagonism was recorded only in one case, when Staphylococcus aureus were treated with a mixture of EEP and ethambutol, suggesting that a chemical bond could have been formed between this anti-tuberculosis antibiotic and one of the active components of the ethanol extract of propolis.

Evaluation of phage assay for rapid phenotypic detection of rifampicin resistance in Mycobacterium tuberculosis

PubMed Central

Yzquierdo, Sergio Luis; Lemus, Dihadenys; Echemendia, Miguel; Montoro, Ernesto; McNerney, Ruth; Martin, Anandi; Palomino, Juan Carlos

2006-01-01

Background Conventional methods for susceptibility testing require several months before results can be reported. However, rapid methods to determine drug susceptibility have been developed recently. Phage assay have been reported as a rapid useful tools for antimicrobial susceptibility testing. The aim of this study was to apply the Phage assay for rapid detection of resistance on Mycobacterium tuberculosis strains in Cuba. Methods Phage D29 assay was performed on 102 M. tuberculosis strains to detect rifampicin resistance. The results were compared with the proportion method (gold standard) to evaluate the sensitivity and specificity of Phage assay. Results Phage assay results were available in 2 days whereas Proportion Methods results were obtain in 42 days. A total of 44 strains were detected as rifampicin resistant by both methods. However, one strains deemed resistant by Proportion Methods was susceptible by Phage assay. The sensitivity and specificity of Phage assay were 97.8 % and 100% respectively. Conclusion Phage assay provides rapid and reliable results for susceptibility testing; it's easy to perform, requires no specialized equipment and is applicable to drug susceptibility testing in low income countries where tuberculosis is a major public health problem. PMID:16630356

Mycobacterium tuberculosis Population in Northwestern Russia: An Update from Russian-EU/Latvian Border Region

PubMed Central

Mokrousov, Igor; Vyazovaya, Anna; Otten, Tatiana; Zhuravlev, Viacheslav; Pavlova, Elena; Tarashkevich, Larisa; Krishevich, Valery; Vishnevsky, Boris; Narvskaya, Olga

2012-01-01

This study aimed to characterize the population structure of Mycobacterium tuberculosis in Pskov oblast in northwestern Russia, to view it in the geographical context, to compare drug resistance properties across major genetic families. Ninety M. tuberculosis strains from tuberculosis (TB) patients, permanent residents in Pskov oblast were subjected to LAM-specific IS6110-PCR and spoligotyping, followed by comparison with SITVITWEB and MIRU-VNTRplus databases. The Beijing genotype (n = 40) was found the most prevalent followed by LAM (n = 18), T (n = 13), Haarlem (n = 10), Ural (n = 5), and Manu2 (n = 1); the family status remained unknown for 3 isolates. The high rate of Beijing genotype and prevalence of LAM family are similar to those in the other Russian settings. A feature specific for M. tuberculosis population in Pskov is a relatively higher rate of Haarlem and T types. Beijing strains were further typed with 12-MIRU (followed by comparison with proprietary global database) and 3 hypervariable loci QUB-3232, VNTR-3820, VNTR-4120. The 12-MIRU typing differentiated 40 Beijing strains into 14 types (HGI = 0.82) while two largest types were M2 (223325153533) prevalent throughout former USSR and M11 (223325173533) prevalent in Russia and East Asia. The use of 3 hypervariable loci increased a discrimination of the Beijing strains (18 profiles, HGI = 0.89). Both major families Beijing and LAM had similar rate of MDR strains (62.5 and 55.6%, respectively) that was significantly higher than in other strains (21.9%; P = 0.001 and 0.03, respectively). The rpoB531 mutations were more frequently found in Beijing strains while LAM drug resistant strains mainly harbored rpoB516 and inhA −15 mutations. Taken together with a high rate of multidrug resistance among Beijing strains from new TB cases (79.3% versus 44.4% in LAM), these findings suggest the critical impact of the Beijing genotype on the current situation with MDR-TB in the Pskov region in northwestern Russia. PMID:22844457

Hyperendemic pulmonary tuberculosis in peri-urban areas of Karachi, Pakistan

PubMed Central

Akhtar, Saeed; White, Franklin; Hasan, Rumina; Rozi, Shafquat; Younus, Mohammad; Ahmed, Faiza; Husain, Sara; Khan, Bilquis Sana

2007-01-01

Background Currently there are very limited empirical data available on the prevalence of pulmonary tuberculosis among residents of marginalized settings in Pakistan. This study assessed the prevalence of pulmonary tuberculosis through active case detection and evaluated predictors of pulmonary tuberculosis among residents of two peri-urban neighbourhoods of Karachi, Pakistan. Methods A cross-sectional study was conducted in two peri-urban neighbourhoods from May 2002 to November 2002. Systematic sampling design was used to select households for inclusion in the study. Consenting subjects aged 15 years or more from selected households were interviewed and, whenever possible, sputum samples were obtained. Sputum samples were subjected to direct microscopy by Ziehl-Neelson method, bacterial culture and antibiotic sensitivity tests. Results The prevalence (per 100,000) of pulmonary tuberculosis among the subjects aged 15 years or more, who participated in the study was 329 (95% confidence interval (CI): 195 – 519). The prevalence (per 100,000) of pulmonary tuberculosis adjusted for non-sampling was 438 (95% CI: 282 – 651). Other than cough, none of the other clinical variables was significantly associated with pulmonary tuberculosis status. Analysis of drug sensitivity pattern of 15 strains of Mycobacterium tuberculosis revealed that one strain was resistant to isoniazid alone, one to streptomycin alone and one was resistant to isoniazid and streptomycin. The remaining 12 strains were susceptible to all five drugs including streptomycin, isoniazid, rifampicin, ethambutol, and pyrazinamide. Conclusion This study of previously undetected tuberculosis cases in an impoverished peri-urban setting reveals the poor operational performance of Pakistan's current approach to tuberculosis control; it also demonstrates a higher prevalence of pulmonary tuberculosis than current national estimates. Public health authorities may wish to augment health education efforts aimed at prompting health-seeking behaviour to facilitate more complete and earlier case detection. Such efforts to improve passive case-finding, if combined with more accessible DOTS infra-structure for treatment of detected cases, may help to diminish the high tuberculosis-related morbidity and mortality in marginalized populations. The economics of implementing a more active approach to case finding in resource-constrained setting also deserve further study. PMID:17477870

Catalase-peroxidase activity has no influence on virulence in a murine model of tuberculosis.

PubMed

Cardona, Pere Joan; Gordillo, Sergi; Amat, Isabel; Díaz, Jorge; Lonca, Joan; Vilaplana, Cristina; Pallarés, Angeles; Llatjós, Roger; Ariza, Aurelio; Ausina, Vicenç

2003-01-01

The capacity to generate a chronic and persistent infection in the experimental murine model of tuberculosis induced aerogenically by a low-dose inoculum was determined in eight isoniazid-resistant clinical strains of Mycobacterium tuberculosis showing different catalase-peroxidase (C-P) activities. Determination of bacillary concentration in lung and spleen and the percentage of pulmonary parenchyma occupied by granulomas were monitored. Data showed no relation between the lack of C-P activity and the ability to develop a persistent infection, highlighting the potential of C-P negative strains to spread through the community.

Doubly end-on azido bridged mixed-valence cobalt trinuclear complex: Spectral study, VTM, inhibitory effect and antimycobacterial activity on human carcinoma and tuberculosis cells

NASA Astrophysics Data System (ADS)

Datta, Amitabha; Das, Kuheli; Sen, Chandana; Karan, Nirmal Kumar; Huang, Jui-Hsien; Lin, Chia-Her; Garribba, Eugenio; Sinha, Chittaranjan; Askun, Tulin; Celikboyun, Pinar; Mane, Sandeep B.

2015-09-01

Doubly end-on azido-bridged mixed-valence trinuclear cobalt complex, [Co3(L)2(N3)6(CH3OH)2] (1) is afforded by employing a potential monoanionic tetradentate-N2O2 Schiff base precursor (2-[{[2-(dimethylamino)ethyl]imino}methyl]-6-methoxyphenol; HL). Single crystal X-ray structure reveals that in 1, the adjacent CoII and CoIII ions are linked by double end-on azido bridges and thus the full molecule is generated by the site symmetry of a crystallographic twofold rotation axis. Complex 1 is subjected on different spectral analysis such as IR, UV-vis, emission and EPR spectroscopy. On variable temperature magnetic study, we observe that during cooling, the χMT values decrease smoothly until 15 K and then reaches to the value 1.56 cm3 K mol-1 at 2 K. Complex 1 inhibits the cell growth on human lung carcinoma (A549 cells), human colorectal (COLO 205 and HT-29 cells), and human heptacellular (PLC5 cells) carcinoma cells. Complex 1 exhibits anti-mycobacterial activity and considerable efficacy on Mycobacterium tuberculosis H37Rv ATCC 27294 and H37Ra ATCC 25177 strains.

Copper-boosting compounds: a novel concept for antimycobacterial drug discovery.

PubMed

Speer, Alexander; Shrestha, Tej B; Bossmann, Stefan H; Basaraba, Randall J; Harber, Gregory J; Michalek, Suzanne M; Niederweis, Michael; Kutsch, Olaf; Wolschendorf, Frank

2013-02-01

We and others recently identified copper resistance as important for virulence of Mycobacterium tuberculosis. Here, we introduce a high-throughput screening assay for agents that induce a copper hypersensitivity phenotype in M. tuberculosis and demonstrate that such copper-boosting compounds are effective against replicating and nonreplicating M. tuberculosis strains.

Live Attenuated Salmonella Vaccines Displaying Regulated Delayed Lysis and Delayed Antigen Synthesis To Confer Protection against Mycobacterium tuberculosis

PubMed Central

Juárez-Rodríguez, María Dolores; Yang, Jiseon; Kader, Rebin; Alamuri, Praveen; Curtiss, Roy

2012-01-01

Live recombinant attenuated Salmonella vaccine (RASV) strains have great potential to induce protective immunity against Mycobacterium tuberculosis by delivering M. tuberculosis antigens. Recently, we reported that, in orally immunized mice, RASV strains delivering the M. tuberculosis early secreted antigenic target 6-kDa (ESAT-6) protein and culture filtrate protein 10 (CFP-10) antigens via the Salmonella type III secretion system (SopE amino-terminal region residues 1 to 80 with two copies of ESAT-6 and one copy of CFP-10 [SopENt80-E2C]) afforded protection against aerosol challenge with M. tuberculosis. Here, we constructed and evaluated an improved Salmonella vaccine against M. tuberculosis. We constructed translational fusions for the synthesis of two copies of ESAT-6 plus CFP-10 fused to the OmpC signal sequence (OmpCSS-E2C) and amino acids 44 to 338 of antigen 85A (Ag85A294) flanked by the signal sequence (SS) and C-terminal peptide (CT) of β-lactamase (BlaSS-Ag85A294-BlaCT) to enable delivery via the Salmonella type II secretion system. The genes expressing these proteins were cloned as an operon transcribed from Ptrc into isogenic Asd+/MurA+ pYA3681 lysis vector derivatives with different replication origins (pBR, p15A, pSC101), resulting in pYA4890, pYA4891, and pYA4892 for SopENt80-E2C/Ag85A294 synthesis and pYA4893 and pYA4894 for OmpCSS-E2C/Ag85A294 synthesis. Mice orally immunized with the RASV χ11021 strain engineered to display regulated delayed lysis and regulated delayed antigen synthesis in vivo and harboring pYA4891, pYA4893, or pYA4894 elicited significantly greater humoral and cellular immune responses, and the RASV χ11021 strain afforded a greater degree of protection against M. tuberculosis aerosol challenge in mice than RASVs harboring any other Asd+/MurA+ lysis plasmid and immunization with M. bovis BCG, demonstrating that RASV strains displaying regulated delayed lysis with delayed antigen synthesis resulted in highly immunogenic delivery vectors for oral vaccination against M. tuberculosis infection. PMID:22144485

Low dose aerosol fitness at the innate phase of murine infection better predicts virulence amongst clinical strains of Mycobacterium tuberculosis.

PubMed

Caceres, Neus; Llopis, Isaac; Marzo, Elena; Prats, Clara; Vilaplana, Cristina; de Viedma, Dario Garcia; Samper, Sofía; Lopez, Daniel; Cardona, Pere-Joan

2012-01-01

Evaluation of a quick and easy model to determine the intrinsic ability of clinical strains to generate active TB has been set by assuming that this is linked to the fitness of Mycobacterium tuberculosis strain at the innate phase of the infection. Thus, the higher the bacillary load, the greater the possibility of inducting liquefaction, and thus active TB, once the adaptive response is set. The virulence of seven clinical Mycobacterium tuberculosis strains isolated in Spain was tested by determining the bacillary concentration in the spleen and lung of mice at weeks 0, 1 and 2 after intravenous (IV) inoculation of 10⁴ CFU, and by determining the growth in vitro until the stationary phase had been reached. Cord distribution automated analysis showed two clear patterns related to the high and low fitness in the lung after IV infection. This pattern was not seen in the in vitro fitness tests, which clearly favored the reference strain (H37Rv). Subsequent determination using a more physiological low-dose aerosol (AER) inoculation with 10² CFU showed a third pattern in which the three best values coincided with the highest dissemination capacity according to epidemiological data. The fitness obtained after low dose aerosol administration in the presence of the innate immune response is the most predictive factor for determining the virulence of clinical strains. This gives support to a mechanism of the induction of active TB derived from the dynamic hypothesis of latent tuberculosis infection.

Apoptosis, production of MMP9, VEGF, TNF-alpha and intracellular growth of M. tuberculosis for different genotypes and different pks5/1 genes.

PubMed

Yorsangsukkamol, Juthaporn; Chaiprasert, Angkana; Palaga, Tanapat; Prammananan, Therdsak; Faksri, Kiatichai; Palittapongarnpim, Prasit; Prayoonwiwat, Narapon

2011-09-01

A previous study of IS6110 RFLP and spoligotyping of M. tuberculosis isolates from 152 Thai patients with tuberculous meningitis revealed a significantly higher percentage (57%) of the Beijing genotype as compared to isolates obtained from pulmonary tuberculosis. We postulated that the M. tuberculosis Beijing genotype is likely to be more virulent than others. Ten M. tuberculosis cerebrospinal fluid (CSF) isolates from five RFLP groups, together with different characteristics of pks15/1, M. tuberculosis H37Rv and M. bovis BCG, were investigated for their virulence in vitro. In this study, THP-1 cells were used as host cells to determine the intracellular growth and the induction of MMP9, VEGF, TNF-alpha and apoptosis. Determinations of the cytokine production and apoptosis were based on available commercial kits using ELISA techniques. No significant difference in intracellular multiplication was found between the M. tuberculosis CSF isolates. Three isolates, consisting of 2 Nonthaburi and 1 heterogeneous isolate, were found to stimulate high TNF-alpha and MMP-9 production during the early infection period.They were isolated from 3 different patients, 2 of whom died with initial stages II and III. This result suggested that there might be an association between TNF-alpha and MMP-9 production that could account for the specific virulent nature of Nonthaburi strains. VEGF production was determined and comparable levels were found in all isolates. No significant apoptosis was detected in M. tuberculosis CSF isolates. No significant differences suggesting that the 2 Beijing strains are more virulent than the others were observed. The predominance of the Beijing strains in cases of tuberculous meningitis (TBM) in Thai patients is not a result of their hypervirulence.

Immunogenicity and protection conferred by Mycobacterium habana in a murine model of pulmonary tuberculosis.

PubMed

Valdés, Iliana; Montoro, Ernesto; Mata-Espinoza, Dulce; Asín, Odalys; Barrios-Payan, Jorge; Francisco-Cruz, Alejandro; Valdivia, José Antonio; Hernández-Pando, Rogelio

2014-01-01

Mycobacterium habana was isolated in Cuba in 1971. Later, was demonstrated its protection capacity in mycobacterial infection. Here we determined the level of virulence, immunogenicity and the efficacy of three different M. habana strains as attenuated live vaccines. Intratracheal infection of BALB/c mice with high dose M. habana TMC 5135 or IPK-337 strains permitted 100% survival and limited tissue damage. Mice infected with M. habana IPK-220 showed lower attenuation, so it was discarded for the vaccination experiments. Strains IPK-337 and TMC 5135 were used as subcutaneous vaccine and compared with BCG. Nude mice vaccinated with strain 5135 showed longer but non-significant survival than BCG vaccinated animals. Cell suspensions from M. habana vaccinated mice produced higher IFNγ after stimulation with mycobacterial antigens than BCG recipients. After four months of challenge with Mycobacterium tuberculosis strain H37Rv, mice vaccinated with BCG substrain Phipps or strain TMC 5135 showed total survival, while 60% survival was exhibited by animals vaccinated with M. habana IPK-337. Both M. habana strains do not prevent the infection with M. tuberculosis but avoided the progression of the experimental disease; strain TMC 5135 showed similar level of protection than BCG. Copyright © 2013 Elsevier Ltd. All rights reserved.

Genetic diversity of the Mycobacterium tuberculosis Beijing family based on multiple genotyping profiles.

PubMed

Liu, Y; Wang, S; Lu, H; Chen, W; Wang, W

2016-06-01

Among the most prevalent Mycobacterium tuberculosis (Mtb) strains worldwide is the Beijing genotype, which has caused large outbreaks of tuberculosis (TB). Characteristics facilitating the dissemination of Beijing family strains remain unknown, but they are presumed to have been acquired through evolution of the lineage. To explore the genetic diversity of the Beijing family Mtb and explore the discriminatory ability of mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) loci in several regions of East Asia, a cross-sectional study was conducted with a total of 163 Beijing strains collected from registered TB patients between 1 June 2009 and 31 November 2010 in Funing County, China. The isolated strains were analysed by 15-MIRU-VNTR loci typing and compared with published MIRU-VNTR profiles of Beijing strains. Synonymous single nucleotide polymorphisms at 10 chromosomal positions were also analysed. The combination of SNP and MIRU-VNTR typing may be used to assess Mtb genotypes in areas dominated by Beijing strains. The modern subfamily in Shanghai overlapped with strains from other countries, whereas the ancient subfamily was genetically differentiated across several countries. Modern subfamilies, especially ST10, were prevalent. Qub11b and four other loci (MIRU 26, Mtub21, Qub26, Mtub04) could be used to discriminate Beijing strains.

Pathology of Camel Tuberculosis and Molecular Characterization of Its Causative Agents in Pastoral Regions of Ethiopia

PubMed Central

Mamo, Gezahegne; Bayleyegn, Gizachew; Sisay Tessema, Tesfaye; Legesse, Mengistu; Medhin, Girmay; Bjune, Gunnar; Abebe, Fekadu; Ameni, Gobena

2011-01-01

A cross sectional study was conducted on 906 apparently healthy camels slaughtered at Akaki and Metehara abattoirs to investigate the pathology of camel tuberculosis (TB) and characterize its causative agents using postmortem examination, mycobacteriological culturing, and multiplex polymerase chain reaction (PCR), region of difference-4 (RD4)-based PCR and spoligotyping. The prevalence of camel TB was 10.04% (91/906) on the basis of pathology and it was significantly higher in females (χ2 = 4.789; P = 0.029). The tropism of TB lesions was significantly different among the lymph nodes (χ2 = 22.697; P = 0.002) and lung lobes (χ2 = 17.901; P = 0.006). Mycobacterial growth was observed in 34% (31/91) of camels with grossly suspicious TB lesions. Upon further molecular characterization using multiplex PCR, 68% (21/31) of the colonies showed a positive signal for the genus Mycobacterium, of which two were confirmed Mycobacterium bovis (M. bovis) by RD4 deletion typing. Further characterization of the two M. bovis at strains level revealed that one of the strains was SB0133 while the other strain was new and had not been reported to the M. bovis database prior to this study. Hence, it has now been reported to the database, and designated as SB1953. In conclusion, the results of the present study have shown that the majority of camel TB lesions are caused by mycobacteria other than Mycobacterium tuberculosis complex. And hence further identification and characterization of these species would be useful towards the efforts made to control TB in camels. PMID:21283668

The Influence of Host and Bacterial Genotype on the Development of Disseminated Disease with Mycobacterium tuberculosis

PubMed Central

Caws, Maxine; Thwaites, Guy; Dunstan, Sarah; Hawn, Thomas R.; Thi Ngoc Lan, Nguyen; Thuong, Nguyen Thuy Thuong; Stepniewska, Kasia; Huyen, Mai Nguyet Thu; Bang, Nguyen Duc; Huu Loc, Tran; Gagneux, Sebastien; van Soolingen, Dick; Kremer, Kristin; van der Sande, Marianne; Small, Peter; Thi Hoang Anh, Phan; Chinh, Nguyen Tran; Thi Quy, Hoang; Thi Hong Duyen, Nguyen; Quang Tho, Dau; Hieu, Nguyen T.; Torok, Estee; Hien, Tran Tinh; Dung, Nguyen Huy; Thi Quynh Nhu, Nguyen; Duy, Phan Minh; van Vinh Chau, Nguyen; Farrar, Jeremy

2008-01-01

The factors that govern the development of tuberculosis disease are incompletely understood. We hypothesized that some strains of Mycobacterium tuberculosis (M. tuberculosis) are more capable of causing disseminated disease than others and may be associated with polymorphisms in host genes responsible for the innate immune response to infection. We compared the host and bacterial genotype in 187 Vietnamese adults with tuberculous meningitis (TBM) and 237 Vietnamese adults with uncomplicated pulmonary tuberculosis. The host genotype of tuberculosis cases was also compared with the genotype of 392 cord blood controls from the same population. Isolates of M. tuberculosis were genotyped by large sequence polymorphisms. The hosts were defined by polymorphisms in genes encoding Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) and Toll-like receptor-2 (TLR-2). We found a significant protective association between the Euro-American lineage of M. tuberculosis and pulmonary rather than meningeal tuberculosis (Odds ratio (OR) for causing TBM 0.395, 95% confidence intervals (C.I.) 0.193–0.806, P = 0.009), suggesting these strains are less capable of extra-pulmonary dissemination than others in the study population. We also found that individuals with the C allele of TLR-2 T597C allele were more likely to have tuberculosis caused by the East-Asian/Beijing genotype (OR = 1.57 [95% C.I. 1.15–2.15]) than other individuals. The study provides evidence that M. tuberculosis genotype influences clinical disease phenotype and demonstrates, for the first time, a significant interaction between host and bacterial genotypes and the development of tuberculosis. PMID:18369480

Whole genome sequencing identifies circulating Beijing-lineage Mycobacterium tuberculosis strains in Guatemala and an associated urban outbreak

PubMed Central

Saelens, Joseph W.; Lau-Bonilla, Dalia; Moller, Anneliese; Medina, Narda; Guzmán, Brenda; Calderón, Maylena; Herrera, Raúl; Sisk, Dana M.; Xet-Mull, Ana M.; Stout, Jason E.; Arathoon, Eduardo; Samayoa, Blanca; Tobin, David M.

2015-01-01

Summary Limited data are available regarding the molecular epidemiology of Mycobacterium tuberculosis (Mtb) strains circulating in Guatemala. Beijing-lineage Mtb strains have gained prevalence worldwide and are associated with increased virulence and drug resistance, but there have been only a few cases reported in Central America. Here we report the first whole genome sequencing of Central American Beijing-lineage strains of Mtb. We find that multiple Beijing-lineage strains, derived from independent founding events, are currently circulating in Guatemala, but overall still represent a relatively small proportion of disease burden. Finally, we identify a specific Beijing-lineage outbreak centered on a poor neighborhood in Guatemala City. PMID:26542222

Activity against Mycobacterium tuberculosis with concomitant induction of cellular immune responses by a tetraaza-macrocycle with acetate pendant arms.

PubMed

David, S; Ordway, D; Arroz, M J; Costa, J; Delgado, R

2001-01-01

The novel tetraaza-macrocyclic compound 3,7,11-tris(carboxymethyl)-3,7,11,17-tetraaza-bicyclo[11.3.1]heptadeca-1(17),13,15-triene, abbreviated as ac3py14, was investigated for its activity against Mycobacterium tuberculosis and for induction of protective cellular immune responses. Perspective results show that ac3py14 and its Fe3+ 1:1 complex, [Fe(ac3py14)], inhibited radiometric growth of several strains of M. tuberculosis. Inhibition with 25 microg/mL varied from 99% for H37Rv to 80% and above for multiple drug-resistant clinical isolates. The capacity of ac3py14 to elicit a beneficial immune response without cellular apoptosis was assessed and compared to the effects of virulent M. tuberculosis. The present study produces evidence that after stimulation with ac3py14 there was significant production of interferon gamma (IFN-gamma), whereas the production of interleukin-5 (IL-5) remained low, and there was development of a memory population (CD45RO). The level of binding of Annexin V, a marker of apoptosis, was not sufficient to result in toxic effects toward alphabeta and gammadelta T cells and CD14+ macrophages. This preliminary study is the first report of a compound that simultaneously exerts an inhibitory effect against M. tuberculosis and induces factors associated with protective immune responses.

The value of microscopic-observation drug susceptibility assay in the diagnosis of tuberculosis and detection of multidrug resistance.

PubMed

Sertel Şelale, Denİz; Uzun, Meltem

2018-01-01

Inexpensive, rapid, and reliable tests for detecting the presence and drug susceptibility of Mycobacterium tuberculosis complex (MTBC) are urgently needed to control the transmission of tuberculosis. In this study, we aimed to assess the accuracy and speed of the microscopic-observation drug susceptibility (MODS) assay in the identification of MTBC and detection of multidrug resistance. Sputum samples from patients suspected to have tuberculosis were simultaneously tested with MODS and conventional culture [Löwenstein-Jensen (LJ) culture, BACTEC MGIT™ 960 (MGIT) system], and drug susceptibility testing (MGIT system) methods. A total of 331 sputum samples were analyzed. Sensitivity and specificity of MODS assay for detection of MTBC strains were 96% and 98.8%, respectively. MODS assay detected multidrug resistant MTBC isolates with 92.3% sensitivity and 96.6% specificity. Median time to culture positivity was similar for MGIT (8 days) and MODS culture (8 days), but was significantly longer with LJ culture (20 days) (p < 0.0001 for both comparisons). Median time to availability of the susceptibility results was significantly (p < 0.0001) shorter with MODS assay (8 days) than MGIT system (20 days). In conclusion, MODS is an inexpensive and rapid test with good performance characteristics for direct diagnosis of tuberculosis and detection of multidrug resistance. © 2017 APMIS. Published by John Wiley & Sons Ltd.

Linking minimum inhibitory concentrations to whole genome sequence-predicted drug resistance in Mycobacterium tuberculosis strains from Romania.

PubMed

Ruesen, Carolien; Riza, Anca Lelia; Florescu, Adriana; Chaidir, Lidya; Editoiu, Cornelia; Aalders, Nicole; Nicolosu, Dragos; Grecu, Victor; Ioana, Mihai; van Crevel, Reinout; van Ingen, Jakko

2018-06-26

Mycobacterium tuberculosis drug resistance poses a major threat to tuberculosis control. Current phenotypic tests for drug susceptibility are time-consuming, technically complex, and expensive. Whole genome sequencing is a promising alternative, though the impact of different drug resistance mutations on the minimum inhibitory concentration (MIC) remains to be investigated. We examined the genomes of 72 phenotypically drug-resistant Mycobacterium tuberculosis isolates from 72 Romanian patients for drug resistance mutations. MICs for first- and second-line drugs were determined using the MycoTB microdilution method. These MICs were compared to macrodilution critical concentration testing by the Mycobacterium Growth Indicator Tube (MGIT) platform and correlated to drug resistance mutations. Sixty-three (87.5%) isolates harboured drug resistance mutations; 48 (66.7%) were genotypically multidrug-resistant. Different drug resistance mutations were associated with different MIC ranges; katG S315T for isoniazid, and rpoB S450L for rifampicin were associated with high MICs. However, several mutations such as in rpoB, rrs and rpsL, or embB were associated with MIC ranges including the critical concentration for rifampicin, aminoglycosides or ethambutol, respectively. Different resistance mutations lead to distinct MICs, some of which may still be overcome by increased dosing. Whole genome sequencing can aid in the timely diagnosis of Mycobacterium tuberculosis drug resistance and guide clinical decision-making.

Whole genome analysis of an MDR Beijing/W strain of Mycobacterium tuberculosis with large genomic deletions associated with resistance to isoniazid.

PubMed

Zhang, Qiufen; Wan, Baoshan; Zhou, Aiping; Ni, Jinjing; Xu, Zhihong; Li, Shuxian; Tao, Jing; Yao, YuFeng

2016-05-15

Mycobacterium tuberculosis (M.tb) is one of the most prevalent bacterial pathogens in the world. With geographical wide spread and hypervirulence, Beijing/W family is the most successful M.tb lineage. China is a country of high tuberculosis (TB) and high multiple drug-resistant TB (MDR-TB) burden, and the Beijing/W family strains take the largest share of MDR strains. To study the genetic basis of Beijing/W family strains' virulence and drug resistance, we performed the whole genome sequencing of M.tb strain W146, a clinical Beijing/W genotype MDR isolated from Wuxi, Jiangsu province, China. Compared with genome sequence of M.tb strain H37Rv, we found that strain W146 lacks three large fragments and the missing of furA-katG operon confers isoniazid resistance. Besides the missing of furA-katG operon, strain W146 harbored almost all known drug resistance-associated mutations. Comparison analysis of single nucleotide polymorphisms (SNPs) and indels between strain W146 and Beijing/W genotype strains and non-Beijing/W genotype strains revealed that strain W146 possessed some unique mutations, which may be related to drug resistance, transmission and pathogenicity. These findings will help to understand the large sequence polymorphisms (LSPs) and the transmission and drug resistance related genetic characteristics of the Beijing/W genotype of M.tb. Copyright © 2016 Elsevier B.V. All rights reserved.

Antibacterial activity of selected Cameroonian dietary spices ethno-medically used against strains of Mycobacterium tuberculosis.

PubMed

Tekwu, Emmanuel Mouafo; Askun, Tülin; Kuete, Victor; Nkengfack, Augustin Ephraim; Nyasse, Barthélémy; Etoa, François-Xavier; Beng, Véronique Penlap

2012-07-13

Tuberculosis (TB) is considered as a re-emerging disease and one of the most important public health problems worldwide. The use or (in most cases) misuse of existint anti-tuberculosis drugs over the years has led to an increasing prevalence of resistant strains, establishing an urgent need to search for new effective agents. Spices are largely used ethno-medically across Africa. The present study aimed to evaluate the in vitro antimycobacterial activities of a total of 20 methanol crude extracts prepared from 20 Cameroonian dietary spices for their ability to inhibit the growth of or kill Mycobacterium tuberculosis strains H(37)Rv (ATCC 27294) and H(37)Ra (ATCC 25177). The antituberculosis screening was performed using the Microplate Alamar Blue Assay (MABA) method to determine the minimum inhibitory concentration (MIC) and the minimum mycobactericidal concentration (MBC). Fifteen (15) plant extracts out of 20 showed varied levels of antimycobacterial activity against the strains M. tuberculosis H(37)Rv and H(37)Ra, with MICs in the range of 2.048-0.016 mg/ml. The extract of Echinops giganteus exhibited the most significant activity with a MIC value of 32 μg/ml and 16 μg/ml, respectively against H(37)Ra and H(37)Rv. To the best of our knowledge, the antimycobacterial activity of the tested spices has not been reported before and therefore our results can be evaluated as the first report about the antimycobacterial properties. The results of this study suggest that Echinops giganteus and Piper guineense could be important sources of bactericidal compounds against M. tuberculosis and could probably be promising candidates that can be further investigated. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

CsoR Is Essential for Maintaining Copper Homeostasis in Mycobacterium tuberculosis

PubMed Central

Marcus, Sarah A.; Sidiropoulos, Sarah W.; Steinberg, Howard; Talaat, Adel M.

2016-01-01

Mycobacterium tuberculosis, a pathogen infecting one third of the world population, faces numerous challenges within the host, including high levels of copper. We have previously shown that M. tuberculosis CsoR is a copper inducible transcriptional regulator. Here we examined the hypothesis that csoR is necessary for maintaining copper homeostasis and surviving under various stress conditions. With an unmarked csoR knockout strain, we were able to characterize the role of csoR in M. tuberculosis as it faced copper and host stress. Growth under high levels of copper demonstrated that M. tuberculosis survives copper stress significantly better in the absence of csoR. Yet under minimal levels of copper, differential expression analysis revealed that the loss of csoR results in a cell wide hypoxia-type stress response with the induction of the DosR regulon. Despite the stress placed on M. tuberculosis by the loss of csoR, survival of the knockout strain was increased compared to wild type during the early chronic stages of mouse infection, suggesting that csoR could play an active role in modulating M. tuberculosis fitness within the host. Overall, analysis of CsoR provided an increased understanding of the M. tuberculosis copper response with implications for other intracellular pathogens harboring CsoR. PMID:26999439

[Discriminatory power of variable number on tandem repeats loci for genotyping Mycobacterium tuberculosis strains in China].

PubMed

Chen, H X; Cai, C; Liu, J Y; Zhang, Z G; Yuan, M; Jia, J N; Sun, Z G; Huang, H R; Gao, J M; Li, W M

2017-06-10

Objective: Using the standard genotype method, variable number of tandem repeats (VNTR), we constructed a VNTR database to cover all provinces and proposed a set of optimized VNTR loci combinations for each province, in order to improve the preventive and control programs on tuberculosis, in China. Methods: A total of 15 loci VNTR was used to analyze 4 116 Mycobacterium tuberculosis strains, isolated from national survey of Drug Resistant Tuberculosis, in 2007. Hunter-Gaston Index (HGI) was also used to analyze the discriminatory power of each VNTR site. A set combination of 12-VNTR, 10-VNTR, 8-VNTR and 5-VNTR was respectively constructed for each province, based on 1) epidemic characteristics of M. tuberculosis lineages in China, with high discriminatory power and genetic stability. Results: Through the completed 15 loci VNTR patterns of 3 966 strains under 96.36 % (3 966/4 116) coverage, we found seven high HGI loci (including QUB11b and MIRU26) as well as low stable loci (including QUB26, MIRU16, Mtub21 and QUB11b) in several areas. In all the 31 provinces, we found an optimization VNTR combination as 10-VNTR loci in Inner Mongolia, Chongqing and Heilongjiang, but with 8-VNTR combination shared in other provinces. Conclusions: It is necessary to not only use the VNTR database for tracing the source of infection and cluster of M. tuberculosis in the nation but also using the set of optimized VNTR combinations in monitoring those local epidemics and M. tuberculosis (genetics in local) population.

Genetically Determined Susceptibility to Tuberculosis in Mice Causally Involves Accelerated and Enhanced Recruitment of Granulocytes

PubMed Central

Keller, Christine; Hoffmann, Reinhard; Lang, Roland; Brandau, Sven; Hermann, Corinna; Ehlers, Stefan

2006-01-01

Classical twin studies and recent linkage analyses of African populations have revealed a potential involvement of host genetic factors in susceptibility or resistance to Mycobacterium tuberculosis infection. In order to identify the candidate genes involved and test their causal implication, we capitalized on the mouse model of tuberculosis, since inbred mouse strains also differ substantially in their susceptibility to infection. Two susceptible and two resistant mouse strains were aerogenically infected with 1,000 CFU of M. tuberculosis, and the regulation of gene expression was examined by Affymetrix GeneChip U74A array with total lung RNA 2 and 4 weeks postinfection. Four weeks after infection, 96 genes, many of which are involved in inflammatory cell recruitment and activation, were regulated in common. One hundred seven genes were differentially regulated in susceptible mouse strains, whereas 43 genes were differentially expressed only in resistant mice. Data mining revealed a bias towards the expression of genes involved in granulocyte pathophysiology in susceptible mice, such as an upregulation of those for the neutrophil chemoattractant LIX (CXCL5), interleukin 17 receptor, phosphoinositide kinase 3 delta, or gamma interferon-inducible protein 10. Following M. tuberculosis challenge in both airways or peritoneum, granulocytes were recruited significantly faster and at higher numbers in susceptible than in resistant mice. When granulocytes were efficiently depleted by either of two regimens at the onset of infection, only susceptible mice survived aerosol challenge with M. tuberculosis significantly longer than control mice. We conclude that initially enhanced recruitment of granulocytes contributes to susceptibility to tuberculosis. PMID:16790804

Novel inhibitors of Mycobacterium tuberculosis growth based on modified pyrimidine nucleosides and their analogues

NASA Astrophysics Data System (ADS)

Shmalenyuk, E. R.; Kochetkov, S. N.; Alexandrova, L. A.

2013-09-01

The review summarizes data on the synthesis and antituberculosis activity of pyrimidine nucleoside derivatives and their analogues. Enzymes from M. tuberculosis as promising targets for prototypes of new-generation drugs are considered. Nucleosides as inhibitors of drug-resistant M. tuberculosis strains are characterized. The bibliography includes 101 references.

[Tuberculosis Laboratory Surveillance Network (TuLSA) study group. The first step for national tuberculosis laboratory surveillance: Ankara, 2011].

PubMed

Sezen, Figen; Albayrak, Nurhan; Özkara, Şeref; Karagöz, Alper; Alp, Alpaslan; Duyar Ağca, Filiz; İnan Süer, Asiye; Müderris, Tuba; Ceyhan, İsmail; Durmaz, Rıza; Ertek, Mustafa

2015-04-01

The most effective method for monitoring country-level drug resistance frequency and to implement the necessary control measures is the establishment of a laboratory-based surveillance system. The aim of this study was to summarize the follow up trend of the drug-resistant tuberculosis (TB) cases, determine the load of resistance and evaluate the capacities of laboratories depending on laboratory quality assurance system for the installation work of National Tuberculosis Laboratory Surveillance Network (TuLSA) which has started in Ankara in 2011. TuLSA studies was carried out under the coordination of National Tuberculosis Reference Laboratory (NRL) with the participation of TB laboratories and dispensaries. Specimens of TB patients, reported from health institutions, were followed in TB laboratories, and the epidemiological information was collected from the dispensaries. One isolate per patient with the drug susceptibility test (DST) results were sent to NRL from TB laboratories and in NRL the isolates were rechecked with the genotypical (MTBDRplus, Hain Lifescience, Germany) and phenotypical (MGIT 960, BD, USA) DST methods. Molecular epidemiological analysis were also performed by spoligotyping and MIRU/VNTR. Second-line DST was applied to the isolates resistant to rifampin. A total of 1276 patients were reported between January 1st to December 31th 2011, and 335 cases were defined as "pulmonary TB from Ankara province". The mean age of those patients was 43.4 ± 20 years, and 67.5% were male. Three hundred seventeen (94.6%) patients were identified as new cases. The average sample number obtained from pulmonary TB cases was 3.26 ± 2.88, and 229 (68.3%) of them was culture positive. DST was applied to all culture positive isolates; 90.4% (207/229) of cases were susceptible to the five drugs tested (ethambutol, isoniazid, pyrazinamide, rifampicin, streptomycin). Eight (3.5%) of the isolates were multidrug-resistant (MDR-TB), while no extensively drug-resistant strains were detected. MDR-TB is likely to occur in 63.3 times more among previously treated cases, and 73.3 times more in legal aliens. The achievement of therapy among pulmonary TB cases was 91.9%. Spoligotyping performed for 221 M.tuberculosis complex isolates, showed that all strains were clustered in nine groups. SIT 41 (105/221; 47.5%) was the most frequent spoligotype detected, and clustering rate based on MIRU-VNTR results were found as 16.3%. All of the clustered strains were sensitive while all of MDR-TB isolates showed specific MIRU-VNTR profiles. In conclusion, TuLSA studies started in Ankara in 2011 and the system is still expanding in the country. Our data obtained with TuLSA have been published as a regional surveillance data in the WHO Global Tuberculosis Report 2011, and as a national surveillance data in Global Tuberculosis Report 2012.

Polyphosphate Deficiency in Mycobacterium tuberculosis Is Associated with Enhanced Drug Susceptibility and Impaired Growth in Guinea Pigs

PubMed Central

Singh, Mamta; Arora, Garima; Kumar, Santosh; Tiwari, Prabhakar; Kidwai, Saqib

2013-01-01

Inorganic polyphosphate (polyP), a linear polymer of hundreds of phosphate residues linked by ATP-like phosphoanhydride bonds, is found in all organisms and performs a wide variety of functions. This study shows that polyP accumulation occurs in Mycobacterium tuberculosis upon exposure to various stress conditions. M. tuberculosis possesses a single homolog of ppk-1, and we have disrupted ppk-1 in the M. tuberculosis genome by allelic replacement. The mutant strain exhibited negligible levels of intracellular polyP, decreased expression of sigF and phoP, and reduced growth in the stationary phase and displayed a survival defect in response to nitrosative stress and in THP-1 macrophages compared to the wild-type strain. We report that reduction in polyP levels is associated with increased susceptibility of M. tuberculosis to certain TB drugs and impairs its ability to cause disease in guinea pigs. These results suggest that polyP contributes to persistence of M. tuberculosis in vitro and plays an important role in the physiology of bacteria residing within guinea pigs. PMID:23585537

Clustering of Mycobacterium tuberculosis strains from foreign-born patients in Korea.

PubMed

Jeon, Christie Y; Kang, Heeyoon; Kim, Mihye; Murray, Megan B; Kim, Heejin; Cho, Eun Hee; Park, Young Kil

2011-12-01

Information on drug resistance and transmission patterns of tuberculosis (TB) in foreign-born patients is lacking in Asia where immigration is increasing. We examined the drug-resistance profiles of 288 Mycobacterium tuberculosis isolates from foreign-born patients in South Korea, and assessed for potential transmission in the host country by analysing their IS6110 genotypes, as well as those of 4780 strains from native Korean TB patients. The prevalence of multidrug-resistant (MDR) TB was 9.7% and 42% among new and previously treated patients, respectively. Chinese nationality was associated with MDR TB (OR(China)=3.0, 95% CI 1.1-9.3). Of the 288 strains, 51 (17.7%) formed 31 clusters, of which 22 were identical to strains from native Koreans. A number of strains belonged to the K family, subtypes known to occur endemically in Korea. MDR TB was common, and clustering patterns showed potential cross-cultural transmission among foreign-born TB patients. Further molecular epidemiological studies of all isolates in the area are needed to determine the extent of international TB transmission in Asia. © 2011 SGM

Genotypic characterisation of Mycobacterium tuberculosis isolates from tuberculous meningitis patients at a tertiary neurocare centre in Southern India.

PubMed

Chandramuki, Akepati; Khanna, Neelam; Shashkina, Elena; Kurepina, Natalia; Mathema, Barun; Kreiswirth, Barry N; Venkataswamy, Manjunatha M

2017-01-01

Specific genotypes of Mycobacterium tuberculosis (MTB) have been reported to cause outbreaks of pulmonary tuberculosis (TB) in geographical areas that are endemic to TB. However, since there is little epidemiological evidence on the association of particular genotypes that cause tuberculous meningitis (TBM), we sought to investigate the association of specific MTB strains with infection of the central nervous system (CNS). We carried out a genetic characterisation of 89 MTB isolates from TBM patients at a Southern Indian tertiary neurocare centre and compared the genotypes with strains of pulmonary TB isolated from Indian immigrants in New York City. We applied the standard methods of genotyping of MTB, namely, IS6110-based restriction fragment length polymorphism and spoligotyping for strain identification, along with principal genetic grouping and single-nucleotide polymorphism cluster analysis. The analysis revealed a high-level of diversity amongst the strain population. The genotypes of the isolates from TBM patients paralleled the pulmonary TB strain population recovered from the Indian immigrants in NYC. We conclude that there is no apparent association between genotypes of MTB and propensity to infect CNS tissue.

Mixed infections in tuberculosis: The missing part in a puzzle.

PubMed

Tarashi, Samira; Fateh, Abolfazl; Mirsaeidi, Mehdi; Siadat, Seyed Davar; Vaziri, Farzam

2017-12-01

The mixed strains infection phenomenon is a major problem posing serious challenges in control of tuberculosis (TB). In patients with mixed infection, several different strains of Mycobacterium tuberculosis can be isolated simultaneously. Although different genotyping methods and various molecular approaches can be employed for detection of mixed infection in clinical samples, the MIRU-VNTR technique is more sensitive with higher discriminative power than many widely used techniques. Furthermore, the recent introduction of whole genome sequencing (WGS) promises to reveal more details about mixed infection with high resolution. WGS has been used for detection of mixed infection with high sensitivity and discriminatory, but the technology is currently limited to developed countries. Mixed infection may involve strains with different susceptibility patterns, which may alter the treatment outcome. In this report, we review the current concepts of mixed strains infection and also infection involving strains with a different susceptibility pattern in TB. We evaluate the importance of identifying mixed infection for diagnosis as well as treatment and highlight the accuracy and clinical utility of direct genotyping of clinical specimens. Copyright © 2017 Elsevier Ltd. All rights reserved.

Correlation between Genotypic and Phenotypic Testing for Resistance to Rifampin in Mycobacterium tuberculosis Clinical Isolates in Haiti: Investigation of Cases with Discrepant Susceptibility Results

PubMed Central

Ocheretina, Oksana; Escuyer, Vincent E.; Mabou, Marie-Marcelle; Royal-Mardi, Gertrude; Collins, Sean; Vilbrun, Stalz C.; Pape, Jean W.; Fitzgerald, Daniel W.

2014-01-01

The World Health Organization has recommended use of molecular-based tests MTBDRplus and GeneXpert MTB/RIF to diagnose multidrug-resistant tuberculosis in developing and high-burden countries. Both tests are based on detection of mutations in the Rifampin (RIF) Resistance-Determining Region of DNA-dependent RNA Polymerase gene (rpoB). Such mutations are found in 95–98% of Mycobacterium tuberculosis strains determined to be RIF-resistant by the “gold standard” culture-based drug susceptibility testing (DST). We report the phenotypic and genotypic characterization of 153 consecutive clinical Mycobacterium tuberculosis strains diagnosed as RIF-resistant by molecular tests in our laboratory in Port-au-Prince, Haiti. 133 isolates (86.9%) were resistant to both RIF and Isoniazid and 4 isolates (2.6%) were RIF mono-resistant in MGIT SIRE liquid culture-based DST. However the remaining 16 isolates (10.5%) tested RIF-sensitive by the assay. Five strains with discordant genotypic and phenotypic susceptibility results had RIF minimal inhibitory concentration (MIC) close to the cut-off value of 1 µg/ml used in phenotypic susceptibility assays and were confirmed as resistant by DST on solid media. Nine strains had sub-critical RIF MICs ranging from 0.063 to 0.5 µg/ml. Finally two strains were pan-susceptible and harbored a silent rpoB mutation. Our data indicate that not only detection of the presence but also identification of the nature of rpoB mutation is needed to accurately diagnose resistance to RIF in Mycobacterium tuberculosis. Observed clinical significance of low-level resistance to RIF supports the re-evaluation of the present critical concentration of the drug used in culture-based DST assays. PMID:24599230

Mouse model of necrotic tuberculosis granulomas develops hypoxic lesions.

PubMed

Harper, Jamie; Skerry, Ciaran; Davis, Stephanie L; Tasneen, Rokeya; Weir, Mariah; Kramnik, Igor; Bishai, William R; Pomper, Martin G; Nuermberger, Eric L; Jain, Sanjay K

2012-02-15

Preclinical evaluation of tuberculosis drugs is generally limited to mice. However, necrosis and hypoxia, key features of human tuberculosis lesions, are lacking in conventional mouse strains. We used C3HeB/FeJ mice, which develop necrotic lesions in response to Mycobacterium tuberculosis infection. Positron emission tomography in live infected animals, postmortem pimonidazole immunohistochemistry, and bacterial gene expression analyses were used to assess whether tuberculosis lesions in C3HeB/FeJ are hypoxic. Efficacy of combination drug treatment, including PA-824, active against M. tuberculosis under hypoxic conditions, was also evaluated. Tuberculosis lesions in C3HeB/FeJ (but not BALB/c) were found to be hypoxic and associated with up-regulation of known hypoxia-associated bacterial genes (P < .001). Contrary to sustained activity reported elsewhere in BALB/c mice, moxifloxacin and pyrazinamide (MZ) combination was not bactericidal beyond 3 weeks in C3HeB/FeJ. Although PA-824 added significant activity, the novel combination of PA-824 and MZ was less effective than the standard first-line regimen in C3HeB/FeJ. We demonstrate that tuberculosis lesions in C3HeB/FeJ are hypoxic. Activities of some key tuberculosis drug regimens in development are represented differently in C3HeB/FeJ versus BALB/c mice. Because C3HeB/FeJ display key features of human tuberculosis, this strain warrants evaluation as a more pathologically relevant model for preclinical studies.

Rapid Test for Identification of a Highly Transmissible Mycobacterium tuberculosis Beijing Strain of Sub-Saharan Origin

PubMed Central

Alonso, Henar; Gavín, Patricia; Hernández-Febles, Melissa; Campos-Herrero, María Isolina; Copado, Rodolfo; Cañas, Fernando; Kremer, Kristin; Caminero, José Antonio; Martín, Carlos; Samper, Sofía

2012-01-01

The development of a rapid test to identify Mycobacterium tuberculosis Beijing isolates and specifically strain GC1237, coming from a sub-Saharan country, is needed due to its alarming wide spread on Gran Canaria Island (Spain). A rapid test that detects IS6110 present between dnaA and dnaN in the Beijing strains and in a specific site for GC1237 (Rv2180c) has been developed. This test would be a useful tool in the surveillance of subsequent cases. PMID:22116140

Principles for designing future regimens for multidrug-resistant tuberculosis.

PubMed

Brigden, Grania; Nyang'wa, Bern-Thomas; du Cros, Philipp; Varaine, Francis; Hughes, Jennifer; Rich, Michael; Horsburgh, C Robert; Mitnick, Carole D; Nuermberger, Eric; McIlleron, Helen; Phillips, Patrick P J; Balasegaram, Manica

2014-01-01

Fewer than 20% of patients with multidrug-resistant (MDR) tuberculosis are receiving treatment and there is an urgent need to scale up treatment programmes. One of the biggest barriers to scale-up is the treatment regimen, which is lengthy, complex, ineffective, poorly tolerated and expensive. For the first time in over 50 years, new drugs have been developed specifically to treat tuberculosis, with bedaquiline and potentially delamanid expected to be available soon for treatment of MDR cases. However, if the new drugs are merely added to the current treatment regimen, the new regimen will be at least as lengthy, cumbersome and toxic as the existing one. There is an urgent need for strategy and evidence on how to maximize the potential of the new drugs to improve outcomes and shorten treatment. We devised eight key principles for designing future treatment regimens to ensure that, once they are proven safe in clinical trials, they will be clinically effective and programmatically practicable. Regimens should contain at least one new class of drug; be broadly applicable for use against MDR and extensively drug-resistant Mycobacterium tuberculosis complex strains; contain three to five effective drugs, each from a different drug class; be delivered orally; have a simple dosing schedule; have a good side-effect profile that allows limited monitoring; last a maximum of 6 months; and have minimal interaction with antiretrovirals. Following these principles will maximize the potential of new compounds and help to overcome the clinical and programmatic disadvantages and scale-up constraints that plague the current regimen.

Genetic diversity, transmission dynamics and drug resistance of Mycobacterium tuberculosis in Angola.

PubMed

Perdigão, João; Clemente, Sofia; Ramos, Jorge; Masakidi, Pedro; Machado, Diana; Silva, Carla; Couto, Isabel; Viveiros, Miguel; Taveira, Nuno; Portugal, Isabel

2017-02-23

Tuberculosis (TB) poses a serious public health problem in Angola. No surveillance data on drug resistance is available and nothing is known regarding the genetic diversity and population structure of circulating Mycobacterium tuberculosis strains. Here, we have genotyped and evaluated drug susceptibility of 89 Mycobacterium tuberculosis clinical isolates from Luanda. Thirty-three different spoligotype profiles corresponding to 24 different Shared International Types (SIT) and 9 orphan profiles were detected. SIT 20 (LAM1) was the most prevalent (n = 16, 18.2%) followed by SIT 42 (LAM9; n = 15, 17.1%). Overall, the M. tuberculosis population structure in this sample was dominated by LAM (64.8%) and T (33.0%) strains. Twenty-four-loci MIRU-VNTR analysis revealed that a total of 13 isolates were grouped in 5 distinct clusters. Drug susceptibility data showed that 22 (24.7%) of the 89 clinical isolates were resistant to one or more antibacillary drugs of which 4 (4.5%) were multidrug resistant. In conclusion, this study demonstrates a high predominance of LAM strains circulating in the Luanda setting and the presence of recent transmission events. The rate and the emergence dynamics of drug resistant TB found in this sample are significant and highlight the need of further studies specifically focused on MDR-TB transmission.

Effect of Lagerstroemia tomentosa and Diospyros virginiana methanolic extracts on different drug-resistant strains of Mycobacterium tuberculosis

PubMed Central

Esfahani, B. Nasr; Hozoorbakhsh, F.; Rashed, Kh.; Havaei, S.A.; Heidari, K.; Moghim, Sh.

2014-01-01

Mycobacterium tuberculosis (MTB) is the causative agent of tuberculosis. The increasing incidence of multi drug resistance tuberculosis (MDR-TB) and extensively drug resistance tuberculosis (XDR-TB) worldwide highlighted the urgent need to search for alternative antimycobacterial agents. More and more people in developing countries utilize traditional medicine for their major primary health care needs. It has been determined that pharmaceutical plant, Lagerstroemia tomentosa and Diospyros virginiana, possesses some antibacterial effect. In this study, the antimycobacterial effects of L. tomentosa and D. virginiana methanolic extracts on sensitive and resistant isolates of MTB were examined. Leaf methanolic extract was prepared using methanol 70%. Sensitivity and resistance of isolates was determined by proportion method. The effects of two different methonolic extract concentrations (20 and 40 μg/ml) of the plants were examined against 6 sensitive and resistant strains of MTB with different patterns of drug resistance. MTB H37Rv (ATCC 27294) was set as control in all culturing and sensitivity testing processes. The results showed that L. tomentosa and D. virginiana methanolic extracts had weak inhibitory effect on different strains of MTB. The highest percentage of inhibition for L. tomentosa and D. virginiana was observed 38% and 33.3%, respectively. PMID:25657789

Genomic analysis of globally diverse Mycobacterium tuberculosis strains provides insights into the emergence and spread of multidrug resistance.

PubMed

Manson, Abigail L; Cohen, Keira A; Abeel, Thomas; Desjardins, Christopher A; Armstrong, Derek T; Barry, Clifton E; Brand, Jeannette; Chapman, Sinéad B; Cho, Sang-Nae; Gabrielian, Andrei; Gomez, James; Jodals, Andreea M; Joloba, Moses; Jureen, Pontus; Lee, Jong Seok; Malinga, Lesibana; Maiga, Mamoudou; Nordenberg, Dale; Noroc, Ecaterina; Romancenco, Elena; Salazar, Alex; Ssengooba, Willy; Velayati, A A; Winglee, Kathryn; Zalutskaya, Aksana; Via, Laura E; Cassell, Gail H; Dorman, Susan E; Ellner, Jerrold; Farnia, Parissa; Galagan, James E; Rosenthal, Alex; Crudu, Valeriu; Homorodean, Daniela; Hsueh, Po-Ren; Narayanan, Sujatha; Pym, Alexander S; Skrahina, Alena; Swaminathan, Soumya; Van der Walt, Martie; Alland, David; Bishai, William R; Cohen, Ted; Hoffner, Sven; Birren, Bruce W; Earl, Ashlee M

2017-03-01

Multidrug-resistant tuberculosis (MDR-TB), caused by drug-resistant strains of Mycobacterium tuberculosis, is an increasingly serious problem worldwide. Here we examined a data set of whole-genome sequences from 5,310 M. tuberculosis isolates from five continents. Despite the great diversity of these isolates with respect to geographical point of isolation, genetic background and drug resistance, the patterns for the emergence of drug resistance were conserved globally. We have identified harbinger mutations that often precede multidrug resistance. In particular, the katG mutation encoding p.Ser315Thr, which confers resistance to isoniazid, overwhelmingly arose before mutations that conferred rifampicin resistance across all of the lineages, geographical regions and time periods. Therefore, molecular diagnostics that include markers for rifampicin resistance alone will be insufficient to identify pre-MDR strains. Incorporating knowledge of polymorphisms that occur before the emergence of multidrug resistance, particularly katG p.Ser315Thr, into molecular diagnostics should enable targeted treatment of patients with pre-MDR-TB to prevent further development of MDR-TB.

Biofilms formed by Mycobacterium tuberculosis on cement, ceramic, and stainless steel surfaces and their controls.

PubMed

Adetunji, Victoria; Kehinde, Aderemi; Bolatito, Olayemi; Chen, Jinru

2014-04-01

This study assessed the biofilms formed by selected strains of Mycobacterium tuberculosis and investigated the efficacy of three different treatments to control the biofilms. Two M. tuberculosis strains were inoculated separately in 150 ml of Middlebrook 7H9-Tween 80 (0.1%) broth with 5% liver extract and 10% oleic albumin dextrose catalase (OADC) supplement, 5% liver extract alone, or 10% OADC alone in sterile jars, each containing a 2-cm2 coupon of cement, ceramic, or stainless steel for biofilm development at 37 °C, with agitation for 2, 3, or 4 weeks. Biofilms on the coupons were exposed to 10 ml of 2% sanitizer A or 0.5% sanitizer B at 28 and 45 °C and to hot water at 85 °C for 5 min. Residual biofilms on treated and untreated coupons were assessed. Both strains of M. tuberculosis formed biofilms on the three surfaces; however, one strain formed more biofilms. More biofilms were formed when media containing 5% liver extract was used. Biofilm mass increased as incubation time increased until the third week. More biofilms were formed on cement than on ceramic and stainless steel coupons. Sanitizing treatments at 45 °C removed more biofilms than those at 28 °C. However, neither treatment completely eliminated the biofilms.

Effect of Active Case Finding on Prevalence and Transmission of Pulmonary Tuberculosis in Dhaka Central Jail, Bangladesh

PubMed Central

Banu, Sayera; Rahman, Md. Toufiq; Uddin, Mohammad Khaja Mafij; Khatun, Razia; Khan, Md. Siddiqur Rahman; Rahman, Md. Mojibur; Uddin, Syed Iftekhar; Ahmed, Tahmeed; Heffelfinger, James D.

2015-01-01

Background Understanding tuberculosis (TB) transmission dynamics is essential for establishing effective TB control strategies in settings where the burden and risk of transmission are high. The objectives of this study were to evaluate the effect of active screening on controlling TB transmission and also to characterize Mycobacterium tuberculosis strains for investigating transmission dynamics in a correctional setting. Methods The study was carried out in Dhaka Central Jail (DCJ), from October 2005 to February 2010. An active case finding strategy for pulmonary TB was established both at the entry point to the prison and inside the prison. Three sputum specimens were collected from all pulmonary TB suspects and subjected to smear microscopy, culture, and drug susceptibility testing as well as genotyping which included deletion analysis, spoligotyping and analysis of mycobacterial interspersed repetitive units (MIRU). Results A total of 60,585 inmates were screened during the study period. We found 466 inmates with pulmonary TB of whom 357 (77%) had positive smear microscopy results and 109 (23%) had negative smear microscopy results but had positive results on culture. The number of pulmonary TB cases declined significantly, from 49 cases during the first quarter to 8 cases in the final quarter of the study period (p=0.001). Deletion analysis identified all isolates as M. tuberculosis and further identified 229 (70%) strains as ‘modern’ and 100 (30%) strains as ‘ancestral’. Analysis of MIRU showed that 347 strains (85%) exhibited unique patterns, whereas 61 strains (15%) clustered into 22 groups. The largest cluster comprised eight strains of the Beijing M. tuberculosis type. The rate of recent transmission was estimated to be 9.6%. Conclusions Implementation of active screening for TB was associated with a decline in TB cases in DCJ. Implementation of active screening in prison settings might substantially reduce the national burden of TB in Bangladesh. PMID:25933377

A country-wide study of spoligotype and drug resistance characteristics of Mycobacterium tuberculosis isolates from children in China.

PubMed

Jiao, Weiwei; Liu, Zhiguang; Han, Rui; Zhao, Xiuqin; Dong, Fang; Dong, Haiyan; Huang, Hairong; Tian, Jianling; Li, Qinjing; Lian, Lulu; Yin, Qingqin; Song, Wenqi; Wan, Kanglin; Shen, A-Dong

2013-01-01

Tuberculosis (TB) is still a big threat to human health, especially in children. However, an isolation of Mycobacterium tuberculosis culture from pediatric cases remains a challenge. In order to provide some scientific basis for children TB control, we investigated the genotyping and drug resistance characteristics of M. tuberculosis isolates from pediatric cases in China. In this study, a total of 440 strains including 90 from children (<15 years), 159 from adolescents (15-18 years) and 191 from adults (>18 years) isolated in 25 provinces across China were subjected to spoligotyping and drug susceptibility testing. As a result, Beijing family strains were shown to remain predominant in China (85.6%, 81.1% and 75.4% in three above groups, respectively), especially among new children cases (91.0% vs. 69.6% in previously treated cases, P=0.03). The prevalence of the Beijing genotype isolates was higher in northern and central China in the total collection (85.1% in northern and 83.9% in central vs. 61.6% in southern China, P<0.001) and a similar trend was seen in all three age groups (P=0.708, <0.001 and 0.025, respectively). In adolescents, the frequencies of isoniazid (INH)-resistant and ethambutol (EMB)-resistant isolates were significantly higher among Beijing strains compared to non-Beijing genotype strains (P=0.028 for INH and P=0.027 for EMB). Furthermore, strong association was observed between resistance to rifampicine (RIF), streptomycin (STR) and multidrug resistance (MDR) among Beijing compared to non-Beijing strains in previously treated cases of children (P=0.01, 0.01 and 0.025, respectively). Beijing family was more prevalent in northern and central China compared to southern China and these strains were predominant in all age groups. The genetic diversity of M. tuberculosis isolates from children was similar to that found in adolescents and adults. Beijing genotype was associated with RIF, STR and MDR resistance in previously treated children.

Effect of active case finding on prevalence and transmission of pulmonary tuberculosis in Dhaka Central Jail, Bangladesh.

PubMed

Banu, Sayera; Rahman, Md Toufiq; Uddin, Mohammad Khaja Mafij; Khatun, Razia; Khan, Md Siddiqur Rahman; Rahman, Md Mojibur; Uddin, Syed Iftekhar; Ahmed, Tahmeed; Heffelfinger, James D

2015-01-01

Understanding tuberculosis (TB) transmission dynamics is essential for establishing effective TB control strategies in settings where the burden and risk of transmission are high. The objectives of this study were to evaluate the effect of active screening on controlling TB transmission and also to characterize Mycobacterium tuberculosis strains for investigating transmission dynamics in a correctional setting. The study was carried out in Dhaka Central Jail (DCJ), from October 2005 to February 2010. An active case finding strategy for pulmonary TB was established both at the entry point to the prison and inside the prison. Three sputum specimens were collected from all pulmonary TB suspects and subjected to smear microscopy, culture, and drug susceptibility testing as well as genotyping which included deletion analysis, spoligotyping and analysis of mycobacterial interspersed repetitive units (MIRU). A total of 60,585 inmates were screened during the study period. We found 466 inmates with pulmonary TB of whom 357 (77%) had positive smear microscopy results and 109 (23%) had negative smear microscopy results but had positive results on culture. The number of pulmonary TB cases declined significantly, from 49 cases during the first quarter to 8 cases in the final quarter of the study period (p=0.001). Deletion analysis identified all isolates as M. tuberculosis and further identified 229 (70%) strains as 'modern' and 100 (30%) strains as 'ancestral'. Analysis of MIRU showed that 347 strains (85%) exhibited unique patterns, whereas 61 strains (15%) clustered into 22 groups. The largest cluster comprised eight strains of the Beijing M. tuberculosis type. The rate of recent transmission was estimated to be 9.6%. Implementation of active screening for TB was associated with a decline in TB cases in DCJ. Implementation of active screening in prison settings might substantially reduce the national burden of TB in Bangladesh.

Mycobacterium tuberculosis causing tuberculous lymphadenitis in Maputo, Mozambique.

PubMed

Viegas, Sofia Omar; Ghebremichael, Solomon; Massawo, Leguesse; Alberto, Matos; Fernandes, Fabíola Couto; Monteiro, Eliane; Couvin, David; Matavele, José Maiane; Rastogi, Nalin; Correia-Neves, Margarida; Machado, Adelina; Carrilho, Carla; Groenheit, Ramona; Källenius, Gunilla; Koivula, Tuija

2015-11-21

The zoonosis bovine tuberculosis (TB) is known to be responsible for a considerable proportion of extrapulmonary TB. In Mozambique, bovine TB is a recognised problem in cattle, but little has been done to evaluate how Mycobacterium bovis has contributed to human TB. We here explore the public health risk for bovine TB in Maputo, by characterizing the isolates from tuberculous lymphadenitis (TBLN) cases, a common manifestation of bovine TB in humans, in the Pathology Service of Maputo Central Hospital, in Mozambique, during one year. Among 110 patients suspected of having TBLN, 49 had a positive culture result. Of those, 48 (98%) were positive for Mycobacterium tuberculosis complex and one for nontuberculous mycobacteria. Of the 45 isolates analysed by spoligotyping and Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR), all were M. tuberculosis. No M. bovis was found. Cervical TBLN, corresponding to 39 (86.7%) cases, was the main cause of TBLN and 66.7% of those where from HIV positive patients. We found that TBLN in Maputo was caused by a variety of M. tuberculosis strains. The most prevalent lineage was the EAI (n = 19; 43.2%). Particular common spoligotypes were SIT 48 (EAI1_SOM sublineage), SIT 42 (LAM 9), SIT 1 (Beijing) and SIT53 (T1), similar to findings among pulmonary cases. M. tuberculosis was the main etiological agent of TBLN in Maputo. M. tuberculosis genotypes were similar to the ones causing pulmonary TB, suggesting that in Maputo, cases of TBLN arise from the same source as pulmonary TB, rather than from an external zoonotic source. Further research is needed on other forms of extrapulmonary TB and in rural areas where there is high prevalence of bovine TB in cattle, to evaluate the risk of transmission of M. bovis from cattle to humans.

Diagnosis of local hepatic tuberculosis through next-generation sequencing: Smarter, faster and better.

PubMed

Ai, Jing-Wen; Li, Yang; Cheng, Qi; Cui, Peng; Wu, Hong-Long; Xu, Bin; Zhang, Wen-Hong

2018-06-01

A 45-year-old man who complained of continuous fever and multiple hepatic masses was admitted to our hospital. Repeated MRI manifestations were similar while each radiological report suggested contradictory diagnosis pointing to infections or malignances respectively. Pathologic examination of the liver tissue showed no direct evidence of either infections or tumor. We performed next-generation sequencing on the liver tissue and peripheral blood to further investigate the possible etiology. High throughput sequencing was performed on the liver lesion tissues using BGISEQ-100 platform, and data was mapped to the Microbial Genome Databases after filtering low quality data and human reads. We identified a total of 299 sequencing reads of Mycobacterium tuberculosis (M. tuberculosis) complex sequences from the liver tissue, including 8, 229 of 4,424,435 of the M. tuberculosis nucleotide sequences, and Mycobacterium africanum, Mycobacterium bovis, and Mycobacterium canettii were also detected due to the 99.9% identical rate among these strains. No specific Mycobacterial tuberculosis nucleotide sequence was detected in the sample of peripheral blood. Patient's symptom quickly recovered after anti-tuberculosis treatment and repeated Ziehl-Neelsen staining of the liver tissue finally identified small numbers of positive bacillus. The diagnosis of this patient was difficult to establish before the next-generation sequencing because of contradictive radiological results and negative pathological findings. More sensitive diagnostic methods are urgently needed. This is the first case reporting hepatic tuberculosis confirmed by the next-generation sequencing, and marks the promising potential of the application of the next-generation sequencing in the diagnosis of hepatic lesions with unknown etiology. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Whole genome sequencing identifies circulating Beijing-lineage Mycobacterium tuberculosis strains in Guatemala and an associated urban outbreak.

PubMed

Saelens, Joseph W; Lau-Bonilla, Dalia; Moller, Anneliese; Medina, Narda; Guzmán, Brenda; Calderón, Maylena; Herrera, Raúl; Sisk, Dana M; Xet-Mull, Ana M; Stout, Jason E; Arathoon, Eduardo; Samayoa, Blanca; Tobin, David M

2015-12-01

Limited data are available regarding the molecular epidemiology of Mycobacterium tuberculosis (Mtb) strains circulating in Guatemala. Beijing-lineage Mtb strains have gained prevalence worldwide and are associated with increased virulence and drug resistance, but there have been only a few cases reported in Central America. Here we report the first whole genome sequencing of Central American Beijing-lineage strains of Mtb. We find that multiple Beijing-lineage strains, derived from independent founding events, are currently circulating in Guatemala, but overall still represent a relatively small proportion of disease burden. Finally, we identify a specific Beijing-lineage outbreak centered on a poor neighborhood in Guatemala City. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Low Dose Aerosol Fitness at the Innate Phase of Murine Infection Better Predicts Virulence amongst Clinical Strains of Mycobacterium tuberculosis

PubMed Central

Caceres, Neus; Llopis, Isaac; Marzo, Elena; Prats, Clara; Vilaplana, Cristina; de Viedma, Dario Garcia; Samper, Sofía; Lopez, Daniel; Cardona, Pere-Joan

2012-01-01

Background Evaluation of a quick and easy model to determine the intrinsic ability of clinical strains to generate active TB has been set by assuming that this is linked to the fitness of Mycobacterium tuberculosis strain at the innate phase of the infection. Thus, the higher the bacillary load, the greater the possibility of inducting liquefaction, and thus active TB, once the adaptive response is set. Methodology/Principal Findings The virulence of seven clinical Mycobacterium tuberculosis strains isolated in Spain was tested by determining the bacillary concentration in the spleen and lung of mice at weeks 0, 1 and 2 after intravenous (IV) inoculation of 104 CFU, and by determining the growth in vitro until the stationary phase had been reached. Cord distribution automated analysis showed two clear patterns related to the high and low fitness in the lung after IV infection. This pattern was not seen in the in vitro fitness tests, which clearly favored the reference strain (H37Rv). Subsequent determination using a more physiological low-dose aerosol (AER) inoculation with 102 CFU showed a third pattern in which the three best values coincided with the highest dissemination capacity according to epidemiological data. Conclusions/Significance The fitness obtained after low dose aerosol administration in the presence of the innate immune response is the most predictive factor for determining the virulence of clinical strains. This gives support to a mechanism of the induction of active TB derived from the dynamic hypothesis of latent tuberculosis infection. PMID:22235258

[Molecular strain typing contribution to epidemiology of tuberculosis in Limousin (1998 to 2006)].

PubMed

Bezanahary, H; Baclet, M-C; Sola, C; Gazaille, V; Turlure, P; Weinbreck, P; Denis, F; Martin, C

2008-06-01

We conducted a molecular epidemiology of Mycobacterium tuberculosis in Limousin, a French area with a low incidence of tuberculosis (4.8/100,000 inhabitants in 2005) to define the molecular diversity and the pattern of transmission. Two hundred and fifty-nine strains were isolated (each strain corresponds to one patient) from 1998 to 2006. Both spoligotyping and MIRU15 were chosen for our study because of their discriminatory power. Only 165 medical records were available: 99M/66F, mean age 56.4 years (14-94), 32.7% foreign-born patients, 16.9% homeless or living in shelters, 21.8% of immunocompromised patients (three HIV positive), 14.5% of alcohol addicts. Pulmonary manifestations were predominant (81.8%) with 45.1% of positive smears. Two strains among the 259 presented a multidrug resistance. Spoligotyping identified 136/259 spoligotypes (110 unique, 26 clusters composed of two to 36 isolates); within these 26 clusters, ST53 (n=36) and ST50 (n=19) were the most frequent. Three major families were observed as follow: T1 (30%), Haarlem (30%) and LAM (20%). MIRU15 identified 28/36 isolates in the ST53 group and 14/19 in the ST50 group. Eleven clusters (32 strains) with identical ST-MIRU15 were obtained with a proved case of recent transmission. Alcohol dependence, immunosuppression and pulmonary infections seem to be involved in transmission factors. M. tuberculosis strains isolated in Limousin are characterized by their high genetic diversity. The rate of recent transmission (8.1%) is low and therefore a reactivation process is predominant in this area.

Specific Proteins in Nontuberculous Mycobacteria: New Potential Tools.

PubMed

Orduña, Patricia; Castillo-Rodal, Antonia I; Mercado, Martha E; Ponce de León, Samuel; López-Vidal, Yolanda

2015-01-01

Nontuberculous mycobacteria (NTM) have been isolated from water, soil, air, food, protozoa, plants, animals, and humans. Although most NTM are saprophytes, approximately one-third of NTM have been associated with human diseases. In this study, we did a comparative proteomic analysis among five NTM strains isolated from several sources. There were different numbers of protein spots from M. gordonae (1,264), M. nonchromogenicum type I (894), M. nonchromogenicum type II (935), M. peregrinum (806), and M. scrofulaceum/Mycobacterium mantenii (1,486) strains, respectively. We identified 141 proteins common to all strains and specific proteins to each NTM strain. A total of 23 proteins were selected for its identification. Two of the common proteins identified (short-chain dehydrogenase/reductase SDR and diguanylate cyclase) did not align with M. tuberculosis complex protein sequences, which suggest that these proteins are found only in the NTM strains. Some of the proteins identified as common to all strains can be used as markers of NTM exposure and for the development of new diagnostic tools. Additionally, the specific proteins to NTM strains identified may represent potential candidates for the diagnosis of diseases caused by these mycobacteria.

Molecular epidemiology of Mycobacterium tuberculosis complex strains isolated from livestock and wild animals in Italy suggests the need for a different eradication strategy for bovine tuberculosis.

PubMed

Amato, B; Di Marco Lo Presti, V; Gerace, E; Capucchio, M T; Vitale, M; Zanghì, P; Pacciarini, M L; Marianelli, C; Boniotti, M B

2018-04-01

Bovine tuberculosis (bTB) is an important zoonosis, which has been re-emerging in different ecological scenarios. In Sicily, Italy, from 2004 to 2014, an anatomopathological survey for tuberculosis-like lesions both in farmed and wild animals was performed. The isolates were genotyped using spoligotyping and Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats (MIRU-VNTR) techniques. High prevalence of lesions was observed for cattle (4%), pigs (4.9%) and wild boars (6.8%), and a total of 625 Mycobacterium bovis isolates were identified. Genotyping analysis showed the presence of 37 different spoligotypes including fifteen spoligotypes not present in other Italian regions and 266 MIRU-VNTR profiles. Spoligotype SB0120 exhibited the highest prevalence in cattle (50%) and pigs (56%) and the highest genetic variety with 126 different MIRU-VNTR profiles. The isolation of M. bovis in a farmer underlines the importance of M. bovis identification during the human TB diagnostic processes. This study supported the use of the genotyping analysis as a valuable tool for the evaluation of the epidemiological role of pigs and other domestic reservoirs such as goats and the role of wildlife in the maintenance of bTB infection. © 2017 Blackwell Verlag GmbH.

Lipoarabinomannan and related glycoconjugates: structure, biogenesis and role in Mycobacterium tuberculosis physiology and host–pathogen interaction

PubMed Central

Mishra, Arun K; Driessen, Nicole N; Appelmelk, Ben J; Besra, Gurdyal S

2011-01-01

Approximately one third of the world's population is infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. This bacterium has an unusual lipid-rich cell wall containing a vast repertoire of antigens, providing a hydrophobic impermeable barrier against chemical drugs, thus representing an attractive target for vaccine and drug development. Apart from the mycolyl–arabinogalactan–peptidoglycan complex, mycobacteria possess several immunomodulatory constituents, notably lipomannan and lipoarabinomannan. The availability of whole-genome sequences of M. tuberculosis and related bacilli over the past decade has led to the identification and functional characterization of various enzymes and the potential drug targets involved in the biosynthesis of these glycoconjugates. Both lipomannan and lipoarabinomannan possess highly variable chemical structures, which interact with different receptors of the immune system during host–pathogen interactions, such as Toll-like receptors-2 and C-type lectins. Recently, the availability of mutants defective in the synthesis of these glycoconjugates in mycobacteria and the closely related bacterium, Corynebacterium glutamicum, has paved the way for host–pathogen interaction studies, as well as, providing attenuated strains of mycobacteria for the development of new vaccine candidates. This review provides a comprehensive account of the structure, biosynthesis and immunomodulatory properties of these important glycoconjugates. PMID:21521247

Rv3852 (H-NS) of Mycobacterium tuberculosis Is Not Involved in Nucleoid Compaction and Virulence Regulation.

PubMed

Odermatt, Nina T; Sala, Claudia; Benjak, Andrej; Kolly, Gaëlle S; Vocat, Anthony; Lupien, Andréanne; Cole, Stewart T

2017-08-15

A handful of nucleoid-associated proteins (NAPs) regulate the vast majority of genes in a bacterial cell. H-NS, the h istone-like n ucleoid- s tructuring protein, is one of these NAPs and protects Escherichia coli from foreign gene expression. Though lacking any sequence similarity with E. coli H-NS, Rv3852 was annotated as the H-NS ortholog in Mycobacterium tuberculosis , as it resembles human histone H1. The role of Rv3852 was thoroughly investigated by immunoblotting, subcellular localization, construction of an unmarked rv3852 deletion in the M. tuberculosis genome, and subsequent analysis of the resulting Δ rv3852 strain. We found that Rv3852 was predominantly present in the logarithmic growth phase with a decrease in protein abundance in stationary phase. Furthermore, it was strongly associated with the cell membrane and not detected in the cytosolic fraction, nor was it secreted. The Δ rv3852 strain displayed no growth defect or morphological abnormalities. Quantitative measurement of nucleoid localization in the Δ rv3852 mutant strain compared to that in the parental H37Rv strain showed no difference in nucleoid position or spread. Infection of macrophages as well as severe combined immunodeficient (SCID) mice demonstrated that loss of Rv3852 had no detected influence on the virulence of M. tuberculosis We thus conclude that M. tuberculosis Rv3852 is not involved in pathogenesis and is not a typical NAP. The existence of an as yet undiscovered Rv3852 ortholog cannot be excluded, although this role is likely played by the well-characterized Lsr2 protein. IMPORTANCE Mycobacterium tuberculosis is the causative agent of the lung infection tuberculosis, claiming more than 1.5 million lives each year. To understand the mechanisms of latent infection, where M. tuberculosis can stay dormant inside the human host, we require deeper knowledge of the basic biology and of the regulatory networks. In our work, we show that Rv3852, previously annotated as H-NS, is not a typical nucleoid-associated protein (NAP) as expected from its initial annotation. Rv3852 from M. tuberculosis has neither influence on nucleoid shape or compaction nor a role in virulence. Our findings reduce the repertoire of identified nucleoid-associated proteins in M. tuberculosis to four transcription regulators and underline the importance of genetic studies to assign a function to bacterial genes. Copyright © 2017 American Society for Microbiology.

Mycobacterium tuberculosis ecology in Venezuela: epidemiologic correlates of common spoligotypes and a large clonal cluster defined by MIRU-VNTR-24

PubMed Central

2009-01-01

Background Tuberculosis remains an endemic public health problem, but the ecology of the TB strains prevalent, and their transmission, can vary by country and by region. We sought to investigate the prevalence of Mycobacterium tuberculosis strains in different regions of Venezuela. A previous study identified the most prevalent strains in Venezuela but did not show geographical distribution nor identify clonal genotypes. To better understand local strain ecology, we used spoligotyping to analyze 1298 M. tuberculosis strains isolated in Venezuela from 1997 to 2006, predominantly from two large urban centers and two geographically distinct indigenous areas, and then studied a subgroup with MIRU-VNTR 24 loci. Results The distribution of spoligotype families is similar to that previously reported for Venezuela and other South American countries: LAM 53%, T 10%, Haarlem 5%, S 1.9%, X 1.2%, Beijing 0.4%, and EAI 0.2%. The six most common shared types (SIT's 17, 93, 605, 42, 53, 20) accounted for 49% of the isolates and were the most common in almost all regions, but only a minority were clustered by MIRU-VNTR 24. One exception was the third most frequent overall, SIT 605, which is the most common spoligotype in the state of Carabobo but infrequent in other regions. MIRU-VNTR homogeneity suggests it is a clonal group of strains and was named the "Carabobo" genotype. Epidemiologic comparisons showed that patients with SIT 17 were younger and more likely to have had specimens positive for Acid Fast Bacilli on microscopy, and patients with SIT 53 were older and more commonly smear negative. Female TB patients tended to be younger than male patients. Patients from the high incidence, indigenous population in Delta Amacuro state were younger and had a nearly equal male:female distribution. Conclusion Six SIT's cause nearly half of the cases of tuberculosis in Venezuela and dominate in nearly all regions. Strains with SIT 17, the most common pattern overall may be more actively transmitted and SIT 53 strains may be less virulent and associated with reactivation of past infections in older patients. In contrast to other common spoligotypes, strains with SIT 605 form a clonal group centered in the state of Carabobo. PMID:19660112

In vitro and in vivo efficacy of β-lactams against replicating and slowly growing/nonreplicating Mycobacterium tuberculosis.

PubMed

Solapure, Suresh; Dinesh, Neela; Shandil, Radha; Ramachandran, Vasanthi; Sharma, Sreevalli; Bhattacharjee, Deepa; Ganguly, Samit; Reddy, Jitendar; Ahuja, Vijaykamal; Panduga, Vijender; Parab, Manish; Vishwas, K G; Kumar, Naveen; Balganesh, Meenakshi; Balasubramanian, V

2013-06-01

Beta-lactams, in combination with beta-lactamase inhibitors, are reported to have activity against Mycobacterium tuberculosis bacteria growing in broth, as well as inside the human macrophage. We tested representative beta-lactams belonging to 3 different classes for activity against replicating M. tuberculosis in broth and nonreplicating M. tuberculosis under hypoxia, as well as against streptomycin-starved M. tuberculosis strain 18b (ss18b) in the presence or absence of clavulanate. Most of the combinations showed bactericidal activity against replicating M. tuberculosis, with up to 200-fold improvement in potency in the presence of clavulanate. None of the combinations, including those containing meropenem, imipenem, and faropenem, killed M. tuberculosis under hypoxia. However, faropenem- and meropenem-containing combinations killed strain ss18b moderately. We tested the bactericidal activities of meropenem-clavulanate and amoxicillin-clavulanate combinations in the acute and chronic aerosol infection models of tuberculosis in BALB/c mice. Based on pharmacokinetic/pharmacodynamic indexes reported for beta-lactams against other bacterial pathogens, a cumulative percentage of a 24-h period that the drug concentration exceeds the MIC under steady-state pharmacokinetic conditions (%TMIC) of 20 to 40% was achieved in mice using a suitable dosing regimen. Both combinations showed marginal reduction in lung CFU compared to the late controls in the acute model, whereas both were inactive in the chronic model.

In Vitro and In Vivo Efficacy of β-Lactams against Replicating and Slowly Growing/Nonreplicating Mycobacterium tuberculosis

PubMed Central

Dinesh, Neela; Shandil, Radha; Ramachandran, Vasanthi; Sharma, Sreevalli; Bhattacharjee, Deepa; Ganguly, Samit; Reddy, Jitendar; Ahuja, Vijaykamal; Panduga, Vijender; Parab, Manish; Vishwas, K. G.; Kumar, Naveen; Balganesh, Meenakshi; Balasubramanian, V.

2013-01-01

Beta-lactams, in combination with beta-lactamase inhibitors, are reported to have activity against Mycobacterium tuberculosis bacteria growing in broth, as well as inside the human macrophage. We tested representative beta-lactams belonging to 3 different classes for activity against replicating M. tuberculosis in broth and nonreplicating M. tuberculosis under hypoxia, as well as against streptomycin-starved M. tuberculosis strain 18b (ss18b) in the presence or absence of clavulanate. Most of the combinations showed bactericidal activity against replicating M. tuberculosis, with up to 200-fold improvement in potency in the presence of clavulanate. None of the combinations, including those containing meropenem, imipenem, and faropenem, killed M. tuberculosis under hypoxia. However, faropenem- and meropenem-containing combinations killed strain ss18b moderately. We tested the bactericidal activities of meropenem-clavulanate and amoxicillin-clavulanate combinations in the acute and chronic aerosol infection models of tuberculosis in BALB/c mice. Based on pharmacokinetic/pharmacodynamic indexes reported for beta-lactams against other bacterial pathogens, a cumulative percentage of a 24-h period that the drug concentration exceeds the MIC under steady-state pharmacokinetic conditions (%TMIC) of 20 to 40% was achieved in mice using a suitable dosing regimen. Both combinations showed marginal reduction in lung CFU compared to the late controls in the acute model, whereas both were inactive in the chronic model. PMID:23507276

Genotypic characteristics of Mycobacterium tuberculosis isolated from household contacts of tuberculosis patients in the Philippines.

PubMed

Sia, Irene G; Buckwalter, Seanne P; Doerr, Kelly A; Lugos, Sonia; Kramer, Rebecca; Orillaza-Chi, Ruth; Quelapio, Maria Imelda; Tupasi, Thelma E; Wengenack, Nancy L

2013-12-05

The Philippines has an extremely high rate of tuberculosis but little is known about M. tuberculosis genotypes and transmission dynamics in this country. The aim of this study was to determine the proportion of household contacts who develop active TB due to direct transmission from an index case in that household. Mycobacterium tuberculosis isolates from household contacts of tuberculosis patients in the Philippines were characterized using restriction-fragment-length polymorphism analysis, spoligotyping, and mycobacterial interspersed repetitive units - variable number tandem repeats typing (12-loci) to determine their utility in elucidating transmission in an area of high tuberculosis prevalence. Drug susceptibility patterns for these isolates were also determined. Spoligotyping and MIRU-VNTR typing results matched in 10 (62.5%) of 16 index patient-household contact pairs while IS6110 fingerprints matched in only six (37.5%) pairs. Only 3/16 (18.8%) index patient-household contact pairs had identical drug susceptibility results. Strain typing of M. tuberculosis isolates from household contacts in the Philippines indicates that transmission of strains does not necessarily occur directly from the index patient living in close proximity in the same household but rather that community-based transmission also frequently occurs. Accurate susceptibility testing of all isolates is necessary to insure optimal care of both the index patients and any culture-positive household contacts.

A Thiolase of Mycobacterium tuberculosis Is Required for Virulence and Production of Androstenedione and Androstadienedione from Cholesterol▿ †

PubMed Central

Nesbitt, Natasha M.; Yang, Xinxin; Fontán, Patricia; Kolesnikova, Irina; Smith, Issar; Sampson, Nicole S.; Dubnau, Eugenie

2010-01-01

Mycobacterium tuberculosis, the causative agent of tuberculosis, is an intracellular pathogen that shifts to a lipid-based metabolism in the host. Moreover, metabolism of the host lipid cholesterol plays an important role in M. tuberculosis infection. We used transcriptional profiling to identify genes transcriptionally regulated by cholesterol and KstR (Rv3574), a TetR-like repressor. The fadA5 (Rv3546) gene, annotated as a lipid-metabolizing thiolase, the expression of which is upregulated by cholesterol and repressed by KstR, was deleted in M. tuberculosis H37Rv. We demonstrated that fadA5 is required for utilization of cholesterol as a sole carbon source in vitro and for full virulence of M. tuberculosis in the chronic stage of mouse lung infection. Cholesterol is not toxic to the fadA5 mutant strain, and, therefore, toxicity does not account for its attenuation. We show that the wild-type strain, H37Rv, metabolizes cholesterol to androst-4-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD) and exports these metabolites into the medium, whereas the fadA5 mutant strain is defective for this activity. We demonstrate that FadA5 catalyzes the thiolysis of acetoacetyl-coenzyme A (CoA). This catalytic activity is consistent with a β-ketoacyl-CoA thiolase function in cholesterol β-oxidation that is required for the production of androsterones. We conclude that the attenuated phenotype of the fadA5 mutant is a consequence of disrupted cholesterol metabolism that is essential only in the persistent stage of M. tuberculosis infection and may be caused by the inability to produce AD/ADD from cholesterol. PMID:19822655

Xpert MTB/RIF for rapid detection of rifampicin-resistant Mycobacterium tuberculosis from pulmonary tuberculosis patients in Southwest Ethiopia.

PubMed

Tadesse, Mulualem; Aragaw, Dossegnaw; Dimah, Belayneh; Efa, Feyisa; Abebe, Gemeda

2016-12-01

Accurate and rapid detection of drug-resistant strains of tuberculosis (TB) is critical for early initiation of treatment and for limiting the transmission of drug-resistant TB. Here, we investigated the accuracy of Xpert MTB/RIF for detection of rifampicin (RIF) resistance, and whether this detection predicts the presence of multidrug resistant (MDR) TB in Southwest Ethiopia. Smear- or culture-positive sputa obtained from TB patients with increased suspicion of drug resistance were included in this study. GenoType MTBDRplus line-probe assays (LPAs) and Xpert MTB/RIF tests were performed on smear-positive sputum specimens and on cultured isolates for smear-negative specimens. We performed routine drug-susceptibility testing using LPA as the reference standard for confirmation of RIF and isoniazid (INH) resistance. First-line drug-susceptibility results were available for 67 Mycobacterium tuberculosis complex-positive sputum specimens using the LPA test, with our preliminary results indicating that 30% (20/67) were MDR-TB, 3% (2/67) were RIF monoresistant, 6% (4/67) were INH monoresistant, and 61% (41/67) were susceptible to both RIF and INH. Relative to routine RIF-susceptibility testing (LPA), Xpert MTB/RIF detected all RIF resistance correctly, with 100% sensitivity and 97.8% specificity and a positive-predictive value of 95.7%. Of the 23 RIF-resistant strains according to Xpert MTB/RIF, 87% (20/23) were resistant to both RIF and INH (MDR), 8.7% (2/23) were RIF monoresistant, and 4.3% (1/23) were sensitive to RIF according to the LPA test. A high proportion of RIF resistance was documented among patients previously categorized as failure cases (50%, 10/20), followed by relapse cases (31.6%, 6/19) and defaulters (28.6%, 2/7). Xpert MTB/RIF was highly effective at identifying RIF-resistant strains in smear- or culture-positive samples. RIF resistance based on Xpert MTB/RIF results could be used to estimate MDR and allow rapid initiation of MDR-TB treatment in regions with high levels of drug-resistant TB. Copyright © 2016.

Stereological analysis of bacterial load and lung lesions in nonhuman primates (rhesus macaques) experimentally infected with Mycobacterium tuberculosis.

PubMed

Luciw, Paul A; Oslund, Karen L; Yang, Xiao-Wei; Adamson, Lourdes; Ravindran, Resmi; Canfield, Don R; Tarara, Ross; Hirst, Linda; Christensen, Miles; Lerche, Nicholas W; Offenstein, Heather; Lewinsohn, David; Ventimiglia, Frank; Brignolo, Laurie; Wisner, Erik R; Hyde, Dallas M

2011-11-01

Infection with Mycobacterium tuberculosis primarily produces a multifocal distribution of pulmonary granulomas in which the pathogen resides. Accordingly, quantitative assessment of the bacterial load and pathology is a substantial challenge in tuberculosis. Such assessments are critical for studies of the pathogenesis and for the development of vaccines and drugs in animal models of experimental M. tuberculosis infection. Stereology enables unbiased quantitation of three-dimensional objects from two-dimensional sections and thus is suited to quantify histological lesions. We have developed a protocol for stereological analysis of the lung in rhesus macaques inoculated with a pathogenic clinical strain of M. tuberculosis (Erdman strain). These animals exhibit a pattern of infection and tuberculosis similar to that of naturally infected humans. Conditions were optimized for collecting lung samples in a nonbiased, random manner. Bacterial load in these samples was assessed by a standard plating assay, and granulomas were graded and enumerated microscopically. Stereological analysis provided quantitative data that supported a significant correlation between bacterial load and lung granulomas. Thus this stereological approach enables a quantitative, statistically valid analysis of the impact of M. tuberculosis infection in the lung and will serve as an essential tool for objectively comparing the efficacy of drugs and vaccines.

Mycobacterium tuberculosis Transcriptome Profiling in Mice with Genetically Different Susceptibility to Tuberculosis.

PubMed

Skvortsov, T A; Ignatov, D V; Majorov, K B; Apt, A S; Azhikina, T L

2013-04-01

Whole transcriptome profiling is now almost routinely used in various fields of biology, including microbiology. In vivo transcriptome studies usually provide relevant information about the biological processes in the organism and thus are indispensable for the formulation of hypotheses, testing, and correcting. In this study, we describe the results of genome-wide transcriptional profiling of the major human bacterial pathogen M. tuberculosis during its persistence in lungs. Two mouse strains differing in their susceptibility to tuberculosis were used for experimental infection with M. tuberculosis. Mycobacterial transcriptomes obtained from the infected tissues of the mice at two different time points were analyzed by deep sequencing and compared. It was hypothesized that the changes in the M. tuberculosis transcriptome may attest to the activation of the metabolism of lipids and amino acids, transition to anaerobic respiration, and increased expression of the factors modulating the immune response. A total of 209 genes were determined whose expression increased with disease progression in both host strains (commonly upregulated genes, CUG). Among them, the genes related to the functional categories of lipid metabolism, cell wall, and cell processes are of great interest. It was assumed that the products of these genes are involved in M. tuberculosis adaptation to the host immune system defense, thus being potential targets for drug development.

Pyrosequencing for Rapid Detection of Mycobacterium tuberculosis Resistance to Rifampin, Isoniazid, and Fluoroquinolones ▿

PubMed Central

Bravo, Lulette Tricia C.; Tuohy, Marion J.; Ang, Concepcion; Destura, Raul V.; Mendoza, Myrna; Procop, Gary W.; Gordon, Steven M.; Hall, Geraldine S.; Shrestha, Nabin K.

2009-01-01

After isoniazid and rifampin (rifampicin), the next pivotal drug class in Mycobacterium tuberculosis treatment is the fluoroquinolone class. Mutations in resistance-determining regions (RDR) of the rpoB, katG, and gyrA genes occur with frequencies of 97%, 50%, and 85% among M. tuberculosis isolates resistant to rifampin, isoniazid, and fluoroquinolones, respectively. Sequences are highly conserved, and certain mutations correlate well with phenotypic resistance. We developed a pyrosequencing assay to determine M. tuberculosis genotypic resistance to rifampin, isoniazid, and fluoroquinolones. We characterized 102 M. tuberculosis clinical isolates from the Philippines for susceptibility to rifampin, isoniazid, and ofloxacin by using the conventional submerged-disk proportion method and validated our pyrosequencing assay using these isolates. DNA was extracted and amplified by using PCR primers directed toward the RDR of the rpoB, katG, and gyrA genes, and pyrosequencing was performed on the extracts. The M. tuberculosis H37Rv strain (ATCC 25618) was used as the reference strain. The sensitivities and specificities of pyrosequencing were 96.7% and 97.3%, 63.8% and 100%, and 70.0% and 100% for the detection of resistance to rifampin, isoniazid, and ofloxacin, respectively. Pyrosequencing is thus a rapid and accurate method for detecting M. tuberculosis resistance to these three drugs. PMID:19846642

High clustering rates of multidrug-resistant Mycobacterium tuberculosis genotypes in Panama

PubMed Central

2013-01-01

Background Tuberculosis continues to be one of the leading causes of death worldwide and in the American region. Although multidrug-resistant tuberculosis (MDR-TB) remains a threat to TB control in Panama, few studies have focused in typing MDR-TB strains. The aim of our study was to characterize MDR Mycobacterium tuberculosis clinical isolates using PCR-based genetic markers. Methods From 2002 to 2004, a total of 231 Mycobacterium tuberculosis isolates from TB cases country-wide were screened for antibiotic resistance, and MDR-TB isolates were further genotyped by double repetitive element PCR (DRE-PCR), (GTG)5-PCR and spoligotyping. Results A total of 37 isolates (0.85%) were resistant to both isoniazid (INH) and rifampicin (RIF). Among these 37 isolates, only two (5.4%) were resistant to all five drugs tested. Dual genotyping using DRE-PCR and (GTG)5-PCR of MDR Mycobacterium tuberculosis isolates revealed eight clusters comprising 82.9% of the MDR-TB strain collection, and six isolates (17.1%) showed unique fingerprints. The spoligotyping of MDR-TB clinical isolates identified 68% as members of the 42 (LAM9) family genotype. Conclusion Our findings suggest that MDR Mycobacterium tuberculosis is highly clustered in Panama’s metropolitan area corresponding to Panama City and Colon City, and our study reveals the genotype distribution across the country. PMID:24053690

Molecular epidemiology of Mycobacterium tuberculosis in Baja California, Mexico: A result of human migration?

PubMed

Flores-López, Carlos A; Zenteno-Cuevas, Roberto; Laniado-Laborín, Rafael; Reynaud, Yann; García-Ortiz, Rosa Alejandra; González-Y-Merchand, Jorge A; Rivera, Sandra; Vázquez-Chacón, Carlos A; Vaughan, Gilberto; Martínez-Guarneros, José Armando; Victoria-Cota, Nelva Lorena; Cruz-Rivera, Mayra; Rastogi, Nalin; Muñiz-Salazar, Raquel

2017-11-01

The State of Baja California (BC) exhibits the highest incidence and prevalence rates of tuberculosis (TB), and multidrug-resistant TB (MDR-TB) in Mexico. However information about the circulation of M. tuberculosis lineages in BC and Mexico as a whole is limited. Here, we describe the genetic relationship and genetic diversity among M. tuberculosis clinical isolates (n=140) collected in BC between October 2009 and April 2011 with other regions of Mexico, the United States, and Latin America. All specimens were genotyped based on 24 mycobacterial interspersed repetitive units (MIRU)-variable number of tandem repeats (VNTR) loci. Population structure and minimum spanning tree (MST) analyses were used to assess the genetic diversity and distribution of BC isolates in comparison to USA and South America strains. Among the nine lineages observed, LAM, Haarlem and S were the most frequent identified in BC. Population structure analysis clustered most BC isolates (41%) into three distinctive groups that included strains from San Diego and South America, whereas other BC strains (22%) clustered with other Mexican strains. A subset of isolates (12%) seemed to be autochthonous of BC, while 25% were cosmopolitan and grouped into multiple clusters. It is highly likely that the TB genetic structure observed in BC is due to human migration. Additional studies are required to determine the mechanism involved in the phylogeographic distribution of M. tuberculosis in Mexico. Implementation of domestic molecular TB surveillance programs is required to better understand the molecular epidemiology of TB not only in the region but at the national level. Copyright © 2016 Elsevier B.V. All rights reserved.

Urease Activity Represents an Alternative Pathway for Mycobacterium tuberculosis Nitrogen Metabolism

PubMed Central

Lin, Wenwei; Mathys, Vanessa; Ang, Emily Lei Yin; Koh, Vanessa Hui Qi; Martínez Gómez, Julia María; Ang, Michelle Lay Teng; Zainul Rahim, Siti Zarina; Tan, Mai Ping; Pethe, Kevin

2012-01-01

Urease represents a critical virulence factor for some bacterial species through its alkalizing effect, which helps neutralize the acidic microenvironment of the pathogen. In addition, urease serves as a nitrogen source provider for bacterial growth. Pathogenic mycobacteria express a functional urease, but its role during infection has yet to be characterized. In this study, we constructed a urease-deficient Mycobacterium tuberculosis strain and confirmed the alkalizing effect of the urease activity within the mycobacterium-containing vacuole in resting macrophages but not in the more acidic phagolysosomal compartment of activated macrophages. However, the urease-mediated alkalizing effect did not confer any growth advantage on M. tuberculosis in macrophages, as evidenced by comparable growth profiles for the mutant, wild-type (WT), and complemented strains. In contrast, the urease-deficient mutant exhibited impaired in vitro growth compared to the WT and complemented strains when urea was the sole source of nitrogen. Substantial amounts of ammonia were produced by the WT and complemented strains, but not with the urease-deficient mutant, which represents the actual nitrogen source for mycobacterial growth. However, the urease-deficient mutant displayed parental colonization profiles in the lungs, spleen, and liver in mice. Together, our data demonstrate a role for the urease activity in M. tuberculosis nitrogen metabolism that could be crucial for the pathogen's survival in nutrient-limited microenvironments where urea is the sole nitrogen source. Our work supports the notion that M. tuberculosis virulence correlates with its unique metabolic versatility and ability to utilize virtually any carbon and nitrogen sources available in its environment. PMID:22645285

Extent and origin of resistance to antituberculosis drugs in the Netherlands, 1993 to 2011.

PubMed

Ruesen, C; van Gageldonk-Lafeber, A B; de Vries, G; Erkens, C G; van Rest, J; Korthals Altes, H; de Neeling, H; Kamst, M; van Soolingen, D

2014-03-20

The elimination of tuberculosis (TB) is threatened by an apparent increase in the level of resistance in Mycobacterium tuberculosis. In the Netherlands, where the majority of TB patients are migrants, resistance may also be increasing. We conducted a retrospective study, using 18,294 M. tuberculosis isolates from TB cases notified between 1993 and 2011. We investigated the trends in antituberculosis drug resistance, focusing on the country of birth of the patients and whether resistance had developed during treatment or was the result of transmission of resistant M. tuberculosis strains. For both scenarios, we determined whether this had happened in or outside the Netherlands. Antituberculosis drug resistance was found in 13% of all cases analysed and showed an increasing trend among patients who had been born in the Netherlands (p<0.001) and a decreasing trend among foreign-born (p=0.02) over the study period. Since 2005, the proportion of M. tuberculosis resistant strains among all strains tested has increased in both groups (p=0.03 and p=0.01, respectively). Overall, we found a significantly increasing trend when excluding streptomycin resistance (p<0.001). The trend was most markedly increased for isoniazid resistance (p = 0.01). Although resistance was mainly due to transmission of resistant strains, mostly outside the Netherlands or before 1993 (when DNA fingerprinting was not systematically performed), in some cases (n=45), resistance was acquired in the Netherlands. We conclude that antituberculosis drug resistance is increasing in the Netherlands, mostly related to migration from high TB-incidence countries, but also to domestic acquisition.

SpolSimilaritySearch - A web tool to compare and search similarities between spoligotypes of Mycobacterium tuberculosis complex.

PubMed

Couvin, David; Zozio, Thierry; Rastogi, Nalin

2017-07-01

Spoligotyping is one of the most commonly used polymerase chain reaction (PCR)-based methods for identification and study of genetic diversity of Mycobacterium tuberculosis complex (MTBC). Despite its known limitations if used alone, the methodology is particularly useful when used in combination with other methods such as mycobacterial interspersed repetitive units - variable number of tandem DNA repeats (MIRU-VNTRs). At a worldwide scale, spoligotyping has allowed identification of information on 103,856 MTBC isolates (corresponding to 98049 clustered strains plus 5807 unique isolates from 169 countries of patient origin) contained within the SITVIT2 proprietary database of the Institut Pasteur de la Guadeloupe. The SpolSimilaritySearch web-tool described herein (available at: http://www.pasteur-guadeloupe.fr:8081/SpolSimilaritySearch) incorporates a similarity search algorithm allowing users to get a complete overview of similar spoligotype patterns (with information on presence or absence of 43 spacers) in the aforementioned worldwide database. This tool allows one to analyze spread and evolutionary patterns of MTBC by comparing similar spoligotype patterns, to distinguish between widespread, specific and/or confined patterns, as well as to pinpoint patterns with large deleted blocks, which play an intriguing role in the genetic epidemiology of M. tuberculosis. Finally, the SpolSimilaritySearch tool also provides with the country distribution patterns for each queried spoligotype. Copyright © 2017 Elsevier Ltd. All rights reserved.

Naphthoquinones isolated from Diospyros anisandra exhibit potent activity against pan-resistant first-line drugs Mycobacterium tuberculosis strains.

PubMed

Uc-Cachón, Andrés Humberto; Borges-Argáez, Rocío; Said-Fernández, Salvador; Vargas-Villarreal, Javier; González-Salazar, Francisco; Méndez-González, Martha; Cáceres-Farfán, Mirbella; Molina-Salinas, Gloria María

2014-02-01

The recent emergence of multidrug-resistant (MDR), extensively drug-resistant (XDR), and totally drug-resistant (TDR) Mycobacterium tuberculosis (MTB) strains have further complicated the control of tuberculosis (TB). There is an urgent need of new molecules candidates to be developed as novel, active, and less toxic anti-tuberculosis (anti-TB) drugs. Medicinal plants have been an excellent source of leads for the development of drugs, particularly as anti-infective agents. In previous studies, the non-polar extract of Diospyros anisandra showed potent anti-TB activity, and three monomeric and five dimeric naphthoquinones have been obtained. In this study, we performed bioguided chemical fractionation and the isolation of eight naphthoquinones from D. anisandra and their evaluation of anti-TB and cytotoxic activities against mammalian cells. The n-hexane crude extract from the stem bark of the plant was obtained by maceration and liquid-liquid fractionation. The isolation of naphthoquinones was carried out by chromatographic methods and identified by gas chromatography and mass spectroscopy data analysis. Anti-TB activity was evaluated against two strains of MTB (H37Rv) susceptible to all five first-line anti-TB drugs and a clinical isolate that is resistant to these medications (pan-resistant, CIBIN 99) by measuring the minimal inhibitory concentration (MIC). Cytotoxicity of naphthoquinones was estimated against two mammalian cells, Vero line and primary cultures of human peripheral blood mononuclear (PBMC) cells, and their selectivity index (SI) was determined. Plumbagin and its dimers maritinone and 3,3'-biplumbagin showed the strongest activity against both MTB strains (MIC = 1.56-3.33 μg/mL). The bioactivity of maritinone and 3,3'-biplumbagin were 32 times more potent than rifampicin against the pan-resistant strain, and both dimers showed to be non-toxic against PBMC and Vero cells. The SI of maritinone and 3,3'-biplumbagin on Vero cells was 74.34 and 194.11 against sensitive and pan-resistant MTB strains, respectively. Maritinone and 3,3'-biplumbagin possess a very interesting potential for development as new drugs against M. tuberculosis, mainly resistant profile strains. Copyright © 2013 Elsevier Ltd. All rights reserved.

[Streptomycin as second-line chemotherapy for tuberculosis].

PubMed

Ruiz, P; Rodríguez-Cano, F; Zerolo, F J; Casal, M

2003-06-01

Streptomycin was the first antibiotic to be used against Mycobacterium tuberculosis. It was used for years in monotherapy regimens, thereby resulting in the appearance of resistance and the relegation of its use. The resistance detected against drugs currently employed has led to a renewed interest in streptomycin. Its mechanism of action is centered on the ribosome, inhibiting the protein synthesis of the microbacteria. Resistance appears when mutations in the genes codifying for rRNA 16S and for protein S12 are produced. We studied the use of streptomycin against 899 M. tuberculosis strains, 713 of which were from pulmonary and 186 from extrapulmonary isolates. The BACTEC 460 TB system was initially employed as was the ESP II system. As controls, the ATCC27294 pattern strains (susceptible to streptomycin, rifampicin, ethambutol and isoniazid) were used. The results showed 2.1% secondary resistance in all the cases. Multiresistance was observed in 12 strains.

Molecular Cloning and Sequence Analysis of the Sta58 Major Antigen Gene of Rickettsia tsutsugamushi: Sequence homology and Antigenic Comparison of Sta58 to the 60-Kilodalton Family of Stress Proteins

DTIC Science & Technology

1990-05-01

Sta58 antigen and the Sta56 strain- GroES, C. burnetii HtpA, Mycobacterium tuberculosis 12- specific major antigen of R. tsutsugamushi (strain Karp...kb HindlIl fragment carrying the gene for the Sta58 tuberculosis, and Mycobacterium smegmatis (65-kDa anti- protein was subjected to DNA sequence...the Hsp6O and HsplO proteins. R. tsu., R. isutsugamushi; M. lep., Mvtcobacteriutn leprae : C. bur., C. burneiii; Synech.. Synechococcus strain 6301; T

Phage display of functional αβ single-chain T-cell receptor molecules specific for CD1b:Ac₂SGL complexes from Mycobacterium tuberculosis-infected cells.

PubMed

Camacho, Frank; Huggett, Jim; Kim, Louise; Infante, Juan F; Lepore, Marco; Perez, Viviana; Sarmiento, María E; Rook, Graham; Acosta, Armando

2013-01-01

The development of molecules specific for M. tuberculosis-infected cells has important implications, as these tools may facilitate understanding of the mechanisms regulating host pathogen interactions in vivo. In addition, development of new tools capable to targeting M. tuberculosis-infected cells may have potential applications to diagnosis, treatment, and prevention of tuberculosis (TB). Due to the lack of CD1b polymorphism, M. tuberculosis lipid-CD1b complexes could be considered as universal tuberculosis infection markers. The aim of the present study was to display on the PIII surface protein of m13 phage, a human αβ single-chain T-cell receptor molecule specific for CD1b:2-stearoyl-3-hydroxyphthioceranoyl-2´-sulfate-α-α´-D-trehalose (Ac₂SGL) which is a complex presented by human cells infected with M. tuberculosis. The results showed the pIII fusion particle was successfully displayed on the phage surface. The study of the recognition of the recombinant phage in ELISA and immunohistochemistry showed the recognition of CD1b:Ac₂SGL complexes and cells in human lung tissue from a tuberculosis patient respectively, suggesting the specific recognition of the lipid-CD1b complex.

[Evaluation of tuberculosis transmission routes in an outbreak among young adults for developing an effective method for contact investigations].

PubMed

Toyota, Makoto

2012-12-01

To evaluate the various transmission routes of tuberculosis in an outbreak among young adults in order to develop an effective method for contact investigations. We reviewed the records of 21 tuberculosis patients involved in an outbreak of tuberculosis; the records were collected by conventional epidemiological studies. Mycobacterium tuberculosis isolates were genotyped using IS6110-based restriction fragment length polymorphism (RFLP). The index patient was a 26-year-old man whose 32-year-old brother was identified as the source patient of tuberculosis through a contact investigation. Investigation of their contacts led to the identification of 10 tuberculosis patients. Further, 5 more patients with only casual contact with the index or source patients developed tuberculosis 18-25 months after identification of the index patient. The RFLP analysis of strains obtained from these 5 patients as well as the index and source patients revealed an identical pattern. Further, 4 persons, among those who had epidemiological links with some of the above-mentioned 5 patients, developed tuberculosis 22-34 months after identification of the index patient. All 21 patients were relatively young. In total, 15 strains obtained from these patients were sent for the RFLP analysis, all of which showed an identical pattern. The epidemiological links were categorized into a household environment, an entertainment area, a university, a music band, and a construction site. Molecular epidemiology can provide insights into the process of tuberculosis transmission, which may otherwise go unrecognized by conventional contact investigations. Additionally, it can play an important role in identifying places of tuberculosis outbreaks and routes of transmission in a contact investigation.

Uptake of Sulfate but Not Phosphate by Mycobacterium tuberculosis Is Slower than That for Mycobacterium smegmatis

PubMed Central

Song, Houhui

2012-01-01

Knowledge of the metabolic pathways used by Mycobacterium tuberculosis during infection is important for understanding its nutrient requirements and host adaptation. However, uptake, the first step in the utilization of nutrients, is poorly understood for many essential nutrients, such as inorganic anions. Here, we show that M. tuberculosis utilizes nitrate as the sole nitrogen source, albeit at lower efficiency than asparagine, glutamate, and arginine. The growth of the porin triple mutant M. smegmatis ML16 in media with limiting amounts of nitrate and sulfate as sole nitrogen and sulfur sources, respectively, was delayed compared to that of the wild-type strain. The uptake of sulfate was 40-fold slower than that of the wild-type strain, indicating that the efficient uptake of these anions is dependent on porins. The uptake by M. tuberculosis of sulfate and phosphate was approximately 40- and 10-fold slower than that of M. smegmatis, respectively, which is consistent with the slower growth of M. tuberculosis. However, the uptake of these anions by M. tuberculosis is orders of magnitude faster than diffusion through lipid membranes, indicating that unknown outer membrane proteins are required to facilitate this process. PMID:22194452

Modeling tuberculosis pathogenesis through ex vivo lung tissue infection.

PubMed

Carranza-Rosales, Pilar; Carranza-Torres, Irma Edith; Guzmán-Delgado, Nancy Elena; Lozano-Garza, Gerardo; Villarreal-Treviño, Licet; Molina-Torres, Carmen; Villarreal, Javier Vargas; Vera-Cabrera, Lucio; Castro-Garza, Jorge

2017-12-01

Tuberculosis (TB) is one of the top 10 causes of death worldwide. Several in vitro and in vivo experimental models have been used to study TB pathogenesis and induction of immune response during Mycobacterium tuberculosis infection. Precision cut lung tissue slices (PCLTS) is an experimental model, in which all the usual cell types of the organ are found, the tissue architecture and the interactions amongst the different cells are maintained. PCLTS in good physiological conditions, monitored by MTT assay and histology, were infected with either virulent Mycobacterium tuberculosis strain H37Rv or the TB vaccine strain Mycobacterium bovis BCG. Histological analysis showed that bacilli infecting lung tissue slices were observed in the alveolar septa, alveolar light spaces, near to type II pneumocytes, and inside macrophages. Mycobacterial infection of PCLTS induced TNF-α production, which is consistent with previous M. tuberculosis in vitro and in vivo studies. This is the first report of using PCLTS as a system to study M. tuberculosis infection. The PCLTS model provides a useful tool to evaluate the innate immune responses and other aspects during the early stages of mycobacterial infection. Copyright © 2017. Published by Elsevier Ltd.

Engineering more stable, selectable marker-free autoluminescent mycobacteria by one step.

PubMed

Yang, Feng; Njire, Moses M; Liu, Jia; Wu, Tian; Wang, Bangxing; Liu, Tianzhou; Cao, Yuanyuan; Liu, Zhiyong; Wan, Junting; Tu, Zhengchao; Tan, Yaoju; Tan, Shouyong; Zhang, Tianyu

2015-01-01

In our previous study, we demonstrated that the use of the autoluminescent Mycobacterium tuberculosis as a reporter strain had the potential to drastically reduce the time, effort, animals and costs consumed in evaluation of the activities of drugs and vaccines in live mice. However, the strains were relatively unstable and lost reporter with time without selection. The kanamycin selection marker used wasn't the best choice as it provides resistance to amino glycosides which are an important class of second line drugs used in tuberculosis treatment. In addition, the marker could limit utility of the strains for screening of new potential drugs or evaluating drug combinations for tuberculosis treatment. Limited selection marker genes for mycobacterial genetic manipulation is a major drawback for such a marker-containing strain in many research fields. Therefore, selectable marker-free, more stable autoluminescent mycobacteria are highly needed. After trying several strategies, we created such mycobacterial strains successfully by using an integrative vector and removing both the resistance maker and integrase genes by Xer site-specific recombination in one step. The corresponding plasmid vectors developed in this study could be very convenient in constructing other selectable marker-free, more stable reporter mycobacteria with diverse applications.

Gallium Disrupts Iron Metabolism of Mycobacteria Residing within Human Macrophages

PubMed Central

Olakanmi, Oyebode; Britigan, Bradley E.; Schlesinger, Larry S.

2000-01-01

Mycobacterium tuberculosis and M. avium complex (MAC) enter and multiply within monocytes and macrophages in phagosomes. In vitro growth studies using standard culture media indicate that siderophore-mediated iron (Fe) acquisition plays a critical role in the growth and metabolism of both M. tuberculosis and MAC. However, the applicability of such studies to conditions within the macrophage phagosome is unclear, due in part to the absence of experimental means to inhibit such a process. Based on the ability of gallium (Ga3+) to concentrate within mononuclear phagocytes and on evidence that Ga disrupts cellular Fe-dependent metabolic pathways by substituting for Fe3+ and failing to undergo redox cycling, we hypothesized that Ga could disrupt Fe acquisition and Fe-dependent metabolic pathways of mycobacteria. We find that Ga(NO3)3 and Ga-transferrin produce an Fe-reversible concentration-dependent growth inhibition of M. tuberculosis strains and MAC grown extracellularly and within human macrophages. Ga is bactericidal for M. tuberculosis growing extracellularly and within macrophages. Finally, we provide evidence that exogenously added Fe is acquired by intraphagosomal M. tuberculosis and that Ga inhibits this Fe acquisition. Thus, Ga(NO3)3 disruption of mycobacterial Fe metabolism may serve as an experimental means to study the mechanism of Fe acquisition by intracellular mycobacteria and the role of Fe in intracellular survival. Furthermore, given the inability of biological systems to discriminate between Ga and Fe, this approach could have broad applicability to the study of Fe metabolism of other intracellular pathogens. PMID:10992462

Interplay between Mutations and Efflux in Drug Resistant Clinical Isolates of Mycobacterium tuberculosis.

PubMed

Machado, Diana; Coelho, Tatiane S; Perdigão, João; Pereira, Catarina; Couto, Isabel; Portugal, Isabel; Maschmann, Raquel De Abreu; Ramos, Daniela F; von Groll, Andrea; Rossetti, Maria L R; Silva, Pedro A; Viveiros, Miguel

2017-01-01

Numerous studies show efflux as a universal bacterial mechanism contributing to antibiotic resistance and also that the activity of the antibiotics subject to efflux can be enhanced by the combined use of efflux inhibitors. Nevertheless, the contribution of efflux to the overall drug resistance levels of clinical isolates of Mycobacterium tuberculosis is poorly understood and still is ignored by many. Here, we evaluated the contribution of drug efflux plus target-gene mutations to the drug resistance levels in clinical isolates of M. tuberculosis . A panel of 17 M. tuberculosis clinical strains were characterized for drug resistance associated mutations and antibiotic profiles in the presence and absence of efflux inhibitors. The correlation between the effect of the efflux inhibitors and the resistance levels was assessed by quantitative drug susceptibility testing. The bacterial growth/survival vs. growth inhibition was analyzed through the comparison between the time of growth in the presence and absence of an inhibitor. For the same mutation conferring antibiotic resistance, different MICs were observed and the different resistance levels found could be reduced by efflux inhibitors. Although susceptibility was not restored, the results demonstrate the existence of a broad-spectrum synergistic interaction between antibiotics and efflux inhibitors. The existence of efflux activity was confirmed by real-time fluorometry. Moreover, the efflux pump genes mmr, mmpL7, Rv1258c, p55 , and efpA were shown to be overexpressed in the presence of antibiotics, demonstrating the contribution of these efflux pumps to the overall resistance phenotype of the M. tuberculosis clinical isolates studied, independently of the genotype of the strains. These results showed that the drug resistance levels of multi- and extensively-drug resistant M. tuberculosis clinical strains are a combination between drug efflux and the presence of target-gene mutations, a reality that is often disregarded by the tuberculosis specialists in favor of the almost undisputed importance of antibiotic target-gene mutations for the resistance in M. tuberculosis .

Molecular epidemiology and evolutionary genetics of Mycobacterium tuberculosis in Taipei.

PubMed

Dou, Horng-Yunn; Tseng, Fan-Chen; Lin, Chih-Wei; Chang, Jia-Ru; Sun, Jun-Ren; Tsai, Wen-Shing; Lee, Shi-Yi; Su, Ih-Jen; Lu, Jang-Jih

2008-12-22

The control of tuberculosis in densely populated cities is complicated by close human-to-human contacts and potential transmission of pathogens from multiple sources. We conducted a molecular epidemiologic analysis of 356 Mycobacterium tuberculosis (MTB) isolates from patients presenting pulmonary tuberculosis in metropolitan Taipei. Classical antibiogram studies and genetic characterization, using mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing and spoligotyping, were applied after culture. A total of 356 isolates were genotyped by standard spoligotyping and the strains were compared with in the international spoligotyping database (SpolDB4). All isolates were also categorized using the 15 loci MIRU-VNTR typing method and combin with NTF locus and RD deletion analyses. Of 356 isolates spoligotyped, 290 (81.4%) displayed known spoligotypes and 66 were not identified in the database. Major spoligotypes found were Beijing lineages (52.5%), followed by Haarlem lineages (13.5%) and EAI plus EAI-like lineages (11%). When MIRU-VNTR was employed, 140 patterns were identified, including 36 clusters by 252 isolates and 104 unique patterns, and the largest cluster comprised 95 isolates from the Beijing family. The combination of spoligotyping and MIRU-VNTR revealed that 236 (67%) of the 356 isolates were clustered in 43 genotypes. Strains of the Beijing family was more likely to be of modern strain and a higher percentage of multiple drug resistance than other families combined (P = 0.08). Patients infected with Beijing strains were younger than those with other strains (mean 58.7 vs. 64.2, p = 0.02). Moreover, 85.3% of infected persons younger than 25 years had Beijing modern strain, suggesting a possible recent spread in the young population by this family of TB strain in Taipei. Our data on MTB genotype in Taipei suggest that MTB infection has not been optimally controlled. Control efforts should be reinforced in view of the high prevalence of the Beijing strain in young population and association with drug resistance.

Molecular epidemiology and evolutionary genetics of Mycobacterium tuberculosis in Taipei

PubMed Central

Dou, Horng-Yunn; Tseng, Fan-Chen; Lin, Chih-Wei; Chang, Jia-Ru; Sun, Jun-Ren; Tsai, Wen-Shing; Lee, Shi-Yi; Su, Ih-Jen; Lu, Jang-Jih

2008-01-01

Background The control of tuberculosis in densely populated cities is complicated by close human-to-human contacts and potential transmission of pathogens from multiple sources. We conducted a molecular epidemiologic analysis of 356 Mycobacterium tuberculosis (MTB) isolates from patients presenting pulmonary tuberculosis in metropolitan Taipei. Classical antibiogram studies and genetic characterization, using mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing and spoligotyping, were applied after culture. Methods A total of 356 isolates were genotyped by standard spoligotyping and the strains were compared with in the international spoligotyping database (SpolDB4). All isolates were also categorized using the 15 loci MIRU-VNTR typing method and combin with NTF locus and RD deletion analyses. Results Of 356 isolates spoligotyped, 290 (81.4%) displayed known spoligotypes and 66 were not identified in the database. Major spoligotypes found were Beijing lineages (52.5%), followed by Haarlem lineages (13.5%) and EAI plus EAI-like lineages (11%). When MIRU-VNTR was employed, 140 patterns were identified, including 36 clusters by 252 isolates and 104 unique patterns, and the largest cluster comprised 95 isolates from the Beijing family. The combination of spoligotyping and MIRU-VNTR revealed that 236 (67%) of the 356 isolates were clustered in 43 genotypes. Strains of the Beijing family was more likely to be of modern strain and a higher percentage of multiple drug resistance than other families combined (P = 0.08). Patients infected with Beijing strains were younger than those with other strains (mean 58.7 vs. 64.2, p = 0.02). Moreover, 85.3% of infected persons younger than 25 years had Beijing modern strain, suggesting a possible recent spread in the young population by this family of TB strain in Taipei. Conclusion Our data on MTB genotype in Taipei suggest that MTB infection has not been optimally controlled. Control efforts should be reinforced in view of the high prevalence of the Beijing strain in young population and association with drug resistance. PMID:19102768

Deep Whole-Genome Sequencing to Detect Mixed Infection of Mycobacterium tuberculosis

PubMed Central

Gan, Mingyu; Liu, Qingyun; Yang, Chongguang; Gao, Qian; Luo, Tao

2016-01-01

Mixed infection by multiple Mycobacterium tuberculosis (MTB) strains is associated with poor treatment outcome of tuberculosis (TB). Traditional genotyping methods have been used to detect mixed infections of MTB, however, their sensitivity and resolution are limited. Deep whole-genome sequencing (WGS) has been proved highly sensitive and discriminative for studying population heterogeneity of MTB. Here, we developed a phylogenetic-based method to detect MTB mixed infections using WGS data. We collected published WGS data of 782 global MTB strains from public database. We called homogeneous and heterogeneous single nucleotide variations (SNVs) of individual strains by mapping short reads to the ancestral MTB reference genome. We constructed a phylogenomic database based on 68,639 homogeneous SNVs of 652 MTB strains. Mixed infections were determined if multiple evolutionary paths were identified by mapping the SNVs of individual samples to the phylogenomic database. By simulation, our method could specifically detect mixed infections when the sequencing depth of minor strains was as low as 1× coverage, and when the genomic distance of two mixed strains was as small as 16 SNVs. By applying our methods to all 782 samples, we detected 47 mixed infections and 45 of them were caused by locally endemic strains. The results indicate that our method is highly sensitive and discriminative for identifying mixed infections from deep WGS data of MTB isolates. PMID:27391214

The Mycobacterium tuberculosis cell-surface glycoprotein apa as a potential adhesin to colonize target cells via the innate immune system pulmonary C-type lectin surfactant protein A.

PubMed

Ragas, Aude; Roussel, Lucie; Puzo, Germain; Rivière, Michel

2007-02-23

Tuberculosis is still a major health problem, and understanding the mechanism by which Mycobacterium tuberculosis (Mtb) invades and colonizes its host target cells remains an important issue for the control of infection. The innate immune system C-type lectins (C-TLs), including the human pulmonary surfactant protein A (PSP-A), have been recently identified as determinant players in the early recognition of the invading pathogen and in mounting the host defense response. Although the antigenic lipoglycan mannosylated lipoarabinomannan is currently considered to be the major C-TL target on the mycobacterial surface, the recognition by some C-TLs of the only mycobacterial species composing the "Mtb complex" indicates that mannosylated lipoarabinomannan cannot account alone for this specificity. Thus, we searched for the mycobacterial molecules targeted by human PSP-A, focusing our attention on the Mtb surface glycoproteins. We developed an original functional proteomic approach based on a lectin blot assay using crude human bronchoalveolar lavage fluid as a source of physiological PSP-A. Combined with selective cell-surface protein extraction and mass spectrometry peptide mapping, this strategy allowed us to identify the Apa (alanine- and proline-rich antigenic) glycoprotein as new potential target for PSP-A. This result was supported by direct binding of PSP-A to purified Apa. Moreover, EDTA addition or deglycosylation of purified Apa samples completely abolished the interaction, demonstrating that the interaction is calcium- and mannose-dependent, as expected. Finally, we provide convincing evidence that Apa, formerly considered as mainly secreted, is associated with the cell wall for a sufficiently long time to aid in the attachment of PSP-A. Because, to date, Apa seems to be restricted to the Mtb complex strains, we propose that it may account for the selective recognition of those strains by PSP-A and other immune system C-TLs containing homologous functional domains.

A Simple and Rapid Identification Method for Mycobacterium bovis BCG with Loop-Mediated Isothermal Amplification.

PubMed

Kouzaki, Yuji; Maeda, Takuya; Sasaki, Hiroaki; Tamura, Shinsuke; Hamamoto, Takaaki; Yuki, Atsushi; Sato, Akinori; Miyahira, Yasushi; Kawana, Akihiko

2015-01-01

Bacillus Calmette-Guérin (BCG) is widely used as a live attenuated vaccine against Mycobacterium tuberculosis and is an agent for standard prophylaxis against the recurrence of bladder cancer. Unfortunately, it can cause severe infectious diseases, especially in immunocompromised patients, and the ability to immediately distinguish BCG from other M. tuberculosis complexes is therefore important. In this study, we developed a simple and easy-to-perform identification procedure using loop-mediated amplification (LAMP) to detect deletions within the region of difference, which is deleted specifically in all M. bovis BCG strains. Reactions were performed at 64 °C for 30 min and successful targeted gene amplifications were detected by real-time turbidity using a turbidimeter and visual inspection of color change. The assay had an equivalent detection limit of 1.0 pg of genomic DNA using a turbidimeter whereas it was 10 pg with visual inspection, and it showed specificity against 49 strains of 44 pathogens, including M. tuberculosis complex. The expected LAMP products were confirmed through identical melting curves in real-time LAMP procedures. We employed the Procedure for Ultra Rapid Extraction (PURE) kit to isolate mycobacterial DNA and found that the highest sensitivity limit with a minimum total cell count of mycobacterium (including DNA purification with PURE) was up to 1 × 10(3) cells/reaction, based on color changes under natural light with FDA reagents. The detection limit of this procedure when applied to artificial serum, urine, cerebrospinal fluid, and bronchoalveolar lavage fluid samples was also about 1 × 10(3) cells/reaction. Therefore, this substitute method using conventional culture or clinical specimens followed by LAMP combined with PURE could be a powerful tool to enable the rapid identification of M. bovis BCG as point-of-care testing. It is suitable for practical use not only in resource-limited situations, but also in any clinical situation involving immunocompromised patients because of its convenience, rapidity, and cost effectiveness.

A Simple and Rapid Identification Method for Mycobacterium bovis BCG with Loop-Mediated Isothermal Amplification

PubMed Central

Kouzaki, Yuji; Maeda, Takuya; Sasaki, Hiroaki; Tamura, Shinsuke; Hamamoto, Takaaki; Yuki, Atsushi; Sato, Akinori; Miyahira, Yasushi; Kawana, Akihiko

2015-01-01

Bacillus Calmette-Guérin (BCG) is widely used as a live attenuated vaccine against Mycobacterium tuberculosis and is an agent for standard prophylaxis against the recurrence of bladder cancer. Unfortunately, it can cause severe infectious diseases, especially in immunocompromised patients, and the ability to immediately distinguish BCG from other M. tuberculosis complexes is therefore important. In this study, we developed a simple and easy-to-perform identification procedure using loop-mediated amplification (LAMP) to detect deletions within the region of difference, which is deleted specifically in all M. bovis BCG strains. Reactions were performed at 64°C for 30 min and successful targeted gene amplifications were detected by real-time turbidity using a turbidimeter and visual inspection of color change. The assay had an equivalent detection limit of 1.0 pg of genomic DNA using a turbidimeter whereas it was 10 pg with visual inspection, and it showed specificity against 49 strains of 44 pathogens, including M. tuberculosis complex. The expected LAMP products were confirmed through identical melting curves in real-time LAMP procedures. We employed the Procedure for Ultra Rapid Extraction (PURE) kit to isolate mycobacterial DNA and found that the highest sensitivity limit with a minimum total cell count of mycobacterium (including DNA purification with PURE) was up to 1 × 103 cells/reaction, based on color changes under natural light with FDA reagents. The detection limit of this procedure when applied to artificial serum, urine, cerebrospinal fluid, and bronchoalveolar lavage fluid samples was also about 1 × 103 cells/reaction. Therefore, this substitute method using conventional culture or clinical specimens followed by LAMP combined with PURE could be a powerful tool to enable the rapid identification of M. bovis BCG as point-of-care testing. It is suitable for practical use not only in resource-limited situations, but also in any clinical situation involving immunocompromised patients because of its convenience, rapidity, and cost effectiveness. PMID:26208001

Transmission of Mycobacterium orygis (M. tuberculosis complex species) from a tuberculosis patient to a dairy cow in New Zealand.

PubMed

Dawson, Kara L; Bell, Anita; Kawakami, R Pamela; Coley, Kathryn; Yates, Gary; Collins, Desmond M

2012-09-01

Mycobacterium orygis, previously called the oryx bacillus, is a member of the Mycobacterium tuberculosis complex and has been reported only recently as a cause of human tuberculosis in patients of South Asian origin. We present the first case documenting the transmission of this organism from a human to a cow.

Preliminary antimycobacterial study on selected Turkish plants (Lamiaceae) against Mycobacterium tuberculosis and search for some phenolic constituents

PubMed Central

2013-01-01

Background The global resurgence of tuberculosis is a significant threat. Lamiaceae members have been used in folk remedies for centuries. This study was designed to assess the in-vitro antimycobacterial activity of eighteen crude extracts from six plants (Lamiaceae) and to characterize their phenolic and flavonoid compounds. Methods Six Turkish medicinal plants of the family Lamiaceae (Stachys tmolea Boiss., Stachys thirkei C. Koch, Ballota acetabulosa (L.) Benth., Thymus sipthorpii Benth., Satureja aintabensis P.H. Davis, and Micromeria juliana (L.) Benth. ex Reich.) were collected in 2009 – 2010. Dried and crushed plant samples were subjected to sequential extraction with petroleum ether, ethyl acetate, and methanol in order of increasing polarity. A broth microdilution method was employed to screen extracts against four mycobacterial strains of Mycobacterium tuberculosis. Phenolic and flavonoid compounds were characterized by liquid chromatography–mass spectrometry. Results S. aintabensis, T. sibthorpii, and M. juliana were found to develop considerable activity against the four strains of M. tuberculosis with the minimal inhibitory concentrations value of 12.5-100 μg/ml. S. aintabensis and T. sibthorpii extracts killed M. tuberculosis with the minimum bactericidal concentration value of 50–800 μg/ml. On the basis of these prominent antimycobacterial activity, we suggest that they could be a source of natural anti-tuberculosis agents. Conclusion S. aintabensis and T. sibthorpii showed activity by killing Mycobacteria strains. The major phenolic compound was rosmarinic for T. sibthorpii and S. aintabensis. Flavonoids might be “a modal” for the drug design. PMID:24359458

Isolation of Mycobacterium tuberculosis Strains with a Silent Mutation in rpoB Leading to Potential Misassignment of Resistance Category ▿

PubMed Central

Alonso, María; Palacios, Juan José; Herranz, Marta; Penedo, Ana; Menéndez, Ángela; Bouza, Emilio; García de Viedma, Darío

2011-01-01

Our study provides an alert regarding the transmission of rifampin-susceptible strains of Mycobacterium tuberculosis with a silent substitution in codon 514 of rpoB. Among 1,450 cases, we identified 12 isolates sharing this mutation and related restriction fragment length polymorphism (RFLP) types. The mutation impaired hybridization with the wild-type probes in three independent commercial assays, which could lead to misassignment of resistance. PMID:21562104

Assessing the Combined Antibacterial Effect of Isoniazid and Rifampin on Four Mycobacterium tuberculosis Strains Using In Vitro Experiments and Response-Surface Modeling

PubMed Central

2017-01-01

ABSTRACT While isoniazid and rifampin have been the cornerstone of tuberculosis therapy caused by drug-susceptible Mycobacterium tuberculosis for more than 40 years, their combined action has never been thoroughly assessed by modern quantitative pharmacology approaches. The aims of this work were to perform in vitro experiments and mathematical modeling of the antibacterial effect of isoniazid and rifampin alone and in combination against various strains of Mycobacterium tuberculosis. After MIC determination of H37Rv and three strains belonging to the Beijing, Euro-American, and Indo-Oceanic lineages, the antibacterial effects of isoniazid and rifampin alone and in combination were studied in static time-kill experiments. A sigmoidal maximum effect model (Hill equation) and a response-surface model were used to describe the effect of the drugs alone and in combination, respectively. The killing effect of isoniazid and rifampin alone were well described by the Hill equation. Rifampin displayed a more concentration-dependent effect than isoniazid around the MIC. The pharmacodynamics parameters of each drug (maximal effect, median effect concentration, and coefficient of sigmoidicity) were quite similar between the four strains. The response-surface model from Minto et al. fit data of combined effect very well with low bias and imprecision (C. F. Minto, T. W. Schnider, T. G. Short, K. M. Gregg, A. Gentilini, Anesthesiology 92:1603–1616, 2000, https://doi.org/10.1097/00000542-200006000-00017). Response-surface modeling showed that the combined action of isoniazid and rifampin was synergistic for the H37Rv, Beijing, and Euro-American strains but only additive for the Indo-Oceanic strain. This study can serve as a motivating example for preclinical evaluation of combined action of antituberculous drugs. PMID:29061753

Activity of LL-37, CRAMP and antimicrobial peptide-derived compounds E2, E6 and CP26 against Mycobacterium tuberculosis.

PubMed

Rivas-Santiago, Bruno; Rivas Santiago, Cesar E; Castañeda-Delgado, Julio E; León-Contreras, Juan C; Hancock, Robert E W; Hernandez-Pando, Rogelio

2013-02-01

Tuberculosis (TB) is a major worldwide health problem in part due to the lack of development of new treatments and the emergence of new strains such as multidrug-resistant (MDR) and extensively drug-resistant strains that are threatening and impairing the control of this disease. In this study, the efficacy of natural and synthetic cationic antimicrobial (host defence) peptides that have been shown often to possess broad-spectrum antimicrobial activity was tested. The natural antimicrobial peptides human LL-37 and mouse CRAMP as well as synthetic peptides E2, E6 and CP26 were tested for their activity against Mycobacterium tuberculosis both in in vitro and in vivo models. The peptides had moderate antimicrobial activities, with minimum inhibitory concentrations ranging from 2 μg/mL to 10 μg/mL. In a virulent model of M. tuberculosis lung infection, intratracheal therapeutic application of these peptides three times a week at doses of ca. 1mg/kg led to significant 3-10-fold reductions in lung bacilli after 28-30 days of treatment. The treatments worked both against the drug-sensitive H37Rv strain and a MDR strain. These results indicate that antimicrobial peptides might constitute a novel therapy against TB. Copyright © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

[A study on genotype of 271 mycobacterium tuberculosis isolates in 6 prefectures in Yunnan Province].

PubMed

Chen, L Y; Yang, X; Ru, H H; Yang, H J; Yan, S Q; Ma, L; Chen, J O; Yang, R; Xu, L

2018-01-06

Objective: To understand the characteristics of genotypes of Mycobacterium tuberculosis isolates in Yunnan province, and provide the molecular epidemiological evidence for prevention and control of tuberculosis in Yunnan Province. Methods: Mycobacterium Tuberculosis isolates were collected from 6 prefectures of Yunnan province in 2014 and their Genetypes of Mycobacterium tuberculosis isolates were obtained using spoligotyping and multiple locus variable numbers of tandem repeats analysis (MLVA). The results of spoligotyping were entered into the SITVITWEB database to obtain the Spoligotyping International Type (SIT) patterns and the sublineages of MTB isolates. The genoyping patterns were clustered with BioNumerics (version 5.0). Results: A total of 271 MTB isolates represented patients were collected from six prefectures in Yunnan province. Out of these patients, 196 (72.3%) were male. The mean age of the patients was (41.9±15.1) years. The most MTB isolates were from Puer, totally 94 iusolates(34.69%). Spoligotyping analysis revealed that 151 (55.72%) MTB isolates belonged to the Beijing genotype, while the other 120 (44.28%) were from non-Beijing genotype; 40 genotypes were consisted of 24 unique genotypes and 16 clusters. The 271 isolates were differentiated into 30 clusters (2 to 17 isolates per cluster) and 177 unique genotypes, showing a clustering rate of 23.62%. Beijing genotype strains showed higher clustering rate than non-Beijing genotype strains (29.14% vs 16.67%). The HGI of 12-locus VNTR in total MTB strains, Beijing genotype strains and non-Beijing genotype was 0.993, 0.982 and 0.995 respectively. Conclusion: The Beijing genotype was the predominant genotype in Yunnan Province, the characteristics of Mycobacterium tuberculosis showed high genetic diversity. The genotyping data reflect the potential recent ongoing transmission in some area, which highlights the urgent need for early diagnosis and treatment of the infectious TB cases, to cut off the transmission and avoid a large TB outbreak.

Ability of innate defence regulator peptides IDR-1002, IDR-HH2 and IDR-1018 to protect against Mycobacterium tuberculosis infections in animal models.

PubMed

Rivas-Santiago, Bruno; Castañeda-Delgado, Julio E; Rivas Santiago, Cesar E; Waldbrook, Matt; González-Curiel, Irma; León-Contreras, Juan C; Enciso-Moreno, Jose Antonio; del Villar, Victor; Mendez-Ramos, Jazmin; Hancock, Robert E W; Hernandez-Pando, Rogelio

2013-01-01

Tuberculosis is an ongoing threat to global health, especially with the emergence of multi drug-resistant (MDR) and extremely drug-resistant strains that are motivating the search for new treatment strategies. One potential strategy is immunotherapy using Innate Defence Regulator (IDR) peptides that selectively modulate innate immunity, enhancing chemokine induction and cell recruitment while suppressing potentially harmful inflammatory responses. IDR peptides possess only modest antimicrobial activity but have profound immunomodulatory functions that appear to be influential in resolving animal model infections. The IDR peptides HH2, 1018 and 1002 were tested for their activity against two M. tuberculosis strains, one drug-sensitive and the other MDR in both in vitro and in vivo models. All peptides showed no cytotoxic activity and only modest direct antimicrobial activity versus M. tuberculosis (MIC of 15-30 µg/ml). Nevertheless peptides HH2 and 1018 reduced bacillary loads in animal models with both the virulent drug susceptible H37Rv strain and an MDR isolate and, especially 1018 led to a considerable reduction in lung inflammation as revealed by decreased pneumonia. These results indicate that IDR peptides have potential as a novel immunotherapy against TB.

Mycobacterium tuberculosis Pili promote adhesion to and invasion of THP-1 macrophages.

PubMed

Ramsugit, Saiyur; Pillay, Manormoney

2014-01-01

Central to the paradigm of the pathogenesis of Mycobacterium tuberculosis is its ability to attach to, enter, and subsequently survive in host macrophages. However, little is known regarding the bacterial adhesins and invasins involved in this interaction with host macrophages. Pili are cell-surface structures produced by certain bacteria and have been implicated in adhesion to and invasion of phagocytes in several species. M. tuberculosis pili (MTP) are encoded by the Rv3312A (mtp) gene. In the present study, we assessed the ability of a Δmtp mutant and an mtp-complemented clinical strain to adhere to and invade THP-1 macrophages in comparison with the parental strain by determining colony-forming units. Both adhesion to and invasion of macrophages, although not reaching significance, were markedly reduced by 42.16% (P = 0.107) and 69.02% (P = 0.052), respectively, in the pili-deficient Δmtp mutant as compared with the wild-type. The pili-overexpressing complemented strain showed significantly higher levels of THP-1 macrophage adhesion (P = 0.000) and invasion (P = 0.040) than the mutant. We, thus, identified a novel adhesin and invasin of M. tuberculosis involved in adhesion to and invasion of macrophages.

Genetic biodiversity of Mycobacterium tuberculosis isolates from patients with pulmonary tuberculosis in India.

PubMed

Singh, Urvashi Balbir; Arora, Jyoti; Suresh, Naga; Pant, Hema; Rana, Tanu; Sola, Christophe; Rastogi, Nalin; Pande, Jitendra Nath

2007-07-01

Spoligotyping was performed on 540 Mycobacterium tuberculosis isolates in order to evaluate the genetic biodiversity of tubercle bacilli in India. One hundred and forty seven patterns were unique and 393 were grouped in 48 clusters. Comparison with an international spoligotype database showed that the most predominant clades among tuberculosis (TB) isolates were Central Asian (CAS) and East-African Indian (EAI) with shared-types (ST) ST26 and ST11 alone being responsible for 34% of all TB cases. Twenty one (3.8%) isolates belonged to the Beijing genotype. Marked variations were observed among circulating strains, STs belonging to CAS family predominated in the North, whereas the EAI family was more common in the Southern India. TB in India is predominantly caused by strains belonging to the principal genetic group 1 (PGG1), suggesting that most of the TB burden in India may be traced to ancestral clones of the tubercle bacilli. This study gives an insight into the global M. tuberculosis genetic biodiversity in India, the predominant spoligotypes and their impact on disease transmission.

Whole genome sequencing of the monomorphic pathogen Mycobacterium bovis reveals local differentiation of cattle clinical isolates.

PubMed

Lasserre, Moira; Fresia, Pablo; Greif, Gonzalo; Iraola, Gregorio; Castro-Ramos, Miguel; Juambeltz, Arturo; Nuñez, Álvaro; Naya, Hugo; Robello, Carlos; Berná, Luisa

2018-01-02

Bovine tuberculosis (bTB) poses serious risks to animal welfare and economy, as well as to public health as a zoonosis. Its etiological agent, Mycobacterium bovis, belongs to the Mycobacterium tuberculosis complex (MTBC), a group of genetically monomorphic organisms featured by a remarkably high overall nucleotide identity (99.9%). Indeed, this characteristic is of major concern for correct typing and determination of strain-specific traits based on sequence diversity. Due to its historical economic dependence on cattle production, Uruguay is deeply affected by the prevailing incidence of Mycobacterium bovis. With the world's highest number of cattle per human, and its intensive cattle production, Uruguay represents a particularly suited setting to evaluate genomic variability among isolates, and the diversity traits associated to this pathogen. We compared 186 genomes from MTBC strains isolated worldwide, and found a highly structured population in M. bovis. The analysis of 23 new M. bovis genomes, belonging to strains isolated in Uruguay evidenced three groups present in the country. Despite presenting an expected highly conserved genomic structure and sequence, these strains segregate into a clustered manner within the worldwide phylogeny. Analysis of the non-pe/ppe differential areas against a reference genome defined four main sources of variability, namely: regions of difference (RD), variable genes, duplications and novel genes. RDs and variant analysis segregated the strains into clusters that are concordant with their spoligotype identities. Due to its high homoplasy rate, spoligotyping failed to reflect the true genomic diversity among worldwide representative strains, however, it remains a good indicator for closely related populations. This study introduces a comprehensive population structure analysis of worldwide M. bovis isolates. The incorporation and analysis of 23 novel Uruguayan M. bovis genomes, sheds light onto the genomic diversity of this pathogen, evidencing the existence of greater genetic variability among strains than previously contemplated.

Infection of great apes and a zoo keeper with the same Mycobacterium tuberculosis spoligotype.

PubMed

Akkerman, Onno W; van der Werf, Tjip S; Rietkerk, Frank; Eger, Tony; van Soolingen, Dick; van der Loo, Kees; van der Zanden, Adri G M

2014-04-01

An animal keeper was diagnosed with pulmonary tuberculosis (TB) after bi-annual screening for latent TB infection in zoo employees. In the same period, several bonobos of the zoo were suffering from TB as well. The Mycobacterium tuberculosis strains from both the animal keeper and the bonobos appeared identical. We provide evidence that the animals infected their keeper.

Internal migration and transmission dynamics of tuberculosis in Shanghai, China: an epidemiological, spatial, genomic analysis.

PubMed

Yang, Chongguang; Lu, Liping; Warren, Joshua L; Wu, Jie; Jiang, Qi; Zuo, Tianyu; Gan, Mingyu; Liu, Mei; Liu, Qingyun; DeRiemer, Kathryn; Hong, Jianjun; Shen, Xin; Colijn, Caroline; Guo, Xiaoqin; Gao, Qian; Cohen, Ted

2018-04-19

Massive internal migration from rural to urban areas poses new challenges for tuberculosis control in China. We aimed to combine genomic, spatial, and epidemiological data to describe the dynamics of tuberculosis in an urban setting with large numbers of migrants. We did a population-based study of culture-positive Mycobacterium tuberculosis isolates in Songjiang, Shanghai. We used whole-genome sequencing to discriminate apparent genetic clusters of M tuberculosis sharing identical variable-number-tandem-repeat (VNTR) patterns, and analysed the relations between proximity of residence and the risk of genomically clustered M tuberculosis. Finally, we used genomic, spatial, and epidemiological data to estimate time of infection and transmission links among migrants and residents. Between Jan 1, 2009, and Dec 31, 2015, 1620 cases of culture-positive tuberculosis were recorded, 1211 (75%) of which occurred among internal migrants. 150 (69%) of 218 people sharing identical VNTR patterns had isolates within ten single-nucleotide polymorphisms (SNPs) of at least one other strain, consistent with recent transmission of M tuberculosis. Pairs of strains collected from individuals living in close proximity were more likely to be genetically similar than those from individuals who lived far away-for every additional km of distance between patients' homes, the odds that genotypically matched strains were within ten SNPs of each other decreased by about 10% (OR 0·89 [95% CI 0·87-0·91]; p<0·0001). We inferred that transmission from residents to migrants occurs as commonly as transmission from migrants to residents, and we estimated that more than two-thirds of migrants in genomic clusters were infected locally after migration. The primary mechanism driving local incidence of tuberculosis in urban centres is local transmission between both migrants and residents. Combined analysis of epidemiological, genomic, and spatial data contributes to a richer understanding of local transmission dynamics and should inform the design of more effective interventions. National Natural Science Foundation of China, National Science and Technology Major Project of China, and US National Institutes of Health. Copyright © 2018 Elsevier Ltd. All rights reserved.

Genetic Diversity and Dynamic Distribution of Mycobacterium tuberculosis Isolates Causing Pulmonary and Extrapulmonary Tuberculosis in Thailand

PubMed Central

Srilohasin, Prapaporn; Tokunaga, Katsushi; Nishida, Nao; Prammananan, Therdsak; Smittipat, Nat; Mahasirimongkol, Surakameth; Chaiyasirinroje, Boonchai; Yanai, Hideki; Palittapongarnpim, Prasit

2014-01-01

This study examined the genetic diversity and dynamicity of circulating Mycobacterium tuberculosis strains in Thailand using nearly neutral molecular markers. The single nucleotide polymorphism (SNP)-based genotypes of 1,414 culture-positive M. tuberculosis isolates from 1,282 pulmonary tuberculosis (PTB) and 132 extrapulmonary TB (EPTB) patients collected from 1995 to 2011 were characterized. Among the eight SNP cluster groups (SCG), SCG2 (44.1%), which included the Beijing (BJ) genotype, and SCG1 (39.4%), an East African Indian genotype, were dominant. Comparisons between the genotypes of M. tuberculosis isolates causing PTB and EPTB in HIV-negative cases revealed similar prevalence trends although genetic diversity was higher in the PTB patients. The identification of 10 reported sequence types (STs) and three novel STs was hypothesized to indicate preferential expansion of the SCG2 genotype, especially the modern BJ ST10 (15.6%) and ancestral BJ ST19 (13.1%). An association between SCG2 and SCG1 genotypes and particular patient age groups implies the existence of different genetic advantages among the bacterial populations. The results revealed that increasing numbers of young patients were infected with M. tuberculosis SCGs 2 and 5, which contrasts with the reduction of the SCG1 genotype. Our results indicate the selection and dissemination of potent M. tuberculosis genotypes in this population. The determination of heterogeneity and dynamic population changes of circulating M. tuberculosis strains in countries using the Mycobacterium bovis BCG (bacillus Calmette-Guérin) vaccine are beneficial for vaccine development and control strategies. PMID:25297330

Rapid, Standardized Method for Determination of Mycobacterium tuberculosis Drug Susceptibility by Use of Mycolic Acid Analysis▿

PubMed Central

Parrish, Nicole; Osterhout, Gerard; Dionne, Kim; Sweeney, Amy; Kwiatkowski, Nicole; Carroll, Karen; Jost, Kenneth C.; Dick, James

2007-01-01

Multidrug-resistant (MDR) Mycobacterium tuberculosis and extrensively drug-resistant (XDR) M. tuberculosis are emerging public health threats whose threats are compounded by the fact that current techniques for testing the susceptibility of M. tuberculosis require several days to weeks to complete. We investigated the use of high-performance liquid chromatography (HPLC)-based quantitation of mycolic acids as a means of rapidly determining drug resistance and susceptibility in M. tuberculosis. Standard susceptibility testing and determination of the MICs of drug-susceptible (n = 26) and drug-resistant M. tuberculosis strains, including MDR M. tuberculosis strains (n = 34), were performed by using the Bactec radiometric growth system as the reference method. The HPLC-based susceptibilities of the current first-line drugs, isoniazid (INH), rifampin (RIF), ethambutol (EMB), and pyrazinamide (PZA), were determined. The vials were incubated for 72 h, and aliquots were removed for HPLC analysis by using the Sherlock mycobacterial identification system. HPLC quantitation of total mycolic acid peaks (TMAPs) was performed with treated and untreated cultures. At 72 h, the levels of agreement of the HPLC method with the reference method were 99.5% for INH, EMB, and PZA and 98.7% for RIF. The inter- and intra-assay reproducibilities varied by drug, with an average precision of 13.4%. In summary, quantitation of TMAPs is a rapid, sensitive, and accurate method for antibiotic susceptibility testing of all first-line drugs currently used against M. tuberculosis and offers the potential of providing susceptibility testing results within hours, rather than days or weeks, for clinical M. tuberculosis isolates. PMID:17913928

Evaluation of the Sensititre MycoTB plate for susceptibility testing of the Mycobacterium tuberculosis complex against first- and second-line agents.

PubMed

Hall, Leslie; Jude, Kurt P; Clark, Shirley L; Dionne, Kim; Merson, Ryan; Boyer, Ana; Parrish, Nicole M; Wengenack, Nancy L

2012-11-01

The Sensititre MycoTB plate (TREK Diagnostic Systems, Cleveland, OH) uses a microtiter plate MIC format for susceptibility testing of Mycobacterium tuberculosis complex isolates against first- and second-line antituberculosis agents. Categorical agreement versus the agar proportion method for 122 M. tuberculosis complex isolates was 94% to 100%.

Evaluation of the Quantamatrix Multiplexed Assay Platform system for simultaneous detection of Mycobacterium tuberculosis and the rifampicin resistance gene using cultured mycobacteria.

PubMed

Wang, Hye-Young; Uh, Young; Kim, Seoyong; Shim, Tae-Sun; Lee, Hyeyoung

2017-08-01

The differentiation of Mycobacterium tuberculosis complex (MTBC) from non-tuberculous mycobacteria (NTM) is of primary importance for infection control and the selection of anti-tuberculosis drugs. Up to date data on rifampicin (RIF)-resistant tuberculosis (TB) is essential for the early management of multidrug-resistant TB. The aim of this study was to evaluate the usefulness of a newly developed multiplexed, bead-based bioassay (Quantamatrix Multiplexed Assay Platform, QMAP) for the rapid differentiation of 23 Mycobacterium species including MTBC and RIF-resistant strains. A total of 314 clinical Mycobacterium isolates cultured from respiratory specimens were used in this study. The sensitivity and specificity of the QMAP system for Mycobacterium species were 100% (95% CI 99.15-100%, p<0.0001) and 97.8% (95% CI 91.86-99.87%, p<0.0001), respectively. The results of conventional drug susceptibility testing and the QMAP Dual-ID assay were completely concordant for all clinical isolates (100%, 95% CI 98.56-100%). Out of 223 M. tuberculosis (MTB) isolates, 196 were pan-susceptible and 27 were resistant to RIF according to QMAP results. All of the mutations in the RIF resistance-determining region detected by the QMAP system were confirmed by rpoB sequence analysis and a REBA MTB-Rifa reverse blot hybridization assay. The majority of the mutations (n=26, 96.3%), including those missing wild-type probe signals, were located in three codons (529-534, 524-529, and 514-520), and 17 (65.4%) of these mutations were detected by three mutation probes (531TTG, 526TAC, and 516GTC). The entire QMAP system assay takes about 3h to complete, while results from the culture-based conventional method can take up to 48-72h. Although improvements to the QMAP system are needed for direct respiratory specimens, it may be useful for rapid screening, not only to identify and accurately discriminate MTBC from NTM, but also to identify RIF-resistant MTB strains in positive culture samples. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Mycobacterium tuberculosis Complex and HIV Co-Infection among Extrapulmonary Tuberculosis Suspected Cases at the University of Gondar Hospital, Northwestern Ethiopia.

PubMed

Fanosie, Alemu; Gelaw, Baye; Tessema, Belay; Tesfay, Wogahta; Admasu, Aschalew; Yitayew, Gashaw

2016-01-01

Extrapulmonary Tuberculosis (EPTB) and Human Immunodeficiency Virus (HIV) infection are interrelated as a result of immune depression. The aim of this study was to determine the prevalence of Mycobacterium tuberculosis complex isolates and the burden of HIV co-infection among EPTB suspected patients. An institution based cross-sectional study was conducted among EPTB suspected patients at the University of Gondar Hospital. Socio-demographic characteristics and other clinical data were collected using a pretested questionnaire. GeneXpert MTB/RIF assay was performed to diagnosis Mycobacterium tuberculosis complex and Rifampicin resistance. All samples were also investigated by cytology and culture. The HIV statuses of all patients were screened initially by KHB, and all positive cases were further re-tested by STAT-pack. Data was analyzed using SPSS version 20 computer software and a P-value of < 0.05 was taken as statistically significant. A total of 141 extrapulmonary suspected patients were enrolled in this study. The overall prevalence of culture confirmed extrapulmonary tuberculosis infection was 29.8%, but the GeneXpert result showed a 26.2% prevalence of Mycobacterium tuberculosis complex infection. The 78.4% prevalence of extrapulmonary tuberculosis infection was found to be higher among the adult population. The prevalence of HIV infection among EPTB suspected patients was 14.1%, while it was 32.4% among GeneXpert-confirmed extrapulmonary TB cases (12/37). Tuberculosis lymphadenitis was the predominant (78.4%) type of EPTB infection followed by tuberculosis cold abscess (10.7%). Adult hood, previous history of contact with known pulmonary tuberculosis patients, and HIV co-infection showed a statistically significant association with extrapulmonary tuberculosis infection (P<0.013). The prevalence of culture confirmed-EPTB infection was high, and a higher EPTB-HIV co-infection was also observed.

[Focus on the role of ventilation and ultraviolet rays in preventing nosocomial transmission of tuberculosis in health care facilities. Groupe de travail sur la prévention de la transmission nosocomiale de la tuberculose (Direction Générale de la Santé)].

PubMed

Desenclos, J C; Abiteboul, D; Bouvet, E; Brucker, G; Demeulemester, R; Haury, B; Huré, P; Leprince, A; Macrez, A; Mayaud, C

1995-01-01

Recent episodes of nosocomial tuberculosis, sometimes due to multiresistant strains, in HIV infected patients in the USA has led to the need for new prevention measures against the transmission of Mycobacterium tuberculosis in health care facilities. Tuberculosis is transmitted in Pflügge droplets generated when contagious persons cough. After drying, the droplets become aerosolized solid particles which are rapidly dispersed by air flow within the patient's room. People exposed to the same air are thus at high risk of being contaminated. If the air pressure in the patient's room is higher than the rest of the facility, the air coming form the room may contaminate personnel and other patients elsewhere in the facility. Infecting particles can be eliminated rapidly if the room air is ventilated outdoors. If the ventilation is strong enough so that air constantly circulates from the corridor into the room, infecting particles can no longer diffuse to the rest of the ward. It is also possible to use ultraviolet C light to disinfect the air, either within the room or within the ventilation system. These two basically simple systems are the fundamental environmental and prevention measures needed to limit tuberculosis spread in health care facilities. These methods are however technically complex, costly and require constant evaluation and maintenance by specialized personnel. In addition the potential side effects of ultraviolet waves could considerably reduce their application. These environmental methods, which are complementary methods, only have a meaning if the elementary measures for preventing the transmission of tuberculosis are correctly applied.(ABSTRACT TRUNCATED AT 250 WORDS)

Draft genome sequence of Mycobacterium tuberculosis strain B9741 of Beijing B0/W lineage from HIV positive patient from Siberia.

PubMed

Shur, K V; Zaychikova, M V; Mikheecheva, N E; Klimina, K M; Bekker, O B; Zhdanova, S N; Ogarkov, O B; Danilenko, V N

2016-12-01

We report a draft genome sequence of Mycobacterium tuberculosis strain B9741 belonging to Beijing B0/W lineage isolated from a HIV patient from Siberia, Russia. This clinical isolate showed MDR phenotype and resistance to isoniazid, rifampin, streptomycin and pyrazinamide. We analyzed SNPs associated with virulence and resistance. The draft genome sequence and annotation have been deposited at GenBank under the accession NZ_LVJJ00000000.

Antimycobacterial activity of new N(1)-[1-[1-aryl-3-[4-(1H-imidazol-1-yl)phenyl]-3-oxo]propyl]-pyridine-2-carboxamidrazone derivatives.

PubMed

Zampieri, Daniele; Mamolo, Maria Grazia; Vio, Luciano; Romano, Maurizio; Skoko, Nataša; Baralle, Marco; Pau, Valentina; De Logu, Alessandro

2016-07-15

N(1)-[1-[1-aryl-3-[4-(1H-imidazol-1-yl)phenyl]-3-oxo]propyl]-pyridine-2-carboxamidrazone derivatives were design, synthesized and tested for their in vitro antimycobacterial activity. The new compounds showed a moderate antimycobacterial activity against the tested strain of Mycobacterium tuberculosis H37Ra and a significant antimycobacterial activity against several mycobacteria other than tuberculosis strains. Copyright © 2016 Elsevier Ltd. All rights reserved.

In vitro antimycobacterial activities of Physalis angulata L.

PubMed

Pietro, R C; Kashima, S; Sato, D N; Januário, A H; França, S C

2000-07-01

The HIV-tuberculosis co-infection has caused an impact on tuberculosis epidemiology all over the world and the efficacies of the therapeutic schemes traditionally prescribed in the treatment of tuberculosis, such as isoniazid, rifampicin and pyrazinamide, have decreased due to the appearance of multidrug-resistant M. tuberculosis strains (MDR). This work is part of research on natural antimicrobial agents from plant extracts through bioassay-guided fractionation, by in vitro determination of the minimum inhibitory concentration (MIC) using the microdilution method with Alamar blue oxidation-reduction dye. Crude CHCl3 Physalis angulata extracts and physalin-containing fractions displayed antimycobacterial activity against Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium kansasii, Mycobacterium malmoense and Mycobacterium intracellulare.

Fragment-Based Drug Discovery in Academia: Experiences From a Tuberculosis Programme

NASA Astrophysics Data System (ADS)

Heikkila, Timo J.; Surade, Sachin; Silvestre, Hernani L.; Dias, Marcio V. B.; Ciulli, Alessio; Bromfield, Karen; Scott, Duncan; Howard, Nigel; Wen, Shijun; Wei, Alvin Hung; Osborne, David; Abell, Chris; Blundell, Tom L.

The problems associated with neglected diseases are often compounded by increasing incidence of antibiotic resistance. Patient negligence and abuse of antibiotics has lead to explosive growth in cases of tuberculosis, with some M. tuberculosis strains becoming virtually untreatable. Structure-based drug development is viewed as cost-effective and time-consuming method for discovery and development of hits to lead compounds. In this review we will discuss the suitability of fragment-based methods for developing new chemotherapeutics against neglected diseases, providing examples from our tuberculosis programme.

Incidence and nature of human tuberculosis due to Mycobacterium africanum in South-East England: 1977-87.

PubMed

Grange, J M; Yates, M D

1989-08-01

A total of 210 new cases of tuberculosis due to Mycobacterium africanum were registered at the South-East Regional Centre for Tuberculosis Bacteriology, Dulwich, between 1977 and 1987 inclusive. This represented 1.25% of bacteriologically-confirmed cases of tuberculosis in South-East England, an incidence slightly higher than that of disease due to M. bovis. Two variants were identified: 150 strains were typed as African I (a type associated with East Africa) and 60 as African II (a type more prevalent in West Africa). Over half the patients infected with African I strains were of Indian subcontinent ethnic origin; patients of African ethnic origin predominated in the African II group while about a fifth o patients infected with either type were of European origin. The European patients with tuberculosis due to M. africanum were notably younger than those in the same region with disease due to other tubercle bacilli. The distribution of lesions due to M. africanum was similar to that due to other tubercle bacilli in the various ethnic groups, except that genito-urinary tuberculosis was uncommon. The importance of a clinical awareness that M. africanum is a highly pathogenic and transmissible tubercle bacillus rather than an opportunist or 'atypical' mycobacterium is stressed.

Cell division inhibitors with efficacy equivalent to isoniazid in the acute murine Mycobacterium tuberculosis infection model

PubMed Central

Knudson, Susan E.; Awasthi, Divya; Kumar, Kunal; Carreau, Alexandra; Goullieux, Laurent; Lagrange, Sophie; Vermet, Hélène; Ojima, Iwao; Slayden, Richard A.

2015-01-01

Objectives The increasing number of clinical strains resistant to one or more of the front-line TB drugs complicates the management of this disease. To develop next-generation benzimidazole-based FtsZ inhibitors with improved efficacy, we employed iterative optimization strategies based on whole bacteria potency, bactericidal activity, plasma and metabolic stability and in vivo efficacy studies. Methods Candidate benzimidazoles were evaluated for potency against Mycobacterium tuberculosis H37Rv and select clinical strains, toxicity against Vero cells and compound stability in plasma and liver microsomes. The efficacy of lead compounds was assessed in the acute murine M. tuberculosis infection model via intraperitoneal and oral routes. Results MICs of SB-P17G-A33, SB-P17G-A38 and SB-P17G-A42 for M. tuberculosis H37Rv and select clinical strains were 0.18–0.39 mg/L. SB-P17G-A38 and SB-P17G-A42 delivered at 50 mg/kg twice daily intraperitoneally or orally demonstrated efficacy in reducing the bacterial load by 5.7–6.3 log10 cfu in the lungs and 3.9–5.0 log10 cfu in the spleen. SB-P17G-A33 delivered at 50 mg/kg twice daily intraperitoneally or orally also reduced the bacterial load by 1.7–2.1 log10 cfu in the lungs and 2.5–3.4 log10 cfu in the spleen. Conclusions Next-generation benzimidazoles with excellent potency and efficacy against M. tuberculosis have been developed. This is the first report on benzimidazole-based FtsZ inhibitors showing an equivalent level of efficacy to isoniazid in an acute murine M. tuberculosis infection model. PMID:26245639

Enhancement of antibiotic activity by efflux inhibitors against multidrug resistant Mycobacterium tuberculosis clinical isolates from Brazil

PubMed Central

Coelho, Tatiane; Machado, Diana; Couto, Isabel; Maschmann, Raquel; Ramos, Daniela; von Groll, Andrea; Rossetti, Maria L.; Silva, Pedro A.; Viveiros, Miguel

2015-01-01

Drug resistant tuberculosis continues to increase and new approaches for its treatment are necessary. The identification of M. tuberculosis clinical isolates presenting efflux as part of their resistant phenotype has a major impact in tuberculosis treatment. In this work, we used a checkerboard procedure combined with the tetrazolium microplate-based assay (TEMA) to study single combinations between antituberculosis drugs and efflux inhibitors (EIs) against multidrug resistant M. tuberculosis clinical isolates using the fully susceptible strain H37Rv as reference. Efflux activity was studied on a real-time basis by a fluorometric method that uses ethidium bromide as efflux substrate. Quantification of efflux pump genes mRNA transcriptional levels were performed by RT-qPCR. The fractional inhibitory concentrations (FIC) indicated synergistic activity for the interactions between isoniazid, rifampicin, amikacin, ofloxacin, and ethidium bromide plus the EIs verapamil, thioridazine and chlorpromazine. The FICs ranged from 0.25, indicating a four-fold reduction on the MICs, to 0.015, 64-fold reduction. The detection of active efflux by real-time fluorometry showed that all strains presented intrinsic efflux activity that contributes to the overall resistance which can be inhibited in the presence of the EIs. The quantification of the mRNA levels of the most important efflux pump genes on these strains shows that they are intrinsically predisposed to expel toxic compounds as the exposure to subinhibitory concentrations of antibiotics were not necessary to increase the pump mRNA levels when compared with the non-exposed counterpart. The results obtained in this study confirm that the intrinsic efflux activity contributes to the overall resistance in multidrug resistant clinical isolates of M. tuberculosis and that the inhibition of efflux pumps by the EIs can enhance the clinical effect of antibiotics that are their substrates. PMID:25972842

Specific Proteins in Nontuberculous Mycobacteria: New Potential Tools

PubMed Central

Orduña, Patricia; Castillo-Rodal, Antonia I.; Mercado, Martha E.; Ponce de León, Samuel; López-Vidal, Yolanda

2015-01-01

Nontuberculous mycobacteria (NTM) have been isolated from water, soil, air, food, protozoa, plants, animals, and humans. Although most NTM are saprophytes, approximately one-third of NTM have been associated with human diseases. In this study, we did a comparative proteomic analysis among five NTM strains isolated from several sources. There were different numbers of protein spots from M. gordonae (1,264), M. nonchromogenicum type I (894), M. nonchromogenicum type II (935), M. peregrinum (806), and M. scrofulaceum/Mycobacterium mantenii (1,486) strains, respectively. We identified 141 proteins common to all strains and specific proteins to each NTM strain. A total of 23 proteins were selected for its identification. Two of the common proteins identified (short-chain dehydrogenase/reductase SDR and diguanylate cyclase) did not align with M. tuberculosis complex protein sequences, which suggest that these proteins are found only in the NTM strains. Some of the proteins identified as common to all strains can be used as markers of NTM exposure and for the development of new diagnostic tools. Additionally, the specific proteins to NTM strains identified may represent potential candidates for the diagnosis of diseases caused by these mycobacteria. PMID:26106621

Genotypic Diversity of Mycobacterium tuberculosis Clinical Isolates in the Multiethnic Area of the Xinjiang Uygur Autonomous Region in China.

PubMed

Liu, Jie; Li, Junlian; Liu, Jiao; Zhao, Xiuqin; Lian, Lulu; Liu, Haican; Lu, Bing; Yu, Qin; Zhang, Jingrui; Qi, Yingcheng; Wan, Kanglin

2017-01-01

Objectives. We studied the genetic diversity of clinical isolates from patients with tuberculosis in the multiethnic area of Xinjiang autonomous region in China. A total of 311 clinical M. tuberculosis isolates were collected in 2006 and 2011 and genotyped by two genotyping methods. All isolates were grouped into 68 distinct spoligotypes using the spoligotyping method. The Beijing family was dominant, followed by T1 and CAS. MIRU-VNTR results showed that a total of 195 different VNTR types were identified. Ten of the 15 loci were highly or moderately discriminant according to their HGDI scores, and 13 loci had good discriminatory power in non-Beijing family strains, whereas only two loci had good discriminatory power in Beijing family strains. Chi-square tests demonstrated that there were no correlations between four characteristics (sex, age, type of case, and treatment history) and the Beijing family. In summary, Beijing family strains were predominant in Xinjiang, and the VNTR-15 China locus-set was suitable for genotyping all Xinjiang strains, but not for the Beijing family strains. Thus, these data suggested that different genotype distributions may exist in different regions; MLVA locus-sets should be adjusted accordingly, with newly added loci to increase resolution if necessary.

Large Sequence Polymorphisms of the Euro-American lineage of Mycobacterium tuberculosis: a phylogenetic reconstruction and evidence for convergent evolution in the DR locus.

PubMed

Rindi, Laura; Lari, Nicoletta; Garzelli, Carlo

2012-10-01

The Euro-American lineage of the Mycobacterium tuberculosis complex consists of 10 sublineages, each defined by a deletion of a large genomic region (RD, region of difference); by spoligotyping, that probes the polymorphism of the Direct Repeat (DR) locus, the Euro-American strains are classified into 5 lineages (T, Haarlem, LAM, S and X) and 34 sublineages, but the relationships between the RD-defined sublineages and the spoligotype groupings are largely unclear. By testing a global sample of 158 Euro-American strains, mutually exclusive deletions of RD115, RD122, RD174, RD182, RD183, RD193, RD219, RD726 or RD761 were found in 122 strains; deletion of RD724, typical of strains from Central Africa, was not found. The RD-defined sublineages, tested for katG463/gyrA95 polymorphism, belonged to Principal Genotypic Group (PGG) 2, with the exception of RD219 sublineage belonging to PGG3; the 36 strains with no deletion were of either PGG2 or 3. Based on these polymorphisms, a phylogenetic reconstruction of the Euro-American lineage, that integrates the previously reported phylogeny, is proposed. Although certain deletions were found to be associated to certain spoligotype lineages (i.e., deletion RD115 to T and LAM, RD174 to LAM, RD182 to Haarlem, RD219 to T), our analysis indicates a general lack of concordance between RD-defined sublineages and spoligotype groupings. Moreover, of the 42 spoligotypes detected among the study strains, sixteen were shared by strains belonging to different RD sublineages. IS6110-RFLP analysis of strains sharing spoligotypes confirmed a poor genetic relatedness between strains of different RD sublineages. These findings provide evidence for the occurrence of a high degree of homoplasy in the DR locus leading to convergent evolution to identical spoligotypes. The incongruence between Large Sequence Polymorphism and spoligotype polymorphism argues against the use of spoligotyping for establishing phylogenetic relationships within the Euro-American lineage. Copyright © 2012 Elsevier B.V. All rights reserved.

Mycobacterium tuberculosis TlyA Protein Negatively Regulates T Helper (Th) 1 and Th17 Differentiation and Promotes Tuberculosis Pathogenesis*

PubMed Central

Rahman, Md. Aejazur; Sobia, Parveen; Dwivedi, Ved Prakash; Bhawsar, Aakansha; Singh, Dhiraj Kumar; Sharma, Pawan; Moodley, Prashini; Van Kaer, Luc; Bishai, William R; Das, Gobardhan

2015-01-01

Mycobacterium tuberculosis, the causative agent of tuberculosis, is an ancient pathogen and a major cause of death worldwide. Although various virulence factors of M. tuberculosis have been identified, its pathogenesis remains incompletely understood. TlyA is a virulence factor in several bacterial infections and is evolutionarily conserved in many Gram-positive bacteria, but its function in M. tuberculosis pathogenesis has not been elucidated. Here, we report that TlyA significantly contributes to the pathogenesis of M. tuberculosis. We show that a TlyA mutant M. tuberculosis strain induces increased IL-12 and reduced IL-1β and IL-10 cytokine responses, which sharply contrasts with the immune responses induced by wild type M. tuberculosis. Furthermore, compared with wild type M. tuberculosis, TlyA-deficient M. tuberculosis bacteria are more susceptible to autophagy in macrophages. Consequently, animals infected with the TlyA mutant M. tuberculosis organisms exhibited increased host-protective immune responses, reduced bacillary load, and increased survival compared with animals infected with wild type M. tuberculosis. Thus, M. tuberculosis employs TlyA as a host evasion factor, thereby contributing to its virulence. PMID:25847237

Production and characterization of a highly pure RNA polymerase holoenzyme from Mycobacterium tuberculosis.

PubMed

Herrera-Asmat, Omar; Lubkowska, Lucyna; Kashlev, Mikhail; Bustamante, Carlos J; Guerra, Daniel G; Kireeva, Maria L

2017-06-01

Recent publications have shown that active RNA polymerase (RNAP) from Mycobacterium tuberculosis (MtbRNAP) can be produced by expressing all four subunits in a single recombinant Escherichia coli strain [1-3]. By reducing the number of plasmids and changing the codon usage of the Mtb genes in the co-expression system published by Banerjee et al. [1], we present a simplified, detailed and reproducible protocol for the purification of recombinant MtbRNAP containing the ω subunit. Moreover, we describe the formation of ternary elongation complexes (TECs) with a short fluorescence-labeled RNA primer and DNA oligonucleotides, suitable for transcription elongation studies. The purification of milligram quantities of the pure and highly active holoenzyme omits ammonium sulfate or polyethylene imine precipitation steps [4] and requires only 5 g of wet cells. Our results indicate that subunit assemblies other than α 2 ββ'ω·σ A can be separated by ion-exchange chromatography on Mono Q column and that assemblies with the wrong RNAP subunit stoichiometry lack transcriptional activity. We show that MtbRNAP TECs can be stalled by NTP substrate deprivation and chased upon the addition of missing NTP(s) without the need of any accessory proteins. Finally, we demonstrate the ability of the purified MtbRNAP to initiate transcription from a promoter and establish that its open promoter complexes are stabilized by the M. tuberculosis protein CarD. Published by Elsevier Inc.

The Tuberculosis Vaccine Candidate Bacillus Calmette-Guérin ΔureC::hly Coexpressing Human Interleukin-7 or -18 Enhances Antigen-Specific T Cell Responses in Mice

PubMed Central

Rao, Martin; Vogelzang, Alexis; Kaiser, Peggy; Schuerer, Stefanie; Kaufmann, Stefan H. E.; Gengenbacher, Martin

2013-01-01

Bacillus Calmette–Guérin (BCG), the only approved tuberculosis vaccine, provides only limited protection. Previously, we generated a recombinant derivative (BCG ΔureC::hly), which secretes the pore-forming toxin listeriolysin O (LLO) of Listeria monocytogenes. This vaccine shows superior protection against tuberculosis in preclinical models and is safe in humans. Here we describe two new vaccine strains which express human interleukin-7 (hIL)-7 or hIL-18 in the genetic background of BCG ΔureC::hly to modulate specific T cell immunity. Both strains exhibited an uncompromised in vitro growth pattern, while inducing a proinflammatory cytokine profile in human dendritic cells (DCs). Human DCs harbouring either strain efficiently promoted secretion of IL-2 by autologous T cells in a coculture system, suggesting superior immunogenicity. BALB/c mice vaccinated with BCG ΔureC::hly, BCG ΔureC::hly_hIL7 or BCG ΔureC::hly_hIL18 developed a more robust Th1 response than after vaccination with parental BCG. Both strains provided significantly better protection than BCG in a murine Mycobacterium tuberculosis challenge model but efficacy remained comparable to that afforded by BCG ΔureC::hly. We conclude that expression of hIL-7 or hIL-18 enhanced specific T cell responses but failed to improve protection over BCG ΔureC::hly in mice. PMID:24236077

Molecular Epidemiology of Pulmonary Tuberculosis in Belgrade, Central Serbia

PubMed Central

Vuković, Dragana; Rüsch-Gerdes, Sabine; Savić, Branislava; Niemann, Stefan

2003-01-01

In order to gain precise data on the actual epidemiology of tuberculosis (TB) in Belgrade, central Serbia, we conducted the molecular epidemiological investigation described herein. IS6110 restriction fragment length polymorphism (RFLP) typing of 176 Mycobacterium tuberculosis isolates was performed. These strains were obtained from 48.4% of all patients diagnosed with culture-proven pulmonary TB from April through September 1998 and from May through October 1999. Clusters containing strains with identical RFLP IS6110 patterns were assumed to have arisen from recent transmission. Of the 176 cases, 55 (31.2%) were grouped into 23 clusters ranging in size from two to six patients. Nearly 80% of clustered patients were directly interviewed, and transmission between family-unrelated contacts was found to be predominant in the study population. Classical contact investigation identified only 2 (3.6%) of the 55 clustered patients. The clustering of TB patients was not associated with any demographic or clinical characteristic other than infection with multidrug-resistant (MDR) M. tuberculosis strains. Nearly 70% of MDR strains were clustered, which indicates active transmission of MDR TB in Belgrade. However, this was not observed by conventional epidemiologic surveillance. In conclusion, the first molecular epidemiologic analysis of TB in the region revealed frequent recent transmission of TB and pointed out significant shortcomings of the current concept for conventional contact tracing. The results presented also demonstrate that transmission of MDR TB in Belgrade is not optimally controlled, and they provide support for the development of improved control strategies, including application of molecular methods. PMID:12958271

Molecular & genetic characteristics of Mycobacterium tuberculosis strains circulating in the southern part of West Siberia

PubMed Central

Pasechnik, Oksana; Dymova, Maya Alexandrovna; Stasenko, Vladimir Leonidovich; Blokh, Aleksey Igorevich; Tatarintseva, Marina Petrovna; Kolesnikova, Lyubov Pavlovna; Filipenko, Maksim Leonidovich

2017-01-01

Background & objectives: A complicated epidemiological situation characterized by significantly high tuberculosis (TB) morbidity is observed in West Siberia. This study was aimed to investigate the genetic characteristics of Mycobacterium tuberculosis circulating in the southern part of West Siberia (in the Omsk region). Methods: From March 2013 to January 2015, 100 isolates of M. tuberculosis were obtained from patients with pulmonary TB living in the Omsk region. Drug susceptibility testing was performed on Lowenstein-Jensen medium (absolute concentration method). Genetic typing of isolates was carried out by variable number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR) typing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The genetic types and characteristics of cluster strains were determined using 15 MIRU-VNTR loci. Results: Thirty six VNTR types were found. Twenty six (26.0%) isolates had a unique profile, and the remaining 74 were grouped in 10 clusters containing from 2 to 23 isolates. The Beijing genotype was found in 72 isolates, 61 (85.0%) of which were part of five clusters that included two large clusters containing 23 isolates. Other genetic families, such as Latin-American Mediterranean (LAM, 11.0%), S family (2.0%) and Haarlem (4.0%), were also detected. The genetic family of 11 isolates could not be determined. Six different VNTR profiles were found in these non-classified isolates. Only 16 per cent of isolates were sensitive to anti-TB drugs. The katG315 (94.8%) and rpoB531 (92.2%) mutations were identified in 77 multidrug-resistant M. tuberculosis isolates. Interpretation & conclusions: This study showed that the M. tuberculosis population in the Omsk region was heterogeneous. The Beijing genotype predominated and was actively spreading. The findings obtained point to the need for the implementation of more effective preventive measures to stop the spread of drug-resistant M. tuberculosis strains. PMID:29168460

Drug resistance characteristics of Mycobacterium tuberculosis isolates to four first-line antituberculous drugs from tuberculosis patients with AIDS in Beijing, China.

PubMed

Gao, Gui-ju; Lian, Lulu; Sun, Yue; Wei, Jianhao; Xiao, Jiang; Wang, Xiaoying; Zhang, Ling; Zhao, Xiuqin; Yang, Di; Zhao, Hong-xin; Zhao, Hui; Wang, Hui-zhu; Wan, Kang-lin; Li, Xing-wang

2015-02-01

The objective of this study was to investigate the drug resistance characteristics of Mycobacterium tuberculosis isolates to four first-line antituberculous drugs (ATDs) from tuberculosis (TB) patients with AIDS in Beijing, China. All M. tuberculosis strains were isolated from specimens from TB patients with AIDS hospitalised between April 2010 and October 2012. Isolates were cultured by mycobacterial culture methods and were identified by multilocus PCR. Drug sensitivity testing was performed by the proportion method with the following first-line ATDs: isoniazid; rifampicin; streptomycin; and ethambutol. Results were compared with the drug resistance status of M. tuberculosis strains isolated from TB patients without HIV infection in Beijing. Among 41 M. tuberculosis isolates from TB patients with AIDS, the rates of total drug resistance (58.5%), initial drug resistance (46.7%) and acquired drug resistance (90.9%) were significantly higher than in TB patients without HIV infection (34.1%, 24.5% and 48.5%, respectively; P<0.05). In TB patients with AIDS, the rates of acquired drug resistance (90.9%) and acquired multidrug-resistant TB (MDR-TB) (54.5%) were significantly higher than the rates of initial drug resistance (46.7%) and initial MDR-TB (10.0%) (P<0.05). In patients with TB without HIV infection, the rate of acquired drug resistance (48.5%) was significantly higher than the rate of initial drug resistance (24.5%) (P<0.05). M. tuberculosis drug resistance in TB patients with AIDS is significantly more serious than in TB patients without HIV infection. These results showed that more attention should be paid to M. tuberculosis drug resistance in AIDS patients. Copyright © 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Prevalence of Mycobacterium tuberculosis Beijing genotype and its association with drug resistance in North India.

PubMed

Mathuria, Jitendra P; Srivastava, Govind N; Sharma, Pragya; Mathuria, Bharat L; Ojha, Sanjay; Katoch, Vishwa M; Anupurba, Shampa

The global presence and rapid dissemination of Beijing genotype of Mycobacterium tuberculosis, makes it an important issue of public health. Its presence and association with multi-drug resistance has been shown in many settings. In present study we tried to find its prevalence and association with drug resistance in North India. One hundred and twenty four M. tuberculosis isolates were analyzed with spoligotyping, further drug susceptibility testing was done by 1% proportional method. Out of these, 11 (8.9%) M. tuberculosis isolates were identified as Beijing and 113 (91.1%) as non-Beijing genotypes. While looking at their drug susceptibility patterns, 6 (54.5%) & 22 (19.5%) were found to be multi drug resistant (MDR) among Beijing and non-Beijing isolates respectively. Our study concluded that the Beijing strains were not so common in north India and these strains do not fully associate with MDR. Copyright © 2017 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.

Heterogeneity of phenotypic characteristics of the modern and ancestral Beijing strains of Mycobacterium tuberculosis.

PubMed

Faksri, Kiatichai; Chaiprasert, Angkana; Pardieu, Clarie; Casali, Nicola; Palaga, Tanapat; Prammananan, Therdsak; Palittapongarnpim, Prasit; Prayoonwiwat, Naraporn; Drobniewski, Francis

2014-06-01

The Beijing strain of Mycobacterium tuberculosis (MTB) is of great concern because this hypervirulent strain has caused numerous tuberculosis outbreaks. However, the mechanisms that allow the MTB Beijing strain to be highly pathogenic remain unclear and previous studies have revealed heterogeneity within this family. To determine the association between some phenotypic characteristics and phylogroups of the Beijing strain of MTB. Eight Beijing strains, 5 modern and 3 ancestral sublineages, were selected from the phylogroups of MTB. The selection was based on copy number of IS6110 at NTF, region of differences, and single nucleotide polymorphisms. The abilities of these strains to grow intracellularly in THP-1 macrophages, to induce apoptosis, necrosis, and cytokines production were examined using quantitative real-time PCR and commercially available ELISA kits, respectively. There were some significant differences between the two sublineages of the Beijing strain of MTB. The ancestral Beijing sublineages showed higher intracellular growth rates (p < 0.05) and necrosis induction rates (p < 0.01) than the modern Beijing sublineages. By contrast, the modern Beijing sublineages induced lower apoptosis and protective cytokine responses, i.e., TNF-α (p < 0.05) and IL-6 (p < 0.01) and higher non-protective IL-10 response. The modern Beijing sublineages may have evolved so that they have greater ability to diminish host defense mechanisms. The slower growth rate and reduced necrosis induction in host cells might allow the bacteria to cause a persistent infection. The results revealed a phylogroup-associated heterogeneity of phenotypes among MTB Beijing sublineages.

Synthesis and evaluation of copper(II) complexes with isoniazid-derived hydrazones as anticancer and antitubercular agents.

PubMed

Firmino, Gisele S S; de Souza, Marcus V N; Pessoa, Claudia; Lourenco, Maria C S; Resende, Jackson A L C; Lessa, Josane A

2016-12-01

In this study, the N,N,O metal chelator 2-pyridinecarboxaldehydeisonicotinoyl hydrazone (HPCIH, 1) and its derivatives 2-acetylpyridine-(HAPIH 2), 2-pyridineformamide-(HPAmIH, 3) and pyrazineformamide-(HPzAmIH, 4) were employed in the synthesis of four copper(II) complexes, [Cu(HPCIH)Cl 2 ]·0.4H 2 O (5), [Cu(HAPIH)Cl 2 ]·1.25H 2 O (6), [Cu(HPAmIH)Cl 2 ]·H 2 O (7) and [Cu(HPzAmIH)Cl 2 ]·1.25H 2 O (8). The compounds were assayed for their action toward Mycobacterium tuberculosis H37Rv ATCC 27294 strain and the human tumor cell lines OVCAR-8 (ovarian cancer), SF-295 (glioblastoma multiforme) and HCT-116 (colon adenocarcinoma). All copper(II) complexes were more effective in reducing growth of HCT-116 and SF-295 cells than the respective free hydrazones at 5 µg/mL, whereas only complex 7 was more cytotoxic toward OVCAR-8 lines than its ligand HPAmIH. 6 proved to be cytotoxic at submicromolar doses, whose IC 50 values (0.39-0.86 µM) are similar to those ones found for doxorubicin (0.23-0.43 µM). Complexes 5 and 6 displayed high activity against M. tuberculosis (MIC = 0.85 and 1.58 µM, respectively), as compared with isoniazid (MIC = 2.27 µM), which suggests the compounds are attractive candidates as antitubercular drugs.

Disulfiram and Copper Ions Kill Mycobacterium tuberculosis in a Synergistic Manner

PubMed Central

Dalecki, Alex G.; Haeili, Mehri; Shah, Santosh; Speer, Alexander; Niederweis, Michael; Kutsch, Olaf

2015-01-01

Tuberculosis is a severe disease affecting millions worldwide. Unfortunately, treatment strategies are hampered both by the prohibitively long treatment regimen and the rise of drug-resistant strains. Significant effort has been expended in the search for new treatments, but few options have successfully emerged, and new treatment modalities are desperately needed. Recently, there has been growing interest in the synergistic antibacterial effects of copper ions (CuII/I) in combination with certain small molecular compounds, and we have previously reported development of a drug screening strategy to harness the intrinsic bactericidal properties of CuII/I. Here, we describe the copper-dependent antimycobacterial properties of disulfiram, an FDA-approved and well-tolerated sobriety aid. Disulfiram was inhibitory to mycobacteria only in the presence of CuII/I and exerted its bactericidal activity well below the active concentration of CuII/I or disulfiram alone. No other physiologically relevant bivalent transition metals (e.g., FeII, NiII, MnII, and CoII) exhibited this effect. We demonstrate that the movement of the disulfiram-copper complex across the cell envelope is porin independent and can inhibit intracellular protein functions. Additionally, the complex is able to synergistically induce intracellular copper stress responses significantly more than CuII/I alone. Our data suggest that by complexing with disulfiram, CuII/I is likely allowed unfettered access to vulnerable intracellular components, bypassing the normally sufficient copper homeostatic machinery. Overall, the synergistic antibacterial activity of CuII/I and disulfiram reveals the susceptibility of the copper homeostasis system of Mycobacterium tuberculosis to chemical attacks and establishes compounds that act in concert with copper as a new class of bacterial inhibitors. PMID:26033731

Dehalogenation of Haloalkanes by Mycobacterium tuberculosis H37Rv and Other Mycobacteria

PubMed Central

Jesenská, Andrea; Sedlác̆ek, Ivo; Damborský, Jir̆í

2000-01-01

Haloalkane dehalogenases convert haloalkanes to their corresponding alcohols by a hydrolytic mechanism. To date, various haloalkane dehalogenases have been isolated from bacteria colonizing environments that are contaminated with halogenated compounds. A search of current databases with the sequences of these known haloalkane dehalogenases revealed the presence of three different genes encoding putative haloalkane dehalogenases in the genome of the human parasite Mycobacterium tuberculosis H37Rv. The ability of M. tuberculosis and several other mycobacterial strains to dehalogenate haloaliphatic compounds was therefore studied. Intact cells of M. tuberculosis H37Rv were found to dehalogenate 1-chlorobutane, 1-chlorodecane, 1-bromobutane, and 1,2-dibromoethane. Nine isolates of mycobacteria from clinical material and four strains from a collection of microorganisms were found to be capable of dehalogenating 1,2-dibromoethane. Crude extracts prepared from two of these strains, Mycobacterium avium MU1 and Mycobacterium smegmatis CCM 4622, showed broad substrate specificity toward a number of halogenated substrates. Dehalogenase activity in the absence of oxygen and the identification of primary alcohols as the products of the reaction suggest a hydrolytic dehalogenation mechanism. The presence of dehalogenases in bacterial isolates from clinical material, including the species colonizing both animal tissues and free environment, indicates a possible role of parasitic microorganisms in the distribution of degradation genes in the environment. PMID:10618227

Ability of Innate Defence Regulator Peptides IDR-1002, IDR-HH2 and IDR-1018 to Protect against Mycobacterium tuberculosis Infections in Animal Models

PubMed Central

Rivas-Santiago, Bruno; Castañeda-Delgado, Julio E.; Rivas Santiago, Cesar E.; Waldbrook, Matt; González-Curiel, Irma; León–Contreras, Juan C.; Enciso-Moreno, Jose Antonio; del Villar, Victor; Mendez-Ramos, Jazmin; Hancock, Robert E. W.; Hernandez-Pando, Rogelio

2013-01-01

Tuberculosis is an ongoing threat to global health, especially with the emergence of multi drug-resistant (MDR) and extremely drug-resistant strains that are motivating the search for new treatment strategies. One potential strategy is immunotherapy using Innate Defence Regulator (IDR) peptides that selectively modulate innate immunity, enhancing chemokine induction and cell recruitment while suppressing potentially harmful inflammatory responses. IDR peptides possess only modest antimicrobial activity but have profound immunomodulatory functions that appear to be influential in resolving animal model infections. The IDR peptides HH2, 1018 and 1002 were tested for their activity against two M. tuberculosis strains, one drug-sensitive and the other MDR in both in vitro and in vivo models. All peptides showed no cytotoxic activity and only modest direct antimicrobial activity versus M. tuberculosis (MIC of 15–30 µg/ml). Nevertheless peptides HH2 and 1018 reduced bacillary loads in animal models with both the virulent drug susceptible H37Rv strain and an MDR isolate and, especially 1018 led to a considerable reduction in lung inflammation as revealed by decreased pneumonia. These results indicate that IDR peptides have potential as a novel immunotherapy against TB. PMID:23555622

Genetic Heterogeneity in Mycobacterium tuberculosis Isolates Reflected in IS6110 Restriction Fragment Length Polymorphism Patterns as Low-Intensity Bands

PubMed Central

de Boer, Annette S.; Kremer, Kristin; Borgdorff, Martien W.; de Haas, Petra E. W.; Heersma, Herre F.; van Soolingen, Dick

2000-01-01

Mycobacterium tuberculosis isolates with identical IS6110 restriction fragment length polymorphism (RFLP) patterns are considered to originate from the same ancestral strain and thus to reflect ongoing transmission. In this study, we investigated 1,277 IS6110 RFLP patterns for the presence of multiple low-intensity bands (LIBs), which may indicate infections with multiple M. tuberculosis strains. We did not find any multiple LIBs, suggesting that multiple infections are rare in the Netherlands. However, we did observe a few LIBs in 94 patterns (7.4%) and examined the nature of this phenomenon. With single-colony cultures it was found that LIBs mostly represent mixed bacterial populations with slightly different RFLP patterns. Mixtures were expressed in RFLP patterns as LIBs when 10 to 30% of the DNA analyzed originated from a bacterial population with another RFLP pattern. Presumably, a part of the LIBs did not represent mixed bacterial populations, as in some clusters all strains exhibited LIBs in their RFLP patterns. The occurrence of LIBs was associated with increased age in patients. This may reflect either a gradual change of the bacterial population in the human body over time or IS6110-mediated genetic adaptation of M. tuberculosis to changes in the environmental conditions during the dormant state or reactivation thereafter. PMID:11101583

Clonality and Micro-Diversity of a Nationwide Spreading Genotype of Mycobacterium tuberculosis in Japan

PubMed Central

Wada, Takayuki; Iwamoto, Tomotada; Tamaru, Aki; Seto, Junji; Ahiko, Tadayuki; Yamamoto, Kaori; Hase, Atushi; Maeda, Shinji; Yamamoto, Taro

2015-01-01

Mycobacterium tuberculosis transmission routes can be estimated from genotypic analysis of clinical isolates from patients. In Japan, still a middle-incidence country of TB, a unique genotype strain designated as ‘M-strain’ has been isolated nationwide recently. To ascertain the history of the wide spread of the strain, 10 clinical isolates from different areas were subjected to genome-wide analysis based on deep sequencers. Results show that all isolates possessed common mutations to those of referential strains. The greatest number of accumulated single nucleotide variants (SNVs) from the oldest coalescence was 13 nucleotides, indicating high clonality of these isolates. When an SNV common to the isolates was used as a surrogate marker of the clone, authentic clonal isolates with variation in a reliable subset of variable number of tandem repeat (VNTR) genotyping method can be selected successfully from clinical isolates populations of M. tuberculosis. When the authentic clones can also be assigned to sub-clonal groups by SNVs derived from the genomic comparison, they are classifiable into three sub-clonal groups with a bias of geographical origins. Feedback from genomic analysis of clinical isolates of M. tuberculosis to genotypic markers will be an efficient strategy for the big data in various settings for public health actions against TB. PMID:25734518

Whole-Transcriptome and -Genome Analysis of Extensively Drug-Resistant Mycobacterium tuberculosis Clinical Isolates Identifies Downregulation of ethA as a Mechanism of Ethionamide Resistance

PubMed Central

de Welzen, Lynne; Eldholm, Vegard; Maharaj, Kashmeel; Manson, Abigail L.; Earl, Ashlee M.

2017-01-01

ABSTRACT Genetics-based drug susceptibility testing has improved the diagnosis of drug-resistant tuberculosis but is limited by our lack of knowledge of all resistance mechanisms. Next-generation sequencing has assisted in identifying the principal genetic mechanisms of resistance for many drugs, but a significant proportion of phenotypic drug resistance is unexplained genetically. Few studies have formally compared the transcriptomes of susceptible and resistant Mycobacterium tuberculosis strains. We carried out comparative whole-genome transcriptomics of extensively drug-resistant (XDR) clinical isolates using RNA sequencing (RNA-seq) to find novel transcription-mediated mechanisms of resistance. We identified a promoter mutation (t to c) at position −11 (t−11c) relative to the start codon of ethA that reduces the expression of a monooxygenase (EthA) that activates ethionamide. (In this article, nucleotide changes are lowercase and amino acid substitutions are uppercase.) Using a flow cytometry-based reporter assay, we show that the reduced transcription of ethA is not due to transcriptional repression by ethR. Clinical strains harboring this mutation were resistant to ethionamide. Other ethA promoter mutations were identified in a global genomic survey of resistant M. tuberculosis strains. These results demonstrate a new mechanism of ethionamide resistance that can cause high-level resistance when it is combined with other ethionamide resistance-conferring mutations. Our study revealed many other genes which were highly up- or downregulated in XDR strains, including a toxin-antitoxin module (mazF5 mazE5) and tRNAs (leuX and thrU). This suggests that global transcriptional modifications could contribute to resistance or the maintenance of bacterial fitness have also occurred in XDR strains. PMID:28993337

An investigation on the population structure of mixed infections of Mycobacterium tuberculosis in Inner Mongolia, China.

PubMed

Wang, Xiaoying; Liu, Haican; Wei, Jianhao; Wu, Xiaocui; Yu, Qin; Zhao, Xiuqin; Lyu, Jianxin; Lou, Yongliang; Wan, Kanglin

2015-12-01

Mixed infections of Mycobacterium tuberculosis strains have attracted more attention due to their increasing frequencies worldwide, especially in the areas of high tuberculosis (TB) prevalence. In this study, we accessed the rates of mixed infections in a setting with high TB prevalence in Inner Mongolia Autonomous Region of China. A total of 384 M. tuberculosis isolates from the local TB hospital were subjected to mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing method. The single clones of the strains with mixed infections were separated by subculturing them on the Löwenstein-Jensen medium. Of these 384 isolates, twelve strains (3.13%) were identified as mixed infections by MIRU-VNTR. Statistical analysis indicated that demographic characteristics and drug susceptibility profiles showed no statistically significant association with the mixed infections. We further subcultured the mixed infection strains and selected 30 clones from the subculture for each mixed infection. Genotyping data revealed that eight (8/12, 66.7%) strains with mixed infections had converted into single infection through subculture. The higher growth rate was associated with the increasing proportion of variant subpopulation through subculture. In conclusion, by using the MIRU-VNTR method, we demonstrate that the prevalence of mixed infections in Inner Mongolia is low. Additionally, our findings reveal that the subculture changes the population structures of mixed infections, and the subpopulation with higher growth rate show better fitness, which is associated with high proportion among the population structure after subculture. This study highlights that the use of clinical specimens, rather than subcultured isolates, is preferred to estimate the prevalence of mixed infections in the specific regions. Copyright © 2015 Elsevier Ltd. All rights reserved.

Polymorphisms of twenty regulatory proteins between Mycobacterium tuberculosis and Mycobacterium bovis

USDA-ARS?s Scientific Manuscript database

Mycobacterium tuberculosis and Mycobacterium bovis are responsible for tuberculosis in humans or animals, respectively. Both species are closely related and belong to the Mycobacterium tuberculosis complex (MTC). M. tuberculosis is the most ancient species from which M. bovis and the other members o...

An acidic sphingomyelinase Type C activity from Mycobacterium tuberculosis.

PubMed

Castro-Garza, Jorge; González-Salazar, Francisco; Quinn, Frederick D; Karls, Russell K; De La Garza-Salinas, Laura Hermila; Guzmán-de la Garza, Francisco J; Vargas-Villarreal, Javier

2016-01-01

Sphingomyelinases (SMases) catalyze the hydrolysis of sphingomyelin to ceramide and phosphorylcholine. Sphingolipids are recognized as diverse and dynamic regulators of a multitude of cellular processes mediating cell cycle control, differentiation, stress response, cell migration, adhesion, and apoptosis. Bacterial SMases are virulence factors for several species of pathogens. Whole cell extracts of Mycobacterium tuberculosis strains H37Rv and CDC1551 were assayed using [N-methyl-(14)C]-sphingomyelin as substrate. Acidic Zn(2+)-dependent SMase activity was identified in both strains. Peak SMase activity was observed at pH 5.5. Interestingly, overall SMase activity levels from CDC1551 extracts are approximately 1/3 of those of H37Rv. The presence of exogenous SMase produced by M. tuberculosis during infection may interfere with the normal host inflammatory response thus allowing the establishment of infection and disease development. This Type C activity is different from previously identified M. tuberculosis SMases. Defining the biochemical characteristics of M. tuberculosis SMases helps to elucidate the roles that these enzymes play during infection and disease. Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

Bovine tuberculosis in Canadian wildlife: An updated history

PubMed Central

Wobeser, Gary

2009-01-01

Mycobacterium bovis infection in wild animals attracted little attention in Canada until the disease was almost eliminated from domestic livestock. Tuberculosis was endemic in plains bison and occurred in elk, moose, and mule deer in Buffalo National Park (BNP), Alberta during the 1920s and 1930s. Bison were moved from BNP to Wood Buffalo National Park (WBNP), where tuberculosis became, and remains, endemic in bison, posing a risk to efforts to restore bison in northern Canada. Tuberculosis was found in a white-tailed deer in Ontario in 1959, and in an infected elk near Riding Mountain National Park (RMNP), Manitoba in 1992. Intense surveillance has resulted in detection of 40 elk, 8 white-tailed deer, and 7 cattle herds infected between 1997 and 2008 in the RMNP area. The strains of M. bovis in the RMNP area are different from strains tested from cattle and bison elsewhere in Canada. Management of tuberculosis in cattle and wild animals is challenging because of uncertainty about the ecology of the disease in various species, difficulty in obtaining samples and population data from wildlife, lack of validated tests, overlapping jurisdictions and authority, and conflicting values and opinions among stakeholders. PMID:20119541

Viral Booster Vaccines Improve Mycobacterium bovis BCG-Induced Protection Against Bovine Tuberculosis

USDA-ARS?s Scientific Manuscript database

Previous work in small animal laboratory models of tuberculosis have shown that vaccination strategies based on heterologous prime-boost protocols using Mycobacterium bovis bacille Calmette-Guerin (BCG) to prime and Modified Vaccinia Ankara strain (MVA85A) or recombinant attenuated adenoviruses (Ad8...

Multidrug resistant tuberculosis diagnosed by synovial fluid analysis.

PubMed

van Zeller, M; Monteiro, R; Ramalho, J; Almeida, I; Duarte, R

2012-01-01

Tuberculosis remains a major public health problem worldwide. HIV co-infection is contributing to an increased incidence of the disease, particularly that caused by multidrug resistant strains of Mycobacterium tuberculosis (MT). We describe an HIV-infected patient with pleural and lymph node tuberculosis diagnosed by pleural effusion characteristics and biopsy specimens, without MT identification, that further presented with knee-joint involvement. Arthrocentesis allowed MT isolation and drug susceptibility testing, resulting in a diagnosis of multidrug-resistant tuberculosis and an appropriate treatment regimen. MT identification and drug susceptibility tests are very important, especially for HIV co-infected patients. Copyright © 2011 Sociedade Portuguesa de Pneumologia. Published by Elsevier España. All rights reserved.

Genetic Diversity of Mycobacterium tuberculosis in Peru and Exploration of Phylogenetic Associations with Drug Resistance

PubMed Central

Sheen, Patricia; Couvin, David; Grandjean, Louis; Zimic, Mirko; Dominguez, Maria; Luna, Giannina; Gilman, Robert H.; Rastogi, Nalin; Moore, David A. J.

2013-01-01

Background There is limited available data on the strain diversity of M tuberculosis in Peru, though there may be interesting lessons to learn from a setting where multidrug resistant TB has emerged as a major problem despite an apparently well-functioning DOTS control programme. Methods Spoligotyping was undertaken on 794 strains of M tuberculosis collected between 1999 and 2005 from 553 community-based patients and 241 hospital-based HIV co-infected patients with pulmonary tuberculosis in Lima, Peru. Phylogenetic and epidemiologic analyses permitted identification of clusters and exploration of spoligotype associations with drug resistance. Results Mean patient age was 31.9 years, 63% were male and 30.4% were known to be HIV+. Rifampicin mono-resistance, isoniazid mono-resistance and multidrug resistance (MDR) were identified in 4.7%, 8.7% and 17.3% of strains respectively. Of 794 strains from 794 patients there were 149 different spoligotypes. Of these there were 27 strains (3.4%) with novel, unique orphan spoligotypes. 498 strains (62.7%) were clustered in the nine most common spoligotypes: 16.4% SIT 50 (clade H3), 12.3% SIT 53 (clade T1), 8.3% SIT 33 (LAM3), 7.4% SIT 42 (LAM9), 5.5% SIT 1 (Beijing), 3.9% SIT 47 (H1), 3.0% SIT 222 (clade unknown), 3.0% SIT1355 (LAM), and 2.8% SIT 92 (X3). Amongst HIV-negative community-based TB patients no associations were seen between drug resistance and specific spoligotypes; in contrast HIV-associated MDRTB, but not isoniazid or rifampicin mono-resistance, was associated with SIT42 and SIT53 strains. Conclusion Two spoligotypes were associated with MDR particularly amongst patients with HIV. The MDR-HIV association was significantly reduced after controlling for SIT42 and SIT53 status; residual confounding may explain the remaining apparent association. These data are suggestive of a prolonged, clonal, hospital-based outbreak of MDR disease amongst HIV patients but do not support a hypothesis of strain-specific propensity for the acquisition of resistance-conferring mutations. PMID:23826083

Bacteriological diagnosis and molecular strain typing of Mycobacterium bovis and Mycobacterium caprae.

PubMed

Gormley, E; Corner, L A L; Costello, E; Rodriguez-Campos, S

2014-10-01

The primary isolation of a Mycobacterium sp. of the Mycobacterium tuberculosis complex from an infected animal provides a definitive diagnosis of tuberculosis. However, as Mycobacterium bovis and Mycobacterium caprae are difficult to isolate, particularly for animals in the early stages of disease, success is dependent on the optimal performance of all aspects of the bacteriological process, from the initial choice of tissue samples at post-mortem examination or clinical samples, to the type of media and conditions used to cultivate the microorganism. Each step has its own performance characteristics, which can contribute to sensitivity and specificity of the procedure, and may need to be optimized in order to achieve the gold standard diagnosis. Having isolated the slow-growing mycobacteria, species identification and fine resolution strain typing are keys to understanding the epidemiology of the disease and to devise strategies to limit transmission of infection. New technologies have emerged that can now even discriminate different isolates from the same animal. In this review we highlight the key factors that contribute to the accuracy of bacteriological diagnosis of M. bovis and M. caprae, and describe the development of advanced genotyping techniques that are increasingly used in diagnostic laboratories for the purpose of supporting detailed epidemiological investigations. Copyright © 2014 Elsevier Ltd. All rights reserved.

Synthesis and antimycobacterial activity of isoniazid derivatives from renewable fatty acids.

PubMed

Rodrigues, Marieli O; Cantos, Jéssica B; D'Oca, Caroline R Montes; Soares, Karina L; Coelho, Tatiane S; Piovesan, Luciana A; Russowsky, Dennis; da Silva, Pedro A; D'Oca, Marcelo G Montes

2013-11-15

This work describes the synthesis of a series of fatty acid hydrazide derivatives of isoniazid (INH). The compounds were tested against Mycobacterium tuberculosis H37Rv (ATCC 27294) as well as INH-resistant (ATCC 35822 and 1896 HF) and rifampicin-resistant (ATCC 35338) M. tuberculosis strains. The fatty acid derivatives of INH showed high antimycobacterial potency against the studied strains, which is desirable for a pharmaceutical compound, suggesting that the increased lipophilicity of isoniazid plays an important role in its antimycobacterial activity. Copyright © 2013 Elsevier Ltd. All rights reserved.

Mycobacterium tuberculosis Complex and HIV Co-Infection among Extrapulmonary Tuberculosis Suspected Cases at the University of Gondar Hospital, Northwestern Ethiopia

PubMed Central

Fanosie, Alemu; Gelaw, Baye; Tessema, Belay; Tesfay, Wogahta; Admasu, Aschalew; Yitayew, Gashaw

2016-01-01

Background Extrapulmonary Tuberculosis (EPTB) and Human Immunodeficiency Virus (HIV) infection are interrelated as a result of immune depression. The aim of this study was to determine the prevalence of Mycobacterium tuberculosis complex isolates and the burden of HIV co-infection among EPTB suspected patients. Method An institution based cross-sectional study was conducted among EPTB suspected patients at the University of Gondar Hospital. Socio-demographic characteristics and other clinical data were collected using a pretested questionnaire. GeneXpert MTB/RIF assay was performed to diagnosis Mycobacterium tuberculosis complex and Rifampicin resistance. All samples were also investigated by cytology and culture. The HIV statuses of all patients were screened initially by KHB, and all positive cases were further re-tested by STAT-pack. Data was analyzed using SPSS version 20 computer software and a P-value of < 0.05 was taken as statistically significant. Results A total of 141 extrapulmonary suspected patients were enrolled in this study. The overall prevalence of culture confirmed extrapulmonary tuberculosis infection was 29.8%, but the GeneXpert result showed a 26.2% prevalence of Mycobacterium tuberculosis complex infection. The 78.4% prevalence of extrapulmonary tuberculosis infection was found to be higher among the adult population. The prevalence of HIV infection among EPTB suspected patients was 14.1%, while it was 32.4% among GeneXpert-confirmed extrapulmonary TB cases (12/37). Tuberculosis lymphadenitis was the predominant (78.4%) type of EPTB infection followed by tuberculosis cold abscess (10.7%). Adult hood, previous history of contact with known pulmonary tuberculosis patients, and HIV co-infection showed a statistically significant association with extrapulmonary tuberculosis infection (P<0.013). Conclusion The prevalence of culture confirmed-EPTB infection was high, and a higher EPTB-HIV co-infection was also observed. PMID:26950547

Virulence of Mycobacterium tuberculosis after Acquisition of Isoniazid Resistance: Individual Nature of katG Mutants and the Possible Role of AhpC.

PubMed

Nieto R, Luisa Maria; Mehaffy, Carolina; Creissen, Elizabeth; Troudt, JoLynn; Troy, Amber; Bielefeldt-Ohmann, Helle; Burgos, Marcos; Izzo, Angelo; Dobos, Karen M

2016-01-01

In the last decade, there were 10 million new tuberculosis cases per year globally. Around 9.5% of these cases were caused by isoniazid resistant (INHr) Mycobacterium tuberculosis (Mtb) strains. Although isoniazid resistance in Mtb is multigenic, mutations in the catalase-peroxidase (katG) gene predominate among the INHr strains. The effect of these drug-resistance-conferring mutations on Mtb fitness and virulence is variable. Here, we assessed differences in bacterial growth, immune response and pathology induced by Mtb strains harboring mutations at the N-terminus of the katG gene. We studied one laboratory and one clinically isolated Mtb clonal pair from different genetic lineages. The INHr strain in each pair had one and two katG mutations with significantly reduced levels of the enzyme and peroxidase activity. Both strains share the V1A mutation, while the double mutant clinical INHr had also the novel E3V katG mutation. Four groups of C57BL/6 mice were infected with one of the Mtb strains previously described. We observed a strong reduction in virulence (reduced bacterial growth), lower induction of proinflammatory cytokines and significantly reduced pathology scores in mice infected with the clinical INHr strain compared to the infection caused by its INHs progenitor strain. On the other hand, there was a subtle reduction of bacteria growth without differences in the pathology scores in mice infected with the laboratory INHr strain. Our results also showed distinct alkyl-hydroperoxidase C (AhpC) levels in the katG mutant strains, which could explain the difference in the virulence profile observed. The difference in the AhpC levels between clonal strains was not related to a genetic defect in the gene or its promoter. Cumulatively, our results indicate that the virulence, pathology and fitness of INHr strains could be negatively affected by multiple mutations in katG, lack of the peroxidase activity and reduced AhpC levels.

Genetic Diversity and Transmission Characteristics of Beijing Family Strains of Mycobacterium tuberculosis in Peru

PubMed Central

Iwamoto, Tomotada; Grandjean, Louis; Arikawa, Kentaro; Nakanishi, Noriko; Caviedes, Luz; Coronel, Jorge; Sheen, Patricia; Wada, Takayuki; Taype, Carmen A.; Shaw, Marie-Anne; Moore, David A. J.; Gilman, Robert H.

2012-01-01

Beijing family strains of Mycobacterium tuberculosis have attracted worldwide attention because of their wide geographical distribution and global emergence. Peru, which has a historical relationship with East Asia, is considered to be a hotspot for Beijing family strains in South America. We aimed to unveil the genetic diversity and transmission characteristics of the Beijing strains in Peru. A total of 200 Beijing family strains were identified from 2140 M. tuberculosis isolates obtained in Lima, Peru, between December 2008 and January 2010. Of them, 198 strains were classified into sublineages, on the basis of 10 sets of single nucleotide polymorphisms (SNPs). They were also subjected to variable number tandem-repeat (VNTR) typing using an international standard set of 15 loci (15-MIRU-VNTR) plus 9 additional loci optimized for Beijing strains. An additional 70 Beijing family strains, isolated between 1999 and 2006 in Lima, were also analyzed in order to make a longitudinal comparison. The Beijing family was the third largest spoligotyping clade in Peru. Its population structure, by SNP typing, was characterized by a high frequency of Sequence Type 10 (ST10), which belongs to a modern subfamily of Beijing strains (178/198, 89.9%). Twelve strains belonged to the ancient subfamily (ST3 [n = 3], ST25 [n = 1], ST19 [n = 8]). Overall, the polymorphic information content for each of the 24 loci values was low. The 24 loci VNTR showed a high clustering rate (80.3%) and a high recent transmission index (RTIn−1 = 0.707). These strongly suggest the active and on-going transmission of Beijing family strains in the survey area. Notably, 1 VNTR genotype was found to account for 43.9% of the strains. Comparisons with data from East Asia suggested the genotype emerged as a uniquely endemic clone in Peru. A longitudinal comparison revealed the genotype was present in Lima by 1999. PMID:23185395

Genetic diversity and transmission characteristics of Beijing family strains of Mycobacterium tuberculosis in Peru.

PubMed

Iwamoto, Tomotada; Grandjean, Louis; Arikawa, Kentaro; Nakanishi, Noriko; Caviedes, Luz; Coronel, Jorge; Sheen, Patricia; Wada, Takayuki; Taype, Carmen A; Shaw, Marie-Anne; Moore, David A J; Gilman, Robert H

2012-01-01

Beijing family strains of Mycobacterium tuberculosis have attracted worldwide attention because of their wide geographical distribution and global emergence. Peru, which has a historical relationship with East Asia, is considered to be a hotspot for Beijing family strains in South America. We aimed to unveil the genetic diversity and transmission characteristics of the Beijing strains in Peru. A total of 200 Beijing family strains were identified from 2140 M. tuberculosis isolates obtained in Lima, Peru, between December 2008 and January 2010. Of them, 198 strains were classified into sublineages, on the basis of 10 sets of single nucleotide polymorphisms (SNPs). They were also subjected to variable number tandem-repeat (VNTR) typing using an international standard set of 15 loci (15-MIRU-VNTR) plus 9 additional loci optimized for Beijing strains. An additional 70 Beijing family strains, isolated between 1999 and 2006 in Lima, were also analyzed in order to make a longitudinal comparison. The Beijing family was the third largest spoligotyping clade in Peru. Its population structure, by SNP typing, was characterized by a high frequency of Sequence Type 10 (ST10), which belongs to a modern subfamily of Beijing strains (178/198, 89.9%). Twelve strains belonged to the ancient subfamily (ST3 [n=3], ST25 [n=1], ST19 [n=8]). Overall, the polymorphic information content for each of the 24 loci values was low. The 24 loci VNTR showed a high clustering rate (80.3%) and a high recent transmission index (RTI(n-1)=0.707). These strongly suggest the active and on-going transmission of Beijing family strains in the survey area. Notably, 1 VNTR genotype was found to account for 43.9% of the strains. Comparisons with data from East Asia suggested the genotype emerged as a uniquely endemic clone in Peru. A longitudinal comparison revealed the genotype was present in Lima by 1999.

Protective Vaccine Efficacy of the Complete Form of PPE39 Protein from Mycobacterium tuberculosis Beijing/K Strain in Mice.

PubMed

Kim, Ahreum; Hur, Yun-Gyoung; Gu, Sunwha; Cho, Sang-Nae

2017-11-01

The aim of this study was to evaluate the protective efficacy of MTBK_24820, a complete form of PPE39 protein derived from a predominant Beijing/K strain of Mycobacterium tuberculosis in South Korea. Mice were immunized with MTKB_24820, M. bovis Bacilli Calmette-Guérin (BCG), or adjuvant prior to a high-dosed Beijing/K strain aerosol infection. After 4 and 9 weeks, bacterial loads were determined and histopathologic and immunologic features in the lungs and spleens of the M. tuberculosis -infected mice were analyzed. Putative immunogenic T-cell epitopes were examined using synthetic overlapping peptides. Successful immunization of MTBK_24820 in mice was confirmed by increased IgG responses ( P < 0.05) and recalled gamma interferon (IFN-γ), interleukin-2 (IL-2), IL-6, and IL-17 responses ( P < 0.05 or P < 0.01) to MTBK_24820. After challenge with the Beijing/K strain, an approximately 0.5 to 1.0 log 10 reduction in CFU in lungs and fewer lung inflammation lesions were observed in MTBK_24820-immunized mice compared to those for control mice. Moreover, MTBK_24820 immunization elicited significantly higher numbers of CD4 + T cells producing protective cytokines, such as IFN-γ and IL-17, in lungs and spleens ( P < 0.01) and CD4 + multifunctional T cells producing IFN-γ, tumor necrosis factor alpha (TNF-α), and/or IL-17 ( P < 0.01) than in control mice, suggesting protection comparable to that of BCG against the hypervirulent Beijing/K strain. The dominant immunogenic T-cell epitopes that induced IFN-γ production were at the N terminus (amino acids 85 to 102 and 217 to 234). Its vaccine potential, along with protective immune responses in vivo , may be informative for vaccine development, particularly in regions where the M. tuberculosis Beijing/K-strain is frequently isolated from TB patients. Copyright © 2017 American Society for Microbiology.

Discovery of anti-microbial and anti-tubercular molecules from Fusarium solani: an endophyte of Glycyrrhiza glabra.

PubMed

Shah, A; Rather, M A; Hassan, Q P; Aga, M A; Mushtaq, S; Shah, A M; Hussain, A; Baba, S A; Ahmad, Z

2017-05-01

Glycyrrhiza glabra is a high-value medicinal plant thriving in biodiversity rich Kashmir Himalaya. The present study was designed to explore the fungal endophytes from G. glabra as a source of bioactive molecules. The extracts prepared from the isolated endophytes were evaluated for anti-microbial activities using broth micro-dilution assay. The endophytic strain coded as A2 exhibiting promising anti-bacterial as well as anti-tuberculosis activity was identified as Fusarium solani by ITS-5.8S ribosomal gene sequencing technique. This strain was subjected to large-scale fermentation followed by isolation of its bioactive compounds using column chromatography. From the results of spectral data analysis and comparison with literature, the molecules were identified as 3,6,9-trihydroxy-7-methoxy-4,4-dimethyl-3,4-dihydro-1H-benzo[g]isochromene-5,10-dione (1), fusarubin (2), 3-O-methylfusarubin (3) and javanicin (4). Compound 1 is reported for the first time from this strain. All the four compounds inhibited the growth of various tested bacterial strains with MIC values in the range of <1 to 256 μg ml -1 . Fusarubin showed good activity against Mycobacterium tuberculosis strain H37Rv with MIC value of 8 μg ml -1 , whereas compounds 1, 3 and 4 exhibited moderate activity with MIC values of 256, 64, 32 μg ml -1 , respectively. To the best of our knowledge, this is the first study that reports significant anti-tuberculosis potential of bioactive molecules from endophytic F. solani evaluated against the virulent strain of M. tuberculosis. This study sets background towards their synthetic intervention for activity enhancement experiments in anti-microbial drug discovery programme. Due to the chemoprofile variation of same endophyte with respect to source plant and ecoregions, further studies are required to explore endophytes of medicinal plants of all unusual biodiversity rich ecoregions for important and or novel bioactive molecules. © 2017 The Society for Applied Microbiology.

Bovine Tuberculosis

USDA-ARS?s Scientific Manuscript database

Tuberculosis (TB) in animals and humans may result from exposure to bacilli within the Mycobacterium tuberculosis complex (i.e., M. tuberculosis, M. bovis, M. africanum, M. pinnipedii, M. microti, M. caprae, or M. canetti). Mycobacterium bovis is the species most often isolated from tuberculous catt...

Bovine tuberculosis

USDA-ARS?s Scientific Manuscript database

Tuberculosis (TB) in animals and humans may result from exposure to bacilli within the Mycobacterium tuberculosis complex (i.e., M. tuberculosis, M. bovis, M. africanum, M. pinnipedii, M. microti, M. caprae, or M. canetti) . Mycobacterium bovis is the species most often isolated from tuberculous cat...

Mycobacterium bovis in Panama, 2013

PubMed Central

Acosta, Fermín; Chernyaeva, Ekatherina; Mendoza, Libardo; Sambrano, Dilcia; Correa, Ricardo; Rotkevich, Mikhail; Tarté, Miroslava; Hernández, Humberto; Velazco, Bredio; de Escobar, Cecilia; de Waard, Jacobus H.

2015-01-01

Panama remains free of zoonotic tuberculosis caused by Mycobacterium bovis. However, DNA fingerprinting of 7 M. bovis isolates from a 2013 bovine tuberculosis outbreak indicated minimal homology with strains previously circulating in Panama. M. bovis dispersion into Panama highlights the need for enhanced genotype testing to track zoonotic infections. PMID:25988479

Rapid detection of rifampin-resistant clinical isolates of Mycobacterium tuberculosis by reverse dot blot hybridization.

PubMed

Guo, Qian; Yu, Yan; Zhu, Yan Ling; Zhao, Xiu Qin; Liu, Zhi Guang; Zhang, Yuan Yuan; Li, Gui Lian; Wei, Jian Hao; Wu, Yi Mou; Wan, Kang Lin

2015-01-01

A PCR-reverse dot blot hybridization (RDBH) assay was developed for rapid detection of rpoB gene mutations in 'hot mutation region' of Mycobacterium tuberculosis (M. tuberculosis). 12 oligonucleotide probes based on the wild-type and mutant genotype rpoB sequences of M. tuberculosis were designed to screen the most frequent wild-type and mutant genotypes for diagnosing RIF resistance. 300 M. tuberculosis clinical isolates were detected by RDBH, conventional drug-susceptibility testing (DST) and DNA sequencing to evaluate the RDBH assay. The sensitivity and specificity of the RDBH assay were 91.2% (165/181) and 98.3% (117/119), respectively, as compared to DST. When compared with DNA sequencing, the accuracy, positive predictive value (PPV) and negative predictive value (NPV) of the RDBH assay were 97.7% (293/300), 98.2% (164/167), and 97.0% (129/133), respectively. Furthermore, the results indicated that the most common mutations were in codons 531 (48.6%), 526 (25.4%), 516 (8.8%), and 511 (6.6%), and the combinative mutation rate was 15 (8.3%). One and two strains of insertion and deletion were found among all strains, respectively. Our findings demonstrate that the RDBH assay is a rapid, simple and sensitive method for diagnosing RIF-resistant tuberculosis. Copyright © 2015 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

Risk factors for pulmonary cavitation in tuberculosis patients from China.

PubMed

Zhang, Liqun; Pang, Yu; Yu, Xia; Wang, Yufeng; Lu, Jie; Gao, Mengqiu; Huang, Hairong; Zhao, Yanlin

2016-10-12

Pulmonary cavitation is one of the most frequently observed clinical characteristics in tuberculosis (TB). The objective of this study was to investigate the potential risk factors associated with cavitary TB in China. A total of 385 smear-positive patients were enrolled in the study, including 192 (49.9%) patients with cavitation as determined by radiographic findings. Statistical analysis revealed that the distribution of patients with diabetes in the cavitary group was significantly higher than that in the non-cavitary group (adjusted odds ratio (OR) (95% confidence interval (CI)):12.08 (5.75-25.35), P<0.001). Similarly, we also found that the proportion of individuals with multidrug-resistant TB in the cavitary group was also higher than that in the non-cavitary group (adjusted OR (95% CI): 2.48 (1.52-4.07), P<0.001). Of the 385 Mycobacterium tuberculosis strains, 330 strains (85.7%) were classified as the Beijing genotype, which included 260 strains that belonged to the modern Beijing sublineage and 70 to the ancient Beijing sublineage. In addition, there were 80 and 31 strains belonging to large and small clusters, respectively. Statistical analysis revealed that cavitary disease was observed more frequently among the large clusters than the small clusters (P=0.037). In conclusion, our findings demonstrate that diabetes and multidrug resistance are risk factors associated with cavitary TB. In addition, there was no significant difference in the cavitary presentation between patients infected with the Beijing genotype strains and those infected with the non-Beijing genotype strains.

A snapshot of the predominant single nucleotide polymorphism cluster groups of Mycobacterium tuberculosis clinical isolates in Delhi, India.

PubMed

Varma-Basil, Mandira; Narang, Anshika; Chakravorty, Soumitesh; Garima, Kushal; Gupta, Shraddha; Kumar Sharma, Naresh; Giri, Astha; Zozio, Thierry; Couvin, David; Hanif, Mahmud; Bhatnagar, Anuj; Menon, Balakrishnan; Niemann, Stefan; Rastogi, Nalin; Alland, David; Bose, Mridula

2016-09-01

Several attempts have been made to associate phylogenetic differences among Mycobacterium tuberculosis strains to variations in the clinical outcome of the disease and to drug resistance. We genotyped 139 clinical isolates of M. tuberculosis obtained from patients of pulmonary tuberculosis in North Delhi region. The isolates were analyzed using nine Single nucleotide polymorphism (SNP) markers, spoligotyping and MIRU-VNTRs; and the results were correlated with their drug susceptibility profile. Results of SNP cluster group (SCG) analysis (available for 138 isolates) showed that the most predominant cluster was SCG 3a, observed in 58.7% (81/138) of the isolates with 44.4% (36/81) of these being drug susceptible, while 16% (13/81) were multidrug resistant (MDR). Of the ancestral cluster SCG 1 observed in 19.5% (27/138) of the isolates, 14.8% (4/27) were MDR while 44.4% (12/27) were drug susceptible. SCG 2 formed 5.79% (8/138) of the isolates and 50% (4/8) of these were multidrug resistant (MDR). Spoligotyping subdivided the strains into 45 shared types (n = 125) and 14 orphan strains. The orphan strains were mostly associated with SCG 3a or SCG 1, reflecting the principal SCGs found in the Indian population. SCG 1 and SCG 2 genotypes were concordant with the East African Indian (EAI) and Beijing families respectively. Central Asian (CAS) clade and its sublineages were predominantly associated with SCG 3a. No consistent association was seen between the SCGs and Harlem, T or X clades. The 15 loci MIRU-VNTR typing revealed 123/136 isolates to be unclustered, while 13 isolates were present in 6 clusters of 2-3 isolates each. However, correlating the cluster analysis with patient details did not suggest any evidence of recent transmission. In conclusion, though our study revealed the preponderance of SCG 1 and 3a in the M. tuberculosis population circulating in the region, the diversity of strains highlights the changes occurring within lineages and reemphasizes the importance of cluster investigations in extended studies. Copyright © 2016 Elsevier Ltd. All rights reserved.

A Target-Based Whole Cell Screen Approach To Identify Potential Inhibitors of Mycobacterium tuberculosis Signal Peptidase

PubMed Central

2016-01-01

The general secretion (Sec) pathway is a conserved essential pathway in bacteria and is the primary route of protein export across the cytoplasmic membrane. During protein export, the signal peptidase LepB catalyzes the cleavage of the signal peptide and subsequent release of mature proteins into the extracellular space. We developed a target-based whole cell assay to screen for potential inhibitors of LepB, the sole signal peptidase in Mycobacterium tuberculosis, using a strain engineered to underexpress LepB (LepB-UE). We screened 72,000 compounds against both the Lep-UE and wild-type (wt) strains. We identified the phenylhydrazone (PHY) series as having higher activity against the LepB-UE strain. We conducted a limited structure–activity relationship determination around a representative PHY compound with differential activity (MICs of 3.0 μM against the LepB-UE strain and 18 μM against the wt); several analogues were less potent against the LepB overexpressing strain. A number of chemical modifications around the hydrazone moiety resulted in improved potency. Inhibition of LepB activity was observed for a number of compounds in a biochemical assay using cell membrane fraction derived from M. tuberculosis. Compounds did not increase cell permeability, dissipate membrane potential, or inhibit an unrelated mycobacterial enzyme, suggesting a specific mode of action related to the LepB secretory mechanism. PMID:27642770

Longitudinal whole genome analysis of pre and post drug treatment Mycobacterium tuberculosis isolates reveals progressive steps to drug resistance.

PubMed

Datta, Gargi; Nieto, Luisa M; Davidson, Rebecca M; Mehaffy, Carolina; Pederson, Caroline; Dobos, Karen M; Strong, Michael

2016-05-01

Tuberculosis (TB) is one of the leading causes of death due to an infectious disease in the world. Understanding the mechanisms of drug resistance has become pivotal in the detection and treatment of newly emerging resistant TB cases. We have analyzed three pairs of Mycobacterium tuberculosis strains pre- and post-drug treatment to identify mutations involved in the progression of resistance to the drugs rifampicin and isoniazid. In the rifampicin resistant strain, we confirmed a mutation in rpoB (S450L) that is known to confer resistance to rifampicin. We discovered a novel L101R mutation in the katG gene of an isoniazid resistant strain, which may directly contribute to isoniazid resistance due to the proximity of the mutation to the katG isoniazid-activating site. Another isoniazid resistant strain had a rare mutation in the start codon of katG. We also identified a number of mutations in each longitudinal pair, such as toxin-antitoxin mutations that may influence the progression towards resistance or may play a role in compensatory fitness. These findings improve our knowledge of drug resistance progression during therapy and provide a methodology to monitor longitudinal strains using whole genome sequencing, polymorphism comparison, and functional annotation. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Recurrence due to Relapse or Reinfection With Mycobacterium tuberculosis: A Whole-Genome Sequencing Approach in a Large, Population-Based Cohort With a High HIV Infection Prevalence and Active Follow-up

PubMed Central

Guerra-Assunção, José Afonso; Houben, Rein M. G. J.; Crampin, Amelia C.; Mzembe, Themba; Mallard, Kim; Coll, Francesc; Khan, Palwasha; Banda, Louis; Chiwaya, Arthur; Pereira, Rui P. A.; McNerney, Ruth; Harris, David; Parkhill, Julian; Clark, Taane G.; Glynn, Judith R.

2015-01-01

Background. Recurrent tuberculosis is a major health burden and may be due to relapse with the original strain or reinfection with a new strain. Methods. In a population-based study in northern Malawi, patients with tuberculosis diagnosed from 1996 to 2010 were actively followed after the end of treatment. Whole-genome sequencing with approximately 100-fold coverage was performed on all available cultures. Results of IS6110 restriction fragment-length polymorphism analyses were available for cultures performed up to 2008. Results. Based on our data, a difference of ≤10 single-nucleotide polymorphisms (SNPs) was used to define relapse, and a difference of >100 SNPs was used to define reinfection. There was no evidence of mixed infections among those classified as reinfections. Of 1471 patients, 139 had laboratory-confirmed recurrences: 55 had relapse, and 20 had reinfection; for 64 type of recurrence was unclassified. Almost all relapses occurred in the first 2 years. Human immunodeficiency virus infection was associated with reinfection but not relapse. Relapses were associated with isoniazid resistance, treatment before 2007, and lineage-3 strains. We identified several gene variants associated with relapse. Lineage-2 (Beijing) was overrepresented and lineage-1 underrepresented among the reinfecting strains (P = .004). Conclusions. While some of the factors determining recurrence depend on the patient and their treatment, differences in the Mycobacterium tuberculosis genome appear to have a role in both relapse and reinfection. PMID:25336729

Isoniazid and rifampicin heteroresistant Mycobacterium tuberculosis isolated from tuberculous meningitis patients in India.

PubMed

Gupta, Renu; Thakur, Rajeev; Kushwaha, Suman; Jalan, Nupur; Rawat, Pumanshi; Gupta, Piyush; Aggarwal, Amitesh; Gupta, Meena; Manchanda, Vikas

2018-01-01

Heteroresistant Mycobacterium tuberculosis (mixture of susceptible and resistant subpopulations) is thought to be a preliminary stage to full resistance and timely detection, initiation of correct treatment is vital for successful anti tubercular therapy. The aim of this study was to detect multi drug resistant (MDR) and heteroresistant M. tuberculosis with the associated gene mutations from patients of tuberculous meningitis. A total of 197 M. tuberculosis isolates from 478 patients of TBM were isolated from July 2012 to July 2015 and subjected to drug susceptibility testing (DST) by BACTEC MGIT and Genotype MTBDR line probe assay (LPA). Heteroresistance was defined as presence of both WT and mutant genes in LPA. Of 197 M. tuberculosis isolates, 11 (5.6%) were MDR, 23 (11.6%), 1 (0.5%) were mono resistant to isoniazid (INH) and rifampicin (RMP) respectively. Heteroresistance was detected in 8 (4%), 2 (1%) isolates to INH and RMP respectively. INH heteroresistant strains had WT bands with mutation band S315T1 whereas RMP heteroresistant strains had WT bands with mutation band S531L. The prevalence of MDR M. tuberculosis was 5.6% in TBM patients with the most common mutation being ΔWT band with S315T1 for INH and ΔWT band with S531T for RMP. MGIT DST was found to be more sensitive for detecting overall resistance in M. tuberculosis but inclusion of LPA not only reduced time for early initiation of appropriate treatment but also enabled detection of heteroresistance in 8 (4%), 2 (1%) isolates for INH and RMP respectively. Copyright © 2017 Tuberculosis Association of India. Published by Elsevier B.V. All rights reserved.

Drug resistant Mycobacterium tuberculosis in Mexico.

PubMed

Zazueta-Beltran, Jorge; León-Sicairos, Claudia; Canizalez-Roman, Adrián

2009-04-30

Tuberculosis (TB) remains a serious public health problem, worsened by an increased frequency of multidrug-resistant (MDR) Mycobacterium tuberculosis strains. The World Health Organization (WHO) and the International Union Against Tuberculosis and Lung Disease (IUATLD) launched the Global Project on Anti-Tuberculosis Drug Resistance Surveillance to measure the prevalence of drug resistance. Data from the global reports on resistance to anti-tuberculosis (anti-TB) drugs have shown that drug resistance still presents worldwide and that MDR-TB is present in almost all the world. Though the Global Project (WHO) has been operating since 1994, very few countries and states have reported new information. Data from repeated surveys employing comparable methodologies over several years are essential to determine with any certainty in which direction the prevalence of drug resistance is moving. Drug-resistant tuberculosis and MDR-TB have been identified in Mexico, even with the existence of a National Tuberculosis Program based on Directly Observed Treatment, Short-course (DOTS). This review discusses available surveillance data on drug susceptibility data for TB in different states of Mexico.

Modelling of Cerebral Tuberculosis: Hope for Continuous Research in Solving the Enigma of the Bottom Billion’s Disease

PubMed Central

Hernández Pando, Rogelio

2011-01-01

Cerebral tuberculosis is a severe type of extrapulmonary disease that is highly predominant in children. It is thought that meningeal tuberculosis, the most common form of cerebral tuberculosis, begins with respiratory infection followed by early haematogenous dissemination to extrapulmonary sites involving the brain. Host genetic susceptibility factors and specific mycobacteria substrains could be involved in the development of this serious form of tuberculosis. In this editorial the different animal models of cerebral tuberculosis are commented, highlighting a recently described murine model in which BALB/c mice were infected by the intratracheal route with clinical isolates, which exhibited rapid dissemination and brain infection. These strains were isolated from the cerebrospinal fluid of patients with meningeal tuberculosis; they showed specific genotype and induced a peculiar immune response in the infected brain. This model could be a useful tool to study host and bacilli factors involved in the pathogenesis of the most severe form of tuberculosis. PMID:22135568

Extensively drug-resistant tuberculosis in a young child after travel to India.

PubMed

Salazar-Austin, Nicole; Ordonez, Alvaro A; Hsu, Alice Jenh; Benson, Jane E; Mahesh, Mahadevappa; Menachery, Elizabeth; Razeq, Jafar H; Salfinger, Max; Starke, Jeffrey R; Milstone, Aaron M; Parrish, Nicole; Nuermberger, Eric L; Jain, Sanjay K

2015-12-01

Extensively drug-resistant (XDR) tuberculosis is becoming increasingly prevalent worldwide, but little is known about XDR tuberculosis in young children. In this Grand Round we describe a 2-year-old child from the USA who developed pneumonia after a 3 month visit to India. Symptoms resolved with empirical first-line tuberculosis treatment; however, a XDR strain of Mycobacterium tuberculosis grew in culture. In the absence of clinical or microbiological markers, low-radiation exposure pulmonary CT imaging was used to monitor treatment response, and guide an individualised drug regimen. Management was complicated by delays in diagnosis, uncertainties about drug selection, and a scarcity of child-friendly formulations. Treatment has been successful so far, and the child is in remission. This report of XDR tuberculosis in a young child in the USA highlights the risks of acquiring drug-resistant tuberculosis overseas, and the unique challenges in management of tuberculosis in this susceptible population. Copyright © 2015 Elsevier Ltd. All rights reserved.

Identification of specific metabolites in culture supernatant of Mycobacterium tuberculosis using metabolomics: exploration of potential biomarkers

PubMed Central

Lau, Susanna KP; Lam, Ching-Wan; Curreem, Shirly OT; Lee, Kim-Chung; Lau, Candy CY; Chow, Wang-Ngai; Ngan, Antonio HY; To, Kelvin KW; Chan, Jasper FW; Hung, Ivan FN; Yam, Wing-Cheong; Yuen, Kwok-Yung; Woo, Patrick CY

2015-01-01

Although previous studies have reported the use of metabolomics for Mycobacterium species differentiation, little is known about the potential of extracellular metabolites of Mycobacterium tuberculosis (MTB) as specific biomarkers. Using an optimized ultrahigh performance liquid chromatography–electrospray ionization–quadruple time of flight–mass spectrometry (UHPLC–ESI–Q–TOF–MS) platform, we characterized the extracellular metabolomes of culture supernatant of nine MTB strains and nine non-tuberculous Mycobacterium (NTM) strains (four M. avium complex, one M. bovis Bacillus Calmette–Guérin (BCG), one M. chelonae, one M. fortuitum and two M. kansasii). Principal component analysis readily distinguished the metabolomes between MTB and NTM. Using multivariate and univariate analysis, 24 metabolites with significantly higher levels in MTB were identified. While seven metabolites were identified by tandem mass spectrometry (MS/MS), the other 17 metabolites were unidentified by MS/MS against database matching, suggesting that they may be potentially novel compounds. One metabolite was identified as dexpanthenol, the alcohol analog of pantothenic acid (vitamin B5), which was not known to be produced by bacteria previously. Four metabolites were identified as 1-tuberculosinyladenosine (1-TbAd), a product of the virulence-associated enzyme Rv3378c, and three previously undescribed derivatives of 1-TbAd. Two derivatives differ from 1-TbAd by the ribose group of the nucleoside while the other likely differs by the base. The remaining two metabolites were identified as a tetrapeptide, Val-His-Glu-His, and a monoacylglycerophosphoglycerol, phosphatidylglycerol (PG) (16∶0/0∶0), respectively. Further studies on the chemical structure and biosynthetic pathway of these MTB-specific metabolites would help understand their biological functions. Studies on clinical samples from tuberculosis patients are required to explore for their potential role as diagnostic biomarkers. PMID:26038762

Multidrug and extensively drug-resistant tuberculosis.

PubMed

Maitre, T; Aubry, A; Jarlier, V; Robert, J; Veziris, N

2017-02-01

The emergence of drug-resistant tuberculosis (TB) compromises global tuberculosis control. The incidence of multidrug-resistant strains (MDR) defined as resistant to the two main antituberculosis drugs, rifampicin and isoniazid, was raised in the 1990s. Ten percent of these strains have developed additional resistance to the main second-line antituberculosis drugs: fluoroquinolones and aminoglycosides. These strains are defined as extensively drug-resistant (XDR). The prognosis of MDR-TB and XDR-TB is poor due to limited therapeutic resources. However, many new innovations may lead to a radical change in this field. Genotypic testing is now able to detect drug resistance within a few hours. Genotypic diagnosis of rifampicin resistance is now recommended in France for each new case of TB. The currently recommended treatment for MDR-TB is long (18-24 months) and toxic. It is, however, on the verge of being replaced by a 9-month treatment. New antituberculosis drugs such as bedaquiline and delamanid should also improve the prognosis of MDR-TB and XDR-TB. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Emerging technologies for monitoring drug-resistant tuberculosis at the point-of-care.

PubMed

Mani, Vigneshwaran; Wang, ShuQi; Inci, Fatih; De Libero, Gennaro; Singhal, Amit; Demirci, Utkan

2014-11-30

Infectious diseases are the leading cause of death worldwide. Among them, tuberculosis (TB) remains a major threat to public health, exacerbated by the emergence of multiple drug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis (Mtb). MDR-Mtb strains are resistant to first-line anti-TB drugs such as isoniazid and rifampicin; whereas XDR-Mtb strains are resistant to additional drugs including at least to any fluoroquinolone and one of the second-line anti-TB injectable drugs such as kanamycin, capreomycin, or amikacin. Clinically, these strains have significantly impacted the management of TB in high-incidence developing countries, where systemic surveillance of TB drug resistance is lacking. For effective management of TB on-site, early detection of drug resistance is critical to initiate treatment, to reduce mortality, and to thwart drug-resistant TB transmission. In this review, we discuss the diagnostic challenges to detect drug-resistant TB at the point-of-care (POC). Moreover, we present the latest advances in nano/microscale technologies that can potentially detect TB drug resistance to improve on-site patient care. Copyright © 2014 Elsevier B.V. All rights reserved.

Emerging Technologies for Monitoring Drug-Resistant Tuberculosis at the Point-of-Care

PubMed Central

Mani, Vigneshwaran; Wang, ShuQi; Inci, Fatih; De Libero, Gennaro; Singhal, Amit; Demirci, Utkan

2014-01-01

Infectious diseases are the leading cause of death worldwide. Among them, tuberculosis (TB) remains a major threat to public health, exacerbated by the emergence of multiple drug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis (Mtb). MDR-Mtb strains are resistant to first-line anti-TB drugs such as isoniazid and rifampicin; whereas XDR-Mtb strains are resistant to additional drugs including at least to any fluoroquinolone and at least one of the second-line anti-TB injectable drugs such as kanamycin, capreomycin, or amikacin. Clinically, these strains have significantly impacted the management of TB in high-incidence developing countries, where systemic surveillance of TB drug resistance is lacking. For effective management of TB on-site, early detection of drug resistance is critical to initiate treatment, to reduce mortality, and to thwart drug-resistant TB transmission. In this review, we discuss the diagnostic challenges to detect drug-resistant TB at the point-of-care (POC). Moreover, we present the latest advances in nano/microscale technologies that can potentially detect TB drug resistance to improve on-site patient care. PMID:24882226

Drug-resistant tuberculosis: time for visionary political leadership.

PubMed

Abubakar, Ibrahim; Zignol, Matteo; Falzon, Dennis; Raviglione, Mario; Ditiu, Lucica; Masham, Susan; Adetifa, Ifedayo; Ford, Nathan; Cox, Helen; Lawn, Stephen D; Marais, Ben J; McHugh, Timothy D; Mwaba, Peter; Bates, Matthew; Lipman, Marc; Zijenah, Lynn; Logan, Simon; McNerney, Ruth; Zumla, Adam; Sarda, Krishna; Nahid, Payam; Hoelscher, Michael; Pletschette, Michel; Memish, Ziad A; Kim, Peter; Hafner, Richard; Cole, Stewart; Migliori, Giovanni Battista; Maeurer, Markus; Schito, Marco; Zumla, Alimuddin

2013-06-01

Two decades ago, WHO declared tuberculosis a global emergency, and invested in the highly cost-effective directly observed treatment short-course programme to control the epidemic. At that time, most strains of Mycobacterium tuberculosis were susceptible to first-line tuberculosis drugs, and drug resistance was not a major issue. However, in 2013, tuberculosis remains a major public health concern worldwide, with prevalence of multidrug-resistant (MDR) tuberculosis rising. WHO estimates roughly 630 000 cases of MDR tuberculosis worldwide, with great variation in the frequency of MDR tuberculosis between countries. In the past 8 years, extensively drug-resistant (XDR) tuberculosis has emerged, and has been reported in 84 countries, heralding the possibility of virtually untreatable tuberculosis. Increased population movement, the continuing HIV pandemic, and the rise in MDR tuberculosis pose formidable challenges to the global control of tuberculosis. We provide an overview of the global burden of drug-resistant disease; discuss the social, health service, management, and control issues that fuel and sustain the epidemic; and suggest specific recommendations for important next steps. Visionary political leadership is needed to curb the rise of MDR and XDR tuberculosis worldwide, through sustained funding and the implementation of global and regional action plans. Copyright © 2013 World Health Organization. Published by Elsevier Ltd/Inc/BV. All rights reserved. Published by Elsevier Ltd. All rights reserved.

GeneXpert MTB/RIF Assay for the Diagnosis of Tuberculous Lymphadenitis on Concentrated Fine Needle Aspirates in High Tuberculosis Burden Settings.

PubMed

Tadesse, Mulualem; Abebe, Gemeda; Abdissa, Ketema; Aragaw, Dossegnaw; Abdella, Kedir; Bekele, Alemayehu; Bezabih, Mesele; Apers, Ludwig; de Jong, Bouke C; Rigouts, Leen

2015-01-01

The diagnosis of tuberculous lymphadenitis (TBL) remains challenging. The routinely used methods (cytology and smear microscopy) have sub-optimal sensitivity. Recently, WHO recommends GeneXpert to be used as the initial diagnostic test in patients suspected of having extra-pulmonary tuberculosis (EPTB). However, this was a conditional recommendation due to very low-quality evidence available and more studies are needed. In this study we evaluated the performance of Xpert for the diagnosis of TBL on concentrated fine needle aspirates (FNA) in Southwest Ethiopia. FNA was collected from presumptive TBL cases. Two smears were prepared from each aspirate and processed for cytology and conventional microscopy. The remaining aspirate was treated with N-acetyl-L-cysteine-NaOH and centrifuged for 15minutes at 3000g. The concentrated sediment was used for culture and Xpert test. Capilia TB-Neo test was used to differentiate M. tuberculosis complex (MTBC) from non-tuberculous mycobacteria (NTM). Composite bacteriological methods (culture and/or smear microscopy) were considered as a reference standard. Out of 143 enrolled suspects, 64.3% (92/143) were confirmed TBL cases by the composite reference standard (CRS). Xpert detected M. tuberculosis complex (MTBC) in 60.1% (86/143) of the presumptive TBL cases. The sensitivity of Xpert compared to CRS was 87.8% [95% CI: 81.0-94.5] and specificity 91.1% [95% CI: 82.8-99.4]. The sensitivity was 27.8% for smear microscopy and 80% for cytology compared to CRS. Cytology showed the lowest specificity (57.8%). Xpert was positive in 4 out of 45 culture- and smear-negative cases. Among 47 cytomorphologically non-TBL cases, 15 were positive on Xpert. More than half of Xpert-positive cases were in the range of very low cut-off threshold values (28

Deletion of the β-Propeller Protein Gene Rv1057 Reduces ESAT-6 Secretion and Intracellular Growth of Mycobacterium tuberculosis.

PubMed

Fu, Jiafang; Zong, Gongli; Zhang, Peipei; Gu, Yuanxin; Cao, Guangxiang

2018-04-01

Rv1057 is the only β-propeller protein in Mycobacterium tuberculosis, but its biological function is still unclear. In this study, we generated a deletion mutant of Rv1057 (D1057) in the virulent M. tuberculosis strain H37Rv and examined the characteristics of the mutant in vitro and in macrophages. We found that deletion of Rv1057 reduces secretion of the major virulence factor ESAT-6 and ESAT-6 stops in the cell envelope fraction during secretion, although ESAT-6 levels were similar in lysates of the mutant and control strains. In infected macrophages, Rv1057 deletion significantly reduced the secretion levels of cytokines IL-1β, IL-10, TNF-α, and INF-γ, but did not affect IL-4 and IL-8. D1057-infected macrophages also release less LDH and produce more nitric oxide (NO) than H37Rv- and D1057com (Rv1057 complemented strain of D1057com)-infected macrophages, indicating that D1057 has the decreased cytotoxicity compared to H37Rv or D1057com. In addition, the capacity of the Rv1057 deletion mutant to grow in macrophages was significantly lower than that of H37Rv and D1057com. Our findings support a role for Rv1057 in ESAT-6 secretion and in modulating the interactions between M. tuberculosis and macrophages.

MTB-DR-RIF 9G test: Detection and discrimination of tuberculosis and multi-drug resistant tuberculosis strains.

PubMed

Song, Keum-Soo; Nimse, Satish Balasaheb; Cho, Nam Hoon; Sung, Nackmoon; Kim, Hee-Jin; Yang, Jeongseong; Kim, Taisun

2015-12-01

This report describes the evaluation of the novel MTB-DR-RIF 9G test for the accurate detection and discrimination of Mycobacterium tuberculosis (MTB) and rifampicin-resistant M. tuberculosis (MTB-DR-RIF) in the clinical samples. The procedure included the amplification of a nucleotide fragment of the rpoB gene of the MTB and MTB-DR-RIF strains and their hybridization with the immobilized probes. The MTB-DR-RIF 9G test was evaluated for its ability to detect and discriminate MTB and MTB-DR-RIF strains in 113 known clinical samples. The accuracy of the MTB-DR-RIF 9G test was determined by comparing its results with sequencing analysis and drug susceptibility testing. The sensitivity and specificity of the MTB-DR-RIF 9G test at 95% confidence interval were found to be 95.4% (89.5-98.5) and 100% (69.2-100), respectively. The positive predictive value and negative predictive value of the MTB-DR-RIF 9G test at 95% confidence interval were found to be 100% (85.0-95.9) and 66.7% (38.4-88.18), respectively. Sequencing analysis of all samples indicated that the mutations present in the regions identified with the MTB-DR-RIF 9G assay can be detected accurately. Copyright © 2015 Elsevier Ltd. All rights reserved.

Induction of β-defensins by l-isoleucine as novel immunotherapy in experimental murine tuberculosis

PubMed Central

Rivas-Santiago, C E; Rivas-Santiago, B; León, D A; Castañeda-Delgado, J; Hernández Pando, R

2011-01-01

Tuberculosis is a worldwide health problem, and multidrug-resistant (MDR) and extensively multidrug-resistant (XMDR) strains are rapidly emerging and threatening the control of this disease. These problems motivate the search for new treatment strategies. One potential strategy is immunotherapy using cationic anti-microbial peptides. The capacity of l-isoleucine to induce beta-defensin expression and its potential therapeutic efficiency were studied in a mouse model of progressive pulmonary tuberculosis. BALB/c mice were infected with Mycobacterium tuberculosis strain H37Rv or with a MDR clinical isolate by the intratracheal route. After 60 days of infection, when disease was in its progressive phase, mice were treated with 250 µg of intratracheal l-isoleucine every 48 h. Bacillary loads were determined by colony-forming units, protein and cytokine gene expression were determined by immunohistochemistry and reverse transcription–quantitative polymerase chain reaction (RT–qPCR), respectively, and tissue damage was quantified by automated morphometry. Administration of l-isoleucine induced a significant increase of beta-defensins 3 and 4 which was associated with decreased bacillary loads and tissue damage. This was seen in animals infected with the antibiotic-sensitive strain H37Rv and with the MDR clinical isolate. Thus, induction of beta-defensins might be a potential therapy that can aid in the control of this significant infectious disease. PMID:21235540

Mutations in ppe38 block PE_PGRS secretion and increase virulence of Mycobacterium tuberculosis.

PubMed

Ates, Louis S; Dippenaar, Anzaan; Ummels, Roy; Piersma, Sander R; van der Woude, Aniek D; van der Kuij, Kim; Le Chevalier, Fabien; Mata-Espinosa, Dulce; Barrios-Payán, Jorge; Marquina-Castillo, Brenda; Guapillo, Carolina; Jiménez, Connie R; Pain, Arnab; Houben, Edith N G; Warren, Robin M; Brosch, Roland; Hernández-Pando, Rogelio; Bitter, Wilbert

2018-02-01

Mycobacterium tuberculosis requires a large number of secreted and exported proteins for its virulence, immune modulation and nutrient uptake. Most of these proteins are transported by the different type VII secretion systems 1,2 . The most recently evolved type VII secretion system, ESX-5, secretes dozens of substrates belonging to the PE and PPE families, which are named for conserved proline and glutamic acid residues close to the amino terminus 3,4 . However, the role of these proteins remains largely elusive 1 . Here, we show that mutations of ppe38 completely block the secretion of two large subsets of ESX-5 substrates, that is, PPE-MPTR and PE_PGRS, together comprising >80 proteins. Importantly, hypervirulent clinical M. tuberculosis strains of the Beijing lineage have such a mutation and a concomitant loss of secretion 5 . Restoration of PPE38-dependent secretion partially reverted the hypervirulence phenotype of a Beijing strain, and deletion of ppe38 in moderately virulent M. tuberculosis increased virulence. This indicates that these ESX-5 substrates have an important role in virulence attenuation. Phylogenetic analysis revealed that deletion of ppe38 occurred at the branching point of the 'modern' Beijing sublineage and is shared by Beijing outbreak strains worldwide, suggesting that this deletion may have contributed to their success and global distribution 6,7 .

Characterisation of a live Salmonella vaccine stably expressing the Mycobacterium tuberculosis Ag85B–ESAT6 fusion protein

PubMed Central

Hall, Lindsay J.; Clare, Simon; Pickard, Derek; Clark, Simon O.; Kelly, Dominic L.F.; Ghany, Moataz Abd El; Hale, Christine; Dietrich, Jes; Andersen, Peter; Marsh, Philip D.; Dougan, Gordon

2009-01-01

A recombinant Salmonella enterica serovar Typhimurium (S. Typhimurium) vaccine strain was constructed that stably expressed the Mycobacterium tuberculosis fusion antigen Ag85B–ESAT6 from the chromosome. Live oral vaccination of mice with the Salmonella/Ag85B–ESAT6 strain generated a potent anti-Ag85B–ESAT6 TH1 response with high antibody titres with a IgG2a-bias and significant IFN-γ production lasting over a 120-day period. When mice primed with the Salmonella/Ag85B–ESAT6 vaccine were mucosally boosted with the Ag85B–ESAT6 antigen and adjuvant the IFN-γ responses increased markedly. To determine the protective efficacy of this vaccine strain, guinea pigs were immunised and followed for a 30-week period after aerosol challenge with M. tuberculosis. The heterologous prime-boost strategy of live Salmonella vaccine followed by a systemic boost of antigen and adjuvant reduced the levels of M. tuberculosis bacteria in the lungs and spleen to the same extent as BCG. Additionally, this vaccination regimen was observed to be statistically equivalent in terms of protection to immunisation with BCG. Thus, live oral priming with the recombinant Salmonella/Ag85B–ESAT6 and boosting with Ag85B–ESAT6 plus the adjuvant LTK63 represents an effective mucosal vaccination regimen. PMID:19755145

Current Methods in the Molecular Typing of Mycobacterium tuberculosis and Other Mycobacteria

PubMed Central

van Ingen, Jakko; Dziadek, Jarosław; Mazur, Paweł K.; Bielecki, Jacek

2014-01-01

In the epidemiology of tuberculosis (TB) and nontuberculous mycobacterial (NTM) diseases, as in all infectious diseases, the key issue is to define the source of infection and to disclose its routes of transmission and dissemination in the environment. For this to be accomplished, the ability of discerning and tracking individual Mycobacterium strains is of critical importance. Molecular typing methods have greatly improved our understanding of the biology of mycobacteria and provide powerful tools to combat the diseases caused by these pathogens. The utility of various typing methods depends on the Mycobacterium species under investigation as well as on the research question. For tuberculosis, different methods have different roles in phylogenetic analyses and person-to-person transmission studies. In NTM diseases, most investigations involve the search for environmental sources or phylogenetic relationships. Here, too, the type of setting determines which methodology is most suitable. Within this review, we summarize currently available molecular methods for strain typing of M. tuberculosis and some NTM species, most commonly associated with human disease. For the various methods, technical practicalities as well as discriminatory power and accomplishments are reviewed. PMID:24527454

Tuberculosis: finding a new potential antimycobacterium derivative in a aldehyde-arylhydrazone-oxoquinoline series.

PubMed

da C Santos, Fernanda; Castro, Helena C; Lourenço, Maria Cristina S; Abreu, Paula A; Batalha, Pedro N; Cunha, Anna C; Carvalho, Guilherme S L; Rodrigues, Carlos R; Medeiros, Cid A; Souza, Simone D; Ferreira, Vitor F; de Souza, Maria C B V

2012-10-01

Tuberculosis (TB) is a contagious disease caused by Mycobacterium tuberculosis, which remains a serious public health problem. The emergence of resistant bacterial strains has continuously increased and new treatment options are currently in need. In this work, we identified a new potential aldehyde-arylhydrazone-oxoquinoline derivative (4e) with interesting chemical structural features that may be important for designing new anti-TB agents. This 1-ethyl-N'-[(1E)-(5-nitro-2-furyl)methylene]-4-oxo-1,4-dihydroquinoline-3-carbohydrazide (4e) presented an in vitro active profile against M. tuberculosis H37Rv strain (minimum inhibitory concentration, MIC = 6.25 μg/mL) better than other acylhydrazones described in the literature (MIC = 12.5 μg/mL) and close to other antitubercular agents currently on the market. The theoretical analysis showed the importance of several structural features that together with the 5-nitro-2-furyl group generated this active compound (4e). This new compound and the analysis of its molecular properties may be useful for designing new and more efficient antibacterial drugs.

Cloning, sequencing, and expression of the apa gene coding for the Mycobacterium tuberculosis 45/47-kilodalton secreted antigen complex.

PubMed

Laqueyrerie, A; Militzer, P; Romain, F; Eiglmeier, K; Cole, S; Marchal, G

1995-10-01

Effective protection against a virulent challenge with Mycobacterium tuberculosis is induced mainly by previous immunization with living attenuated mycobacteria, and it has been hypothesized that secreted proteins serve as major targets in the specific immune response. To identify and purify molecules present in culture medium filtrate which are dominant antigens during effective vaccination, a two-step selection procedure was used to select antigens able to interact with T lymphocytes and/or antibodies induced by immunization with living bacteria and to counterselect antigens interacting with the immune effectors induced by immunization with dead bacteria. A Mycobacterium bovis BCG 45/47-kDa antigen complex, present in BCG culture filtrate, has been previously identified and isolated (F. Romain, A. Laqueyrerie, P. Militzer, P. Pescher, P. Chavarot, M. Lagranderie, G. Auregan, M. Gheorghiu, and G. Marchal, Infect. Immun. 61:742-750, 1993). Since the cognate antibodies recognize the very same antigens present in M. tuberculosis culture medium filtrates, a project was undertaken to clone, express, and sequence the corresponding gene of M. tuberculosis. An M. tuberculosis shuttle cosmid library was transferred in Mycobacterium smegmatis and screened with a competitive enzyme-linked immunosorbent assay to detect the clones expressing the proteins. A clone containing a 40-kb DNA insert was selected, and by means of subcloning in Escherichia coli, a 2-kb fragment that coded for the molecules was identified. An open reading frame in the 2,061-nucleotide sequence codes for a secreted protein with a consensus signal peptide of 39 amino acids and a predicted molecular mass of 28,779 Da. The gene was referred to as apa because of the high percentages of proline (21.7%) and alanine (19%) in the purified protein. Southern hybridization analysis of digested total genomic DNA from M. tuberculosis (reference strains H37Rv and H37Ra) indicated that the apa gene was present as a single copy on the genome. The N-terminal identity or homology of the M. tuberculosis and M. bovis BCG purified molecules and their similar global and deduced amino acid compositions demonstrated the perfect correspondence between the molecular and chemical analyses. The presence of a high percentage of proline (21.7%) was confirmed and explained the apparent higher molecular mass (45/47 kDa) determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis resulting from the increased rigidity of molecules due to proline residues.(ABSTRACT TRUNCATED AT 400 WORDS)

Monocarbonyl analogs of curcumin inhibit growth of antibiotic sensitive and resistant strains of Mycobacterium tuberculosis

PubMed Central

Baldwin, Patrick R.; Reeves, Analise Z.; Powell, Kimberly R.; Napier, Ruth J.; Swimm, Alyson I.; Sun, Aiming; Giesler, Kyle; Bommarius, Bettina; Shinnick, Thomas M.; Snyder, James P.; Liotta, Dennis C.; Kalman, Daniel

2016-01-01

Tuberculosis (TB) is a major public health concern worldwide with over 2 billion people currently infected. The rise of strains of Mycobacterium tuberculosis (Mtb) that are resistant to some or all first and second line antibiotics, including multidrug-resistant (MDR), extensively drug resistant (XDR) and totally drug resistant (TDR) strains, is of particular concern and new anti-TB drugs are urgently needed. Curcumin, a natural product used in traditional medicine in India, exhibits anti-microbial activity that includes Mtb, however it is relatively unstable and suffers from poor bioavailability. To improve activity and bioavailability, mono-carbonyl analogs of curcumin were synthesized and screened for their capacity to inhibit the growth of Mtb and the related Mycobacterium marinum (Mm). Using disk diffusion and liquid culture assays, we found several analogs that inhibit in vitro growth of Mm and Mtb, including rifampicin-resistant strains. Structure activity analysis of the analogs indicated that Michael acceptor properties are critical for inhibitory activity. However, no synergistic effects were evident between the monocarbonyl analogs and rifampicin on inhibiting growth. Together, these data provide a structural basis for the development of analogs of curcumin with pronounced anti-mycobacterial activity and provide a roadmap to develop additional structural analogs that exhibit more favorable interactions with other anti-TB drugs. PMID:25618016

Clinical Isolates of Mycobacterium tuberculosis Differ in Their Ability to Induce Respiratory Burst and Apoptosis in Neutrophils as a Possible Mechanism of Immune Escape

PubMed Central

Romero, María M.; Balboa, Luciana; Basile, Juan I.; López, Beatriz; Ritacco, Viviana; de la Barrera, Silvia S.; Sasiain, María C.; Barrera, Lucía; Alemán, Mercedes

2012-01-01

Tuberculosis pathogenesis was earlier thought to be mainly related to the host but now it appears to be clear that bacterial factors are also involved. Genetic variability of Mycobacterium tuberculosis (Mtb) could be slight but it may lead to sharp phenotypic differences. We have previously reported that nonopsonized Mtb H37Rv induce apoptosis of polymorphonuclear neutrophils (PMNs) by a mechanism that involves the p38 pathway. Here we evaluated the capability to induce PMN apoptosis of two prevalent Mtb lineages in Argentina, the Latin America and Mediterranean (LAM), and Haarlem, using the H37Rv as a reference strain. Results showed that LAM strains strongly induced apoptosis of PMN which correlated with the induction of reactive oxygen species (ROS) production and p38 activation. Interestingly, the highly prosperous multidrug-resistant M strain, belonging to the Haarlem lineage, lacked the ability to activate and to induce PMN apoptosis as a consequence of (1) a weak ROS production and (2) the contribution of antiapoptotic mechanisms mediated at least by ERK. Although with less skill, M is able to enter the PMN so that phenotypic differences could lead PMN to be a reservoir allowing some pathogens to prevail and persist over other strains in the community. PMID:22778761

Structural Basis for Catalysis of a Tetrameric Class IIa Fructose 1,6-Bisphosphate Aldolase from Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pegan, Scott D.; Ruskseree, Kamolchanok; Franzblau, Scott G.

2009-03-04

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), currently infects one-third of the world's population in its latent form. The emergence of multidrug-resistant and extensive drug-resistant strains has highlighted the need for new pharmacological targets within M. tuberculosis. The class IIa fructose 1,6-bisphosphate aldolase (FBA) enzyme from M. tuberculosis (MtFBA) has been proposed as one such target since it is upregulated in latent TB. Since the structure of MtFBA has not been determined and there is little information available on its reaction mechanism, we sought to determine the X-ray structure of MtFBA in complex with its substrates. By lowering themore » pH of the enzyme in the crystalline state, we were able to determine a series of high-resolution X-ray structures of MtFBA bound to dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, and fructose 1,6-bisphosphate at 1.5, 2.1, and 1.3 {angstrom}, respectively. Through these structures, it was discovered that MtFBA belongs to a novel tetrameric class of type IIa FBAs. The molecular details at the interface of the tetramer revealed important information for better predictability of the quaternary structures among the FBAs based on their primary sequences. These X-ray structures also provide interesting and new details on the reaction mechanism of class II FBAs. Substrates and products were observed in geometries poised for catalysis; in addition, unexpectedly, the hydroxyl-enolate intermediate of dihydroxyacetone phosphate was also captured and resolved structurally. These concise new details offer a better understanding of the reaction mechanisms for FBAs in general and provide a structural basis for inhibitor design efforts aimed at this class of enzymes.« less

Structural basis for catalysis of a tetrameric, class IIa fructose 1,6-bisphosphate aldolase from M. tuberculosis

PubMed Central

Pegan, Scott D.; Rukseree, Kamolchanok; Franzblau, Scott G.; Mesecar, Andrew D.

2009-01-01

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), currently infects one third of the world’s population in its latent form. The emergence of multidrug resistant strains, MDR-TB and XDR-TB has highlighted the need for new pharmacological targets within M. tuberculosis. The Class IIa fructose 1,6-bisphosphate aldolase (FBA) enzyme from M. tuberculosis (MtFBA) has been proposed as one such target since its upregulated in latent TB. Since the structure of MtFBA had not been determined and since there was little information available on its reaction mechanism, we sought to determine the X-ray structure of MtFBA in complex with its substrates. By lowering the pH of the enzyme in the crystalline state, we were able to determine a series of high-resolution X-ray structures of MtFBA bound to dihydroxyacetonephosphate (DHAP), glyceraldehyde-3-phosphate (G3P), and fructose 1,6-bisphosphate (FBP) at 1.5 Å, 2.1 Å, and 1.3 Å respectively. Through these structures it was discovered that MtFBA belongs to a novel tetrameric class of the type IIa FBAs. The molecular details at the interface of the tetramer revealed important information for being able to better predict the quaternary structures among the FBAs based on their primary sequences. These X-ray structures also provide interesting and new details on the reaction mechanism of class II FBAs. Not only were the substrates and products observed in geometries poised for catalysis, but unexpectedly the hydroxyl enolate intermediate of DHAP was also captured and resolved structurally. These concise new details provide a better understanding of the reaction mechanisms for FBAs in general and provide a structural basis for inhibitor design efforts aimed at this class of enzymes. PMID:19167403

Gene Deletions in Mycobacterium bovis BCG Stimulate Increased CD8+ T Cell Responses

PubMed Central

Panas, Michael W.; Sixsmith, Jaimie D.; White, KeriAnn; Korioth-Schmitz, Birgit; Shields, Shana T.; Moy, Brian T.; Lee, Sunhee; Schmitz, Joern E.; Jacobs, William R.; Porcelli, Steven A.; Haynes, Barton F.; Letvin, Norman L.

2014-01-01

Mycobacteria, the etiological agents of tuberculosis and leprosy, have coevolved with mammals for millions of years and have numerous ways of suppressing their host's immune response. It has been suggested that mycobacteria may contain genes that reduce the host's ability to elicit CD8+ T cell responses. We screened 3,290 mutant Mycobacterium bovis bacillus Calmette Guerin (BCG) strains to identify genes that decrease major histocompatibility complex (MHC) class I presentation of mycobacterium-encoded epitope peptides. Through our analysis, we identified 16 mutant BCG strains that generated increased transgene product-specific CD8+ T cell responses. The genes disrupted in these mutant strains had disparate predicted functions. Reconstruction of strains via targeted deletion of genes identified in the screen recapitulated the enhanced immunogenicity phenotype of the original mutant strains. When we introduced the simian immunodeficiency virus (SIV) gag gene into several of these novel BCG strains, we observed enhanced SIV Gag-specific CD8+ T cell responses in vivo. This study demonstrates that mycobacteria carry numerous genes that act to dampen CD8+ T cell responses and suggests that genetic modification of these genes may generate a novel group of recombinant BCG strains capable of serving as more effective and immunogenic vaccine vectors. PMID:25287928

Gene deletions in Mycobacterium bovis BCG stimulate increased CD8+ T cell responses.

PubMed

Panas, Michael W; Sixsmith, Jaimie D; White, KeriAnn; Korioth-Schmitz, Birgit; Shields, Shana T; Moy, Brian T; Lee, Sunhee; Schmitz, Joern E; Jacobs, William R; Porcelli, Steven A; Haynes, Barton F; Letvin, Norman L; Gillard, Geoffrey O

2014-12-01

Mycobacteria, the etiological agents of tuberculosis and leprosy, have coevolved with mammals for millions of years and have numerous ways of suppressing their host's immune response. It has been suggested that mycobacteria may contain genes that reduce the host's ability to elicit CD8(+) T cell responses. We screened 3,290 mutant Mycobacterium bovis bacillus Calmette Guerin (BCG) strains to identify genes that decrease major histocompatibility complex (MHC) class I presentation of mycobacterium-encoded epitope peptides. Through our analysis, we identified 16 mutant BCG strains that generated increased transgene product-specific CD8(+) T cell responses. The genes disrupted in these mutant strains had disparate predicted functions. Reconstruction of strains via targeted deletion of genes identified in the screen recapitulated the enhanced immunogenicity phenotype of the original mutant strains. When we introduced the simian immunodeficiency virus (SIV) gag gene into several of these novel BCG strains, we observed enhanced SIV Gag-specific CD8(+) T cell responses in vivo. This study demonstrates that mycobacteria carry numerous genes that act to dampen CD8(+) T cell responses and suggests that genetic modification of these genes may generate a novel group of recombinant BCG strains capable of serving as more effective and immunogenic vaccine vectors. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Immunogenicity and protective efficacy of the Mycobacterium tuberculosis fadD26 mutant

PubMed Central

Infante, E; Aguilar, L D; Gicquel, B; Pando, R Hernandez

2005-01-01

The Mycobacterium tuberculosis fadD26 mutant has impaired synthesis of phthiocerol dimycocerosates (DIM) and is attenuated in BALB/c mice. Survival analysis following direct intratracheal infection confirmed the attenuation: 60% survival at 4 months post-infection versus 100% mortality at 9 weeks post-infection with the wild-type strain. The fadD26 mutant induced less pneumonia and larger DTH reactions. It induced lower but progressive production of interferon (IFN)-γ, interleukin (IL)-4 and tumour necrosis factor (TNF)-α. Used as a subcutaneous vaccine 60 days before intratracheal challenge with a hypervirulent strain of M. tuberculosis (Beijing code 9501000), the mutant induced a higher level of protection than did Bacille Calmette–Guérin (BCG). Seventy per cent of the mice vaccinated with the fadD26 mutant survived at 16 weeks after challenge compared to 30% of those vaccinated with BCG. Similarly, there was less tissue damage (pneumonia) and lower colony-forming units (CFU) in the mice vaccinated with the fadD26 mutant compared to the findings in mice vaccinated with BCG. These data suggest that DIM synthesis is important for the pathogenicity of M. tuberculosis, and that inactivation of DIM synthesis can increase the immunogenicity of live vaccines, and increase their ability to protect against tuberculosis. PMID:15958066

One-tube loop-mediated isothermal amplification combined with restriction endonuclease digestion and ELISA for colorimetric detection of resistance to isoniazid, ethambutol and streptomycin in Mycobacterium tuberculosis isolates.

PubMed

Lee, Mei-Feng; Chen, Yen-Hsu; Hsu, Hui-Jine; Peng, Chien-Fang

2010-10-01

In this study, we designed a simple and rapid colorimetric detection method, a one-tube loop-mediated isothermal amplification (LAMP)-PCR-hybridization-restriction endonuclease-ELISA [one-tube LAMP-PCR-HY-RE-ELISA] system, to detect resistance to isoniazid, ethambutol and streptomycin in strains of Mycobacterium tuberculosis isolated from clinical specimens. The clinical performance of this method for detecting isoniazid-resistant, ethambutol-resistant and streptomycin-resistant isolates of M. tuberculosis showed 98.9%, 94.3% and 93.8%, respectively. This assay is rapid and convenient that can be performed within one working day. One-tube LAMP-PCR-HY-RE-ELISA system was designed based on hot spot point mutations in target drug-resistant genes, using LAMP-PCR, hybridization, digestion with restriction endonuclease and colorimetric method of ELISA. In this study, LAMP assay was used to amplify DNA from drug-resistant M. tuberculosis, and ELISA was used for colorimetrical determination. This assay will be a useful tool for rapid diagnosis of mutant codons in strains of M. tuberculosis for isoniazid at katG 315 and katG 463, ethambutol at embB 306 and embB 497, and streptomycin at rpsL 43. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

Strong In Vitro Activities of Two New Rifabutin Analogs against Multidrug-Resistant Mycobacterium tuberculosis ▿ †

PubMed Central

García, Ana-Belén; Palacios, Juan J.; Ruiz, María-Jesús; Barluenga, José; Aznar, Fernando; Cabal, María-Paz; García, José María; Díaz, Natalia

2010-01-01

Two new rifabutin analogs, RFA-1 and RFA-2, show high in vitro antimycobacterial activities against Mycobacterium tuberculosis. MIC values of RFA-1 and RFA-2 were ≤0.02 μg/ml against rifamycin-susceptible strains and 0.5 μg/ml against a wide selection of multidrug-resistant strains, compared to ≥50 μg/ml for rifampin and 10 μg/ml for rifabutin. Molecular dynamic studies indicate that the compounds may exert tighter binding to mutants of RNA polymerase that have adapted to the rifamycins. PMID:20855731

Evaluation of the immunogenic capability of the BCG strains BCGΔBCG1419c and BCGΔBCG1416c in a three-dimensional human lung tissue model.

PubMed

Parasa, Venkata Ramanarao; Rose, Jeronimo; Castillo-Diaz, Luis Alberto; Aceves-Sánchez, Michel de Jesús; Vega-Domínguez, Perla Jazmín; Lerm, Maria; Flores-Valdez, Mario Alberto

2018-03-27

Tuberculosis (TB) still remains as an unmet global threat. The current vaccine is not fully effective and novel alternatives are needed. Here, two vaccine candidate strains derived from BCG carrying deletions in the BCG1416c or BCG1419c genes were analysed for their capacity to modulate the cytokine/chemokine profile and granuloma formation in a human lung tissue model (LTM). We show that the clustering of monocytes, reminiscent of early granuloma formation, in LTMs infected with BCG strains was similar for all of them. However, BCGΔBCG1419c, like M. tuberculosis, was capable of inducing the production of IL-6 in contrast to the other BCG strains. This work suggests that LTM could be a useful ex vivo assay to evaluate the potential immunogenicity of novel TB vaccine candidates. Copyright © 2018 Elsevier Ltd. All rights reserved.

Use of immunochromatographic assay for rapid identification of Mycobacterium tuberculosis complex from liquid culture

PubMed Central

Považan, Anika; Vukelić, Anka; Savković, Tijana; Kurucin, Tatjana

2012-01-01

A new, simple immunochromatographic assay for rapid identification of Mycobacterium tuberculosis complex in liquid cultures has been developed. The principle of the assay is binding of the Mycobacterium tuberculosis complex specific antigen to the monoclonal antibody conjugated on the test strip. The aim of this study is evaluation of the performance of immunochromatographic assay in identification of Mycobacterium tuberculosis complex in primary positive liquid cultures of BacT/Alert automated system. A total of 159 primary positive liquid cultures were tested using the immunochromatographic assay (BD MGIT TBc ID) and the conventional subculture, followed by identification using biochemical tests. Of 159 positive liquid cultures, using the conventional method, Mycobacterium tuberculos is was identified in 119 (74.8%), nontuberculous mycobacteria were found in 4 (2.5%), 14 (8.8%) cultures were contaminated and 22 (13.8%) cultures were found to be negative. Using the immunochromatographic assay, Mycobacterium tuberculosis complex was detected in 118 (74.2%) liquid cultures, and 41 (25.8%) tests were negative. Sensitivity, specificity, positive and negative predictive values of the test were 98.3%; 97.5%; 99.15%; 95.12%, respectively. The value of kappa test was 0.950, and McNemar test was 1.00. The immunochromatographic assay is a simple and rapid test which represents a suitable alternative to the conventional subculture method for the primary identification of Mycobacterium tuberculosis complex in liquid cultures of BacT/Alert automated system. PMID:22364301

An Upstream Truncation of the furA-katG Operon Confers High-Level Isoniazid Resistance in a Mycobacterium tuberculosis Clinical Isolate with No Known Resistance-Associated Mutations

PubMed Central

Yam, Wing Cheong; Zhang, Ying; Kao, Richard Y. T.

2014-01-01

Although the major causes of isoniazid (INH) resistance in Mycobacterium tuberculosis are confined to structural mutations in katG and promoter mutations in the mabA-inhA operon, a significant proportion of INH-resistant strains have unknown resistance mechanisms. Recently, we identified a high-level INH-resistant M. tuberculosis clinical isolate, GB005, with no known resistance-associated mutations. A comprehensive study was performed to investigate the molecular basis of drug resistance in this strain. Although no mutations were found throughout the katG and furA-katG intergenic region, the katG expression and the catalase activity were greatly diminished compared to those in H37Rv (P < 0.01). Northern blotting revealed that the katG transcript from the isolate was smaller than that of H37Rv. Sequencing analysis of furA and upstream genes discovered a 7.2-kb truncation extended from the 96th base preceding the initiation codon of katG. Complementation of the M. tuberculosis Δ(furA-katG) strain with katG and different portions of the truncated region identified a 134-bp upstream fragment of furA that was essential for full catalase activity and INH susceptibility in M. tuberculosis. The promoter activity of this fragment was also shown to be stronger than that of the furA-katG intergenic region (P < 0.01). Collectively, these findings demonstrate that deletion of the 134-bp furA upstream fragment is responsible for the reduction in katG expression, resulting in INH resistance in GB005. To our knowledge, this is the first report showing that deletion of the upstream region preceding the furA-katG operon causes high-level INH resistance in a clinical isolate of M. tuberculosis. PMID:25092698

Acquisition of second-line drug resistance and extensive drug resistance during recent transmission of Mycobacterium tuberculosis in rural China.

PubMed

Hu, Y; Mathema, B; Zhao, Q; Chen, L; Lu, W; Wang, W; Kreiswirth, B; Xu, B

2015-12-01

Multidrug-resistant tuberculosis (MDR-TB) is prevalent in countries with a high TB burden, like China. As little is known about the emergence and spread of second-line drug (SLD) -resistant TB, we investigate the emergence and transmission of SLD-resistant Mycobacterium tuberculosis in rural China. In a multi-centre population-based study, we described the bacterial population structure and the transmission characteristics of SLD-resistant TB using Spoligotyping in combination with genotyping based on 24-locus MIRU-VNTR (mycobacterial interspersed repetitive unit-variable-number tandem repeat) plus four highly variable loci for the Beijing family, in four rural Chinese regions with diverse geographic and socio-demographic characteristics. Transmission networks among genotypically clustered patients were constructed using social network analysis. Of 1332 M. tuberculosis patient isolates recovered, the Beijing family represented 74.8% of all isolates and an association with MDR and simultaneous resistance between first-line drugs and SLDs. The genotyping analysis revealed that 189 isolates shared MIRU-VNTR patterns in 78 clusters with clustering rate and recent transmission rate of 14.2% and 8.3%, respectively. Fifty-three SLD-resistant isolates were observed in 31 clusters, 30 of which contained the strains with different drug susceptibility profiles and genetic mutations. In conjunction with molecular data, socio-network analysis indicated a key role of Central Township in the transmission across a highly interconnected network where SLD resistance accumulation occurred during transmission. SLD-resistant M. tuberculosis has been spreading in rural China with Beijing family being the dominant strains. Primary transmission of SLD-resistant strains in the population highlights the importance of routine drug susceptibility testing and effective anti-tuberculosis regimens for drug-resistant TB. Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Clinical, molecular and drug sensitivity pattern of mycobacterial isolates from extra-pulmonary tuberculosis cases in Addis Ababa, Ethiopia.

PubMed

Korma, Workneh; Mihret, Adane; Hussien, Jemal; Anthony, Richard; Lakew, Mekuria; Aseffa, Abraham

2015-10-26

In conjunction with the spread of HIV infection, tuberculosis (TB) remains a major cause of illness and death worldwide. The Ethiopian national report reveals that extra pulmonary tuberculosis is on the rise and that case detection rate is exceeding that of smear positive or negative cases in many parts of the country. Different studies indicated that host and/or pathogen related factors are associated with the rise of extra pulmonary cases. However, the reason for this is not clearly known in our setting. Specimens were taken from clinically suspected extra pulmonary patients and confirmed by cytology, histopathology and culture. Deletion typing and Spoligotyping was utilized to identify the strains. The isolates were then assigned to lineage using conformal Bayesian network (rules model) algorithm and dendrograms were drawn using UPGMA methods. In addition, drug sensitivity test was done using the indirect proportion and 24 well plate methods. Out of the 200 clinically suspected extra pulmonary tuberculosis patients, 106 (53 %) were between 15 and 35 years of age and 167 (83.5 %) were new while 33 (16.5 %) were retreatment cases. The culture yield was 29.5 % (59). Of these only one was M. bovis and 58 were M. tuberculosis strains with 31 different spoligotype patterns grouped into seven clusters. The largest cluster (ST53) comprised 12 (20.3 %) isolates. There was higher clustering of CAS isolates in TBLN than in any other form of extra pulmonary tuberculosis cases. Resistance to rifampicin was higher (22 %) than that for INH, STM and EMB (8.1 %, 5 % and 3 % respectively). Out of the 37 isolates tested for resistance, only 2 isolates were resistant for both STM and INH and no MDR strain was found. There is an ongoing active recent transmission among extra pulmonary tuberculosis in the study areas as shown by the presence of clusters. Although no MDR case was observed, there is a risk of emergence of MDR as noted from the high proportion of resistance to rifampicin. Detailed study at population level is recommended to monitor its trend.

Recurrent tuberculosis in an urban area in China: Relapse or exogenous reinfection?

PubMed

Shen, Xin; Yang, Chongguang; Wu, Jie; Lin, Senlin; Gao, Xu; Wu, Zheyuan; Tian, Jiyun; Gan, Mingyu; Luo, Tao; Wang, Lili; Yu, Chenlei; Mei, Jian; Pan, Qichao; DeRiemer, Kathryn; Yuan, ZhengAn; Gao, Qian

2017-03-01

Recurrent tuberculosis is an important indicator of the effectiveness of tuberculosis control and can occur by relapse or exogenous reinfection. We conducted a retrospective cohort study on all bacteriologically confirmed tuberculosis cases that were successfully treated between 2000 and 2012 in Shanghai, an urban area with a high number but a low prevalence rate of tuberculosis cases and a low prevalence of HIV infection. Genotyping the Mycobacterium tuberculosis from clinical isolates was used to distinguish between relapse and reinfection. In total, 5.3% (710/13,417) of successfully treated cases had a recurrence, a rate of 7.55 (95% CI 7.01-8.13) episodes per 1000 person-years, more than 18 times the rate of tuberculosis in the general population. Patients who were male, age 30-59, retreatment cases, had cavitation, diabetes, drug-resistant or multidrug-resistant tuberculosis in their initial episode of tuberculosis, were at high risk for a recurrence. Among 141 recurrent cases that had paired isolates, 59 (41.8%) had different genotypes, indicating reinfection with a different strain. Patients who completed treatment were still at high risk of another episode of tuberculosis and exogenous reinfection contributed a significant proportion of the recurrent tuberculosis cases. Targeted control strategies are needed to prevent new tuberculosis infections in this setting. Copyright © 2017 Elsevier Ltd. All rights reserved.

Recurrent tuberculosis in an urban area in China: relapse or exogenous reinfection?

PubMed Central

Shen, Xin; Yang, Chongguang; Wu, Jie; Lin, Senlin; Gao, Xu; Wu, Zheyuan; Tian, Jiyun; Gan, Mingyu; Luo, Tao; Wang, Lili; Yu, Chenlei; Mei, Jian; Pan, Qichao; DeRiemer, Kathryn; Yuan, ZhengAn; Gao, Qian

2017-01-01

Recurrent tuberculosis is an important indicator of the effectiveness of tuberculosis control and can occur by relapse or exogenous reinfection. We conducted a retrospective cohort study on all bacteriologically confirmed tuberculosis cases that were successfully treated between 2000 and 2012 in Shanghai, an urban area with a high number but a low prevalence rate of tuberculosis cases and a low prevalence of HIV infection. Genotyping the Mycobacterium tuberculosis from clinical isolates was used to distinguish between relapse and reinfection. In total, 5.3% (710/13,417) of successfully treated cases had a recurrence, a rate of 7.55 (95% CI 7.01–8.13) episodes per 1000 person-years, more than 18 times the rate of tuberculosis in the general population. Patients who were male, age 30–59, retreatment cases, had cavitation, diabetes, drug-resistant or multidrug-resistant tuberculosis in their initial episode of tuberculosis, were at high risk for a recurrence. Among 141 recurrent cases that had paired isolates, 59 (41.8%) had different genotypes, indicating reinfection with a different strain. Patients who completed treatment were still at high risk of another episode of tuberculosis and exogenous reinfection contributed a significant proportion of the recurrent tuberculosis cases. Targeted control strategies are needed to prevent new tuberculosis infections in this setting. PMID:28237039

Tuberculosis in domestic livestock: pathogenesis, transmission, and vaccination

USDA-ARS?s Scientific Manuscript database

The Mycobacterium tuberculosis complex includes agents such as M. tuberculosis and M. bovis, the cause of tuberculosis in most animals and a zoonotic pathogen. Mycobacterium bovis has one of the broadest host ranges of any pathogen, infecting most mammals, including humans. Models are used to study ...

Is IQG-607 a Potential Metallodrug or Metallopro-Drug With a Defined Molecular Target in Mycobacterium tuberculosis?

PubMed Central

Abbadi, Bruno L.; Rodrigues-Junior, Valnês da Silva; Dadda, Adilio da Silva; Pissinate, Kenia; Villela, Anne D.; Campos, Maria M.; Lopes, Luiz G. de França; Bizarro, Cristiano V.; Machado, Pablo; Sousa, Eduardo H. S.; Basso, Luiz A.

2018-01-01

The emergence of strains of Mycobacterium tuberculosis resistant to isoniazid (INH) has underscored the need for the development of new anti-tuberculosis agents. INH is activated by the mycobacterial katG-encoded catalase-peroxidase, forming an acylpyridine fragment that is covalently attached to the C4 of NADH. This isonicotinyl-NAD adduct inhibits the activity of 2-trans-enoyl-ACP(CoA) reductase (InhA), which plays a role in mycolic acid biosynthesis. A metal-based INH analog, Na3[FeII(CN)5(INH)]·4H2O, IQG-607, was designed to have an electronic redistribution on INH moiety that would lead to an intramolecular electron transfer to bypass KatG activation. HPLC and EPR studies showed that the INH moiety can be oxidized by superoxide or peroxide yielding similar metabolites and isonicotinoyl radical only when associated to IQG-607, thereby supporting redox-mediated drug activation as a possible mechanism of action. However, IQG-607 was shown to inhibit the in vitro activity of both wild-type and INH-resistant mutant InhA enzymes in the absence of KatG activation. IQG-607 given by the oral route to M. tuberculosis-infected mice reduced lung lesions. Experiments using early and late controls of infection revealed a bactericidal activity for IQG-607. HPLC and voltammetric methods were developed to quantify IQG-607. Pharmacokinetic studies showed short half-life, high clearance, moderate volume of distribution, and low oral bioavailability, which was not altered by feeding. Safety and toxic effects of IQG-607 after acute and 90-day repeated oral administrations in both rats and minipigs showed occurrence of mild to moderate toxic events. Eight multidrug-resistant strains (MDR-TB) were resistant to IQG-607, suggesting an association between katG mutation and increasing MIC values. Whole genome sequencing of three spontaneous IQG-607-resistant strains harbored katG gene mutations. MIC measurements and macrophage infection experiments with a laboratorial strain showed that katG mutation is sufficient to confer resistance to IQG-607 and that the macrophage intracellular environment cannot trigger the self-activation mechanism. Reduced activity of IQG-607 against an M. tuberculosis strain overexpressing S94A InhA mutant protein suggested both the need for KatG activation and InhA as its target. Further efforts are suggested to be pursued toward attempting to translate IQG-607 into a chemotherapeutic agent to treat tuberculosis. PMID:29765372

[Tuberculosis and HIV infection: experience of the national tuberculosis prevention program in Djibouti: 1990-1996].

PubMed

Renoux, E; Matan, A Barreh; Sevre, J P; Mohamed Ali, I; Chami, D; Vincent, V

2002-01-01

Based on analysis of data collected from the national tuberculosis prevention program in Djibouti between 1990 and 1996, the authors analyzed the relationship between HIV infection and tuberculosis. The study cohort comprised a total of 22,000 patients including 14,000 with documented HIV infection. Although HIV infection probably worsened the situation, it was neither the only nor the main factor involved in the resurgence of tuberculosis. Demographic growth, higher population density, and increasing poverty as well as the quality of the national tuberculosis prevention program must be taken into account. The incidence of smear-negative tuberculosis was not significantly higher in HIV-infected patients (incidence of smear positive cases, > 92%). Extrapulmonary tuberculosis especially of pleural involvement was more common (15% versus 9.4%). Treatment was effective in HIV-infected patients. If directly observed (DOT) therapy was used, there was no risk of emergence of multidrug-resistant tuberculosis strains. Drug side-effects associated with the protocols used in Djibouti were not greater in HIV-infected patients. Most additional mortality observed in HIV-infected tuberculosis patients (10.5% versus 2%) was due to progression of HIV infection.

Spatio-Temporal Distribution of Mycobacterium tuberculosis Complex Strains in Ghana.

PubMed

Yeboah-Manu, Dorothy; Asare, P; Asante-Poku, A; Otchere, I D; Osei-Wusu, S; Danso, E; Forson, A; Koram, K A; Gagneux, Sebastien

2016-01-01

There is a perception that genomic differences in the species/lineages of the nine species making the Mycobacterium tuberculosis complex (MTBC) may affect the efficacy of distinct control tools in certain geographical areas. We therefore analyzed the prevalence and spatial distribution of MTBC species and lineages among isolates from pulmonary TB cases over an 8-year period, 2007-2014. Mycobacterial species isolated by culture from consecutively recruited pulmonary tuberculosis patients presenting at selected district/sub-district health facilities were confirmed as MTBC by IS6110 and rpoß PCR and further assigned lineages and sub lineages by spoligotyping and large sequence polymorphism PCR (RDs 4, 9, 12, 702, 711) assays. Patient characteristics, residency, and risks were obtained with a structured questionnaire. We used SaTScan and ArcMap analyses to identify significantly clustered MTBC lineages within selected districts and spatial display, respectively. Among 2,551 isolates, 2,019 (79.1%), 516 (20.2%) and 16 (0.6%) were identified as M. tuberculosis sensu stricto (MTBss), M. africanum (Maf), 15 M. bovis and 1 M. caprae, respectively. The proportions of MTBss and Maf were fairly constant within the study period. Maf spoligotypes were dominated by Spoligotype International Type (SIT) 331 (25.42%), SIT 326 (15.25%) and SIT 181 (14.12%). We found M. bovis to be significantly higher in Northern Ghana (1.9% of 212) than Southern Ghana (0.5% of 2339) (p = 0.020). Using the purely spatial and space-time analysis, seven significant MTBC lineage clusters (p< 0.05) were identified. Notable among the clusters were Ghana and Cameroon sub-lineages found to be associated with north and south, respectively. This study demonstrated that overall, 79.1% of TB in Ghana is caused by MTBss and 20% by M. africanum. Unlike some West African Countries, we did not observe a decline of Maf prevalence in Ghana.

Physiology of Mycobacteria

PubMed Central

Cook, Gregory M.; Berney, Michael; Gebhard, Susanne; Heinemann, Matthias; Cox, Robert A.; Danilchanka, Olga; Niederweis, Michael

2013-01-01

Mycobacterium tuberculosis is a prototrophic, metabolically flexible bacterium that has achieved a spread in the human population that is unmatched by any other bacterial pathogen. The success of M. tuberculosis as a pathogen can be attributed to its extraordinary stealth and capacity to adapt to environmental changes throughout the course of infection. These changes include: nutrient deprivation, hypoxia, various exogenous stress conditions and, in the case of the pathogenic species, the intraphagosomal environment. Knowledge of the physiology of M. tuberculosis during this process has been limited by the slow growth of the bacterium in the laboratory and other technical problems such as cell aggregation. Advances in genomics and molecular methods to analyse the M. tuberculosis genome have revealed that adaptive changes are mediated by complex regulatory networks and signals, resulting in temporal gene expression coupled to metabolic and energetic changes. An important goal for bacterial physiologists will be to elucidate the physiology of M. tuberculosis during the transition between the diverse conditions encountered by M. tuberculosis. This review covers the growth of the mycobacterial cell and how environmental stimuli are sensed by this bacterium. Adaptation to different environments is described from the viewpoint of nutrient acquisition, energy generation and regulation. To gain quantitative understanding of mycobacterial physiology will require a systems biology approach and recent efforts in this area are discussed. “It is now 100 years since the first mycobacterium was isolated by Hansen (1874). Somewhat ironically, this was the leprosy bacillus, Mycobacterium leprae, which even today is still resisting all attempts to cultivate it in the laboratory. The tubercle bacillus, M. tuberculosis was not discovered until eight years later (Koch, 1882) and this has remained an object of intensive investigation ever since. The widespread interest in the mycobacteria of course stems from the diseases they cause and, lest it be imagined that tuberculosis is a disease which has now been largely conquered and that leprosy is of relatively rare occurrence, current estimates for the number of case of tuberculosis and leprosy in the world today are 20,000,000 and 11,000,000, respectively (Bechelli and Dominguez, 1972). The annual estimated mortality rate is equally dramatic, namely 3,000,000 (World Health Organization, 1974). Also causing unease is the continuing isolation from tubercular patients of strains already resistant to one or more chemotherapeutic agent”. C. Ratledge (1976). PMID:19573696

A comparative modeling and molecular docking study on Mycobacterium tuberculosis targets involved in peptidoglycan biosynthesis.

PubMed

Fakhar, Zeynab; Naiker, Suhashni; Alves, Claudio N; Govender, Thavendran; Maguire, Glenn E M; Lameira, Jeronimo; Lamichhane, Gyanu; Kruger, Hendrik G; Honarparvar, Bahareh

2016-11-01

An alarming rise of multidrug-resistant Mycobacterium tuberculosis strains and the continuous high global morbidity of tuberculosis have reinvigorated the need to identify novel targets to combat the disease. The enzymes that catalyze the biosynthesis of peptidoglycan in M. tuberculosis are essential and noteworthy therapeutic targets. In this study, the biochemical function and homology modeling of MurI, MurG, MraY, DapE, DapA, Alr, and Ddl enzymes of the CDC1551 M. tuberculosis strain involved in the biosynthesis of peptidoglycan cell wall are reported. Generation of the 3D structures was achieved with Modeller 9.13. To assess the structural quality of the obtained homology modeled targets, the models were validated using PROCHECK, PDBsum, QMEAN, and ERRAT scores. Molecular dynamics simulations were performed to calculate root mean square deviation (RMSD) and radius of gyration (Rg) of MurI and MurG target proteins and their corresponding templates. For further model validation, RMSD and Rg for selected targets/templates were investigated to compare the close proximity of their dynamic behavior in terms of protein stability and average distances. To identify the potential binding mode required for molecular docking, binding site information of all modeled targets was obtained using two prediction algorithms. A docking study was performed for MurI to determine the potential mode of interaction between the inhibitor and the active site residues. This study presents the first accounts of the 3D structural information for the selected M. tuberculosis targets involved in peptidoglycan biosynthesis.

Burden of Multidrug Resistant Mycobacterium tuberculosis Among New Cases in Al-Madinah Al-Monawarah, Saudi Arabia.

PubMed

Elhassan, Mogahid M; Hemeg, Hassan A; Elmekki, Miskelyemen A; Turkistani, Khalid A; Abdul-Aziz, Ahmed A

2017-01-01

The pattern of Mycobacterium tuberculosis susceptibility to first line drugs and multidrug resistance in Al-Madinah Al-Munawarah, a seasonally overcrowded are during Hajj and Omrah, is not well studied. This study aimed to investigate anti-tuberculosis drug resistance and its distribution among new cases in Al-Madinah Al-Monawarah. Study subjects included 622 patients with first time confirmed TB referred to the central tuberculosis laboratory in Al-Madinah between January 2012 and December 2014. Out of the 622 isolates, 99 (15.9%) were Mycobacteria Other Than Tuberculosis (MOTTS) and 25 (4.0%), three of which (12%) were children under five years of age, revealed multidrug resistance (MDR). Monoresistance to isoniazid (H) was (1.8%), to rifampin (R) was (1.4%), to streptomycin (S) was (1.9 %) to ethambutol (E) was (1.1 %) and to pyrazinamide (Z) was (2.1%). Being among the new cases, multidrug resistant tuberculosis (MDR TB) is supposed to be caused by strains which are originally multidrug resistant. Neither nationality nor gender was found to be associated with MDR TB. Since 12% of MDR cases were among children, a probability of primary infection with MDR strains is to be considered. Moreover, mass gathering during Hajj and Omrah seasons does not seem to increase the burden of MDR in the region. However, further investigation is needed to molecularly characterize MDR isolates and their phylogenetics and geographical origin. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Molecular epidemiology of tuberculosis: achievements and challenges to current knowledge.

PubMed Central

Murray, Megan; Nardell, Edward

2002-01-01

Over the past 10 years, molecular methods have become available with which to strain-type Mycobacterium tuberculosis. They have allowed researchers to study certain important but previously unresolved issues in the epidemiology of tuberculosis (TB). For example, some unsuspected microepidemics have been revealed and it has been shown that the relative contribution of recently acquired disease to the TB burden in many settings is far greater than had been thought. These findings have led to the strengthening of TB control. Other research has demonstrated the existence and described the frequency of exogenous reinfection in areas of high incidence. Much recent work has focused on the phenotypic variation among strains and has evaluated the relative transmissibility, virulence, and immunogenicity of different lineages of the organism. We summarize the recent achievements in TB epidemiology associated with the introduction of DNA fingerprinting techniques, and consider the implications of this technology for the design and analysis of epidemiological studies. PMID:12132006

Improved Phenoxyalkylbenzimidazoles with Activity against Mycobacterium tuberculosis Appear to Target QcrB

PubMed Central

2017-01-01

The phenoxy alkyl benzimidazoles (PABs) have good antitubercular activity. We expanded our structure–activity relationship studies to determine the core components of PABs required for activity. The most potent compounds had minimum inhibitory concentrations against Mycobacterium tuberculosis in the low nanomolar range with very little cytotoxicity against eukaryotic cells as well as activity against intracellular bacteria. We isolated resistant mutants against PAB compounds, which had mutations in either Rv1339, of unknown function, or qcrB, a component of the cytochrome bc1 oxidase of the electron transport chain. QcrB mutant strains were resistant to all PAB compounds, whereas Rv1339 mutant strains were only resistant to a subset, suggesting that QcrB is the target. The discovery of the target for PAB compounds will allow for the improved design of novel compounds to target intracellular M. tuberculosis. PMID:29035551

Genetic profiling of Mycobacterium bovis strains from slaughtered cattle in Eritrea

PubMed Central

Hlokwe, Tiny; Rutten, Victor P. M. G.; Allepuz, Alberto; Cadmus, Simeon; Muwonge, Adrian; Robbe-Austerman, Suelee; Michel, Anita L.

2018-01-01

Mycobacterium bovis (M.bovis) is the main causative agent for bovine tuberculosis (BTB) and can also be the cause of zoonotic tuberculosis in humans. In view of its zoonotic nature, slaughterhouse surveillance, potentially resulting in total or partial condemnation of the carcasses and organs, is conducted routinely. Spoligotyping, VNTR profiling, and whole genome sequencing (WGS) of M. bovis isolated from tissues with tuberculosis-like lesions collected from 14 cattle at Eritrea’s largest slaughterhouse in the capital Asmara, were conducted.The 14 M. bovis isolates were classified into three different spoligotype patterns (SB0120, SB0134 and SB0948) and six VNTR profiles. WGS results matched those of the conventional genotyping methods and further discriminated the six VNTR profiles into 14 strains. Furthermore, phylogenetic analysis of the M. bovis isolates suggests two independent introductions of BTB into Eritrea possibly evolving from a common ancestral strain in Europe.This molecular study revealed the most important strains of M. bovis in Eritrea and their (dis)similarities with the strains generally present in East Africa and Europe, as well as potential routes of introduction of M. bovis. Though the sample size is small, the current study provides important information as well as platform for future in-depth molecular studies on isolates from both the dairy and the traditional livestock sectors in Eritrea and the region. This study provides information onthe origin of some of the M. bovis strains in Eritrea, its genetic diversity, evolution and patterns of spread between dairy herds. Such information is essential in the development and implementation of future BTB control strategy for Eritrea. PMID:29664901

Bovine Tuberculosis Vaccine Efficacy Studies: Neonatal Calves and White-tailed Deer

USDA-ARS?s Scientific Manuscript database

Introduction Tuberculosis (TB) in humans and animals may result from exposure to bacilli within the Mycobacterium tuberculosis complex (i.e., M. tuberculosis, M. bovis, M. africanum, M. pinnipedi, M. microti, M. caprae, or M. canetti)(#1). Mycobacterium bovis is the species most often isolated from ...

Drug development against tuberculosis: Impact of alkaloids.

PubMed

Mishra, Shardendu K; Tripathi, Garima; Kishore, Navneet; Singh, Rakesh K; Singh, Archana; Tiwari, Vinod K

2017-09-08

Despite of the advances made in the treatment and management, tuberculosis (TB) still remains one of main public health problem. The contrary effects of first and second-line anti-tuberculosis drugs have generated extended research interest in natural products in the hope of devising new antitubercular leads. Interestingly, plethoras of natural products have been discovered to exhibit activity towards various resistant strains of M. tuberculosis. Extensive applications of alkaloids in the field of therapeutics is well-established and nowday's researches being pursued to develop new potent drugs from natural sources for tuberculosis. Alkaloids are categorized in quite a few groups according to their structures and isolation from both terrestrial and marine sources. These new drugs might be a watershed in the battle against tuberculosis. This review summarizes alkaloids, which were found active against Mycobacteria since last ten years with special attention on the study of structure-activity relationship (SAR) and mode of action with their impact in drug discovery and development against tuberculosis. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Tuberculosis in Sardinia: An investigation into the relationship between natives and immigrants.

PubMed

Ruggeri, Melania; Molicotti, Paola; Cubeddu, Marina; Cannas, Sara; Bua, Alessandra; Zanetti, Stefania

2016-09-01

Tuberculosis (TB) has had a recrudescence in the last few decades in Italy as a result of many factors, among which migration from countries where TB is endemic is one of them. In Sardinia, a major island of Italy, there was no knowledge of the mechanisms of transmission of TB in the immigrant subpopulation and the impact it may have on the native subpopulation and on the community as a whole. Therefore, a molecular epidemiological study was carried out to get a clearer picture of the number and genetic features of Mycobacterium tuberculosis strains isolated from immigrants and from natives in Sardinia. Two groups of clinical isolates of M. tuberculosis, one collected from immigrants and the other one from Sardinians, were analyzed in this study. The genotyping was executed through the variable number tandem repeat-mycobacterial interspersed repetitive units technique and a first-line antimycobacterial drug-susceptibility test was also carried out. Thirty-six clinical isolates from immigrants and 25 from Sardinians were analyzed. Variable number tandem repeat-mycobacterial interspersed repetitive units technique showed that all of them belonged to different strains and there was a quite high allelic diversity among them. Moreover, data collected allowed the finding of, with a good approximation, the phylogenetic relations among the strains isolated and the best-known phylogenetic groups. The study pointed out that since every strain is different, there was no TB transmission in any of the subpopulations and between immigrants and natives. This showed that the presence of immigrants was not a risk factor for contracting TB in the community. Copyright © 2016 Asian-African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.

Wild-type MIC distributions for aminoglycoside and cyclic polypeptide antibiotics used for treatment of Mycobacterium tuberculosis infections.

PubMed

Juréen, P; Angeby, K; Sturegård, E; Chryssanthou, E; Giske, C G; Werngren, J; Nordvall, M; Johansson, A; Kahlmeter, G; Hoffner, S; Schön, T

2010-05-01

The aminoglycosides and cyclic polypeptides are essential drugs in the treatment of multidrug-resistant tuberculosis, underscoring the need for accurate and reproducible drug susceptibility testing (DST). The epidemiological cutoff value (ECOFF) separating wild-type susceptible strains from non-wild-type strains is an important but rarely used tool for indicating susceptibility breakpoints against Mycobacterium tuberculosis. In this study, we established wild-type MIC distributions on Middlebrook 7H10 medium for amikacin, kanamycin, streptomycin, capreomycin, and viomycin using 90 consecutive clinical isolates and 21 resistant strains. Overall, the MIC variation between and within runs did not exceed +/-1 MIC dilution step, and validation of MIC values in Bactec 960 MGIT demonstrated good agreement. Tentative ECOFFs defining the wild type were established for all investigated drugs, including amikacin and viomycin, which currently lack susceptibility breakpoints for 7H10. Five out of seven amikacin- and kanamycin-resistant isolates were classified as susceptible to capreomycin according to the current critical concentration (10 mg/liter) but were non-wild type according to the ECOFF (4 mg/liter), suggesting that the critical concentration may be too high. All amikacin- and kanamycin-resistant isolates were clearly below the ECOFF for viomycin, and two of them were below the ECOFF for streptomycin, indicating that these two drugs may be considered for treatment of amikacin-resistant strains. Pharmacodynamic indices (peak serum concentration [Cmax]/MIC) were more favorable for amikacin and viomycin compared to kanamycin and capreomycin. In conclusion, our data emphasize the importance of establishing wild-type MIC distributions for improving the quality of drug susceptibility testing against Mycobacterium tuberculosis.

Wild-Type MIC Distributions for Aminoglycoside and Cyclic Polypeptide Antibiotics Used for Treatment of Mycobacterium tuberculosis Infections▿

PubMed Central

Juréen, P.; Ängeby, K.; Sturegård, E.; Chryssanthou, E.; Giske, C. G.; Werngren, J.; Nordvall, M.; Johansson, A.; Kahlmeter, G.; Hoffner, S.; Schön, T.

2010-01-01

The aminoglycosides and cyclic polypeptides are essential drugs in the treatment of multidrug-resistant tuberculosis, underscoring the need for accurate and reproducible drug susceptibility testing (DST). The epidemiological cutoff value (ECOFF) separating wild-type susceptible strains from non-wild-type strains is an important but rarely used tool for indicating susceptibility breakpoints against Mycobacterium tuberculosis. In this study, we established wild-type MIC distributions on Middlebrook 7H10 medium for amikacin, kanamycin, streptomycin, capreomycin, and viomycin using 90 consecutive clinical isolates and 21 resistant strains. Overall, the MIC variation between and within runs did not exceed ±1 MIC dilution step, and validation of MIC values in Bactec 960 MGIT demonstrated good agreement. Tentative ECOFFs defining the wild type were established for all investigated drugs, including amikacin and viomycin, which currently lack susceptibility breakpoints for 7H10. Five out of seven amikacin- and kanamycin-resistant isolates were classified as susceptible to capreomycin according to the current critical concentration (10 mg/liter) but were non-wild type according to the ECOFF (4 mg/liter), suggesting that the critical concentration may be too high. All amikacin- and kanamycin-resistant isolates were clearly below the ECOFF for viomycin, and two of them were below the ECOFF for streptomycin, indicating that these two drugs may be considered for treatment of amikacin-resistant strains. Pharmacodynamic indices (peak serum concentration [Cmax]/MIC) were more favorable for amikacin and viomycin compared to kanamycin and capreomycin. In conclusion, our data emphasize the importance of establishing wild-type MIC distributions for improving the quality of drug susceptibility testing against Mycobacterium tuberculosis. PMID:20237102

Whole-Genome Analysis of Mycobacterium tuberculosis from Patients with Tuberculous Spondylitis, Russia.

PubMed

Chernyaeva, Ekaterina; Rotkevich, Mikhail; Krasheninnikova, Ksenia; Yurchenko, Andrey; Vyazovaya, Anna; Mokrousov, Igor; Solovieva, Natalia; Zhuravlev, Viacheslav; Yablonsky, Piotr; O'Brien, Stephen J

2018-03-01

Whole-genome analysis of Mycobacterium tuberculosis isolates collected in Russia (N = 71) from patients with tuberculous spondylitis supports a detailed characterization of pathogen strain distributions and drug resistance phenotype, plus distinguished occurrence and association of known resistance mutations. We identify known and novel genome determinants related to bacterial virulence, pathogenicity, and drug resistance.

Drug-Resistant Tuberculosis: A Genetic Analysis Using Online Bioinformatics Tools

ERIC Educational Resources Information Center

Taylor, Jessica M.; Davidson, Rebecca M.; Strong, Michael

2014-01-01

Tuberculosis (TB) continues to be a serious global health problem, resulting in >1.4 million deaths each year. Of increasing concern is the evolution of antibiotic resistant strains of the bacterium that causes TB. Using this real-world scenario, we created a 90-minute activity for high school or undergraduate students to use online…

Magnitude and sources of bias in the detection of mixed strain M. tuberculosis infection.

PubMed

Plazzotta, Giacomo; Cohen, Ted; Colijn, Caroline

2015-03-07

High resolution tests for genetic variation reveal that individuals may simultaneously host more than one distinct strain of Mycobacterium tuberculosis. Previous studies find that this phenomenon, which we will refer to as "mixed infection", may affect the outcomes of treatment for infected individuals and may influence the impact of population-level interventions against tuberculosis. In areas where the incidence of TB is high, mixed infections have been found in nearly 20% of patients; these studies may underestimate the actual prevalence of mixed infection given that tests may not be sufficiently sensitive for detecting minority strains. Specific reasons for failing to detect mixed infections would include low initial numbers of minority strain cells in sputum, stochastic growth in culture and the physical division of initial samples into parts (typically only one of which is genotyped). In this paper, we develop a mathematical framework that models the study designs aimed to detect mixed infections. Using both a deterministic and a stochastic approach, we obtain posterior estimates of the prevalence of mixed infection. We find that the posterior estimate of the prevalence of mixed infection may be substantially higher than the fraction of cases in which it is detected. We characterize this bias in terms of the sensitivity of the genotyping method and the relative growth rates and initial population sizes of the different strains collected in sputum. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Genotypic characteristics of Mycobacterium tuberculosis circulating in Xinjiang, China.

PubMed

Yuan, Li; Mi, Ligu; Li, Yongxiang; Zhang, Hui; Zheng, Fang; Li, Zhuoya

2016-02-01

Tuberculosis (TB), a chronic infectious disease caused by Mycobacterium tuberculosis (MTB), poses a serious threat to human health. We investigated the genotypes of MTB in the high prevalence province Xinjiang, China. From March 2010 to May 2013, 381 MTB isolates from patients with pulmonary TB were analyzed by molecular typing of 24 mycobacterial interspersed repetitive unit-variable number tandem repeat loci and PCR detection of the deleted regions of difference of the Beijing/W lineage and its sublineages. These isolates were shown to be highly polymorphic and to be composed of 345 unique genotypes, including 30 genotype clusters consisting of 2 or 3 strains and 315 individual genotypes. The genotype clustering rate was 17.32% and recent transmission index was low (9.45%). The Beijing/W lineage strains accounted for 57.48% of the isolates, and this predominant family strain was further subdivided into four sublineages: 181 (69.86%), 207 (14.61%), 105 (10.96%), and 150 (4.56%). The Beijing/W lineage (especially sublineage 181) strains were predominant and were associated with the transmissibility of TB in Xinjiang. Based on our data, we hypothesize that the circulating MTB strains in Xinjiang have significant genetic diversity and that the majority of the TB in Xinjiang may be explained by non-recent transmission emerging by endogenous reactivation. The possibility of outbreak is low, and current measures to control TB should first focus on standardized treatment of TB patients to prevent reactivation of latent infections.

Modular programming for tuberculosis control, the "AuTuMN" platform.

PubMed

Trauer, James McCracken; Ragonnet, Romain; Doan, Tan Nhut; McBryde, Emma Sue

2017-08-07

Tuberculosis (TB) is now the world's leading infectious killer and major programmatic advances will be needed if we are to meet the ambitious new End TB Targets. Although mathematical models are powerful tools for TB control, such models must be flexible enough to capture the complexity and heterogeneity of the global TB epidemic. This includes simulating a disease that affects age groups and other risk groups differently, has varying levels of infectiousness depending upon the organ involved and varying outcomes from treatment depending on the drug resistance pattern of the infecting strain. We adopted sound basic principles of software engineering to develop a modular software platform for simulation of TB control interventions ("AuTuMN"). These included object-oriented programming, logical linkage between modules and consistency of code syntax and variable naming. The underlying transmission dynamic model incorporates optional stratification by age, risk group, strain and organ involvement, while our approach to simulating time-variant programmatic parameters better captures the historical progression of the epidemic. An economic model is overlaid upon this epidemiological model which facilitates comparison between new and existing technologies. A "Model runner" module allows for predictions of future disease burden trajectories under alternative scenario situations, as well as uncertainty, automatic calibration, cost-effectiveness and optimisation. The model has now been used to guide TB control strategies across a range of settings and countries, with our modular approach enabling repeated application of the tool without the need for extensive modification for each application. The modular construction of the platform minimises errors, enhances readability and collaboration between multiple programmers and enables rapid adaptation to answer questions in a broad range of contexts without the need for extensive re-programming. Such features are particularly important in simulating an epidemic as complex and diverse as TB.

Failure of BACTEC™ MGIT 960™ to detect Mycobacterium tuberculosis complex within a 42-day incubation period.

PubMed

Mahomed, Sharana; Dlamini-Mvelase, Nomonde R; Dlamini, Moses; Mlisana, Koleka

2017-01-01

For the optimal recovery of Mycobacterium tuberculosis from the BACTEC™ Mycobacterium Growth Indicator Tube 960™ system, an incubation period of 42-56 days is recommended by the manufacturer. Due to logistical reasons, it is common practice to follow an incubation period of 42 days. We undertook a retrospective study to document positive Mycobacterium Growth Indicator Tube cultures beyond the 42-day incubation period. In total, 98/110 (89%) were positive for M. tuberculosis complex. This alerted us to M. tuberculosis growth detection failure at 42 days.

Genome analysis of smooth tubercle bacilli provides insights into ancestry and pathoadaptation of the etiologic agent of tuberculosis

PubMed Central

Supply, Philip; Marceau, Michael; Mangenot, Sophie; Roche, David; Rouanet, Carine; Khanna, Varun; Majlessi, Laleh; Criscuolo, Alexis; Tap, Julien; Pawlik, Alexandre; Fiette, Laurence; Orgeur, Mickael; Fabre, Michel; Parmentier, Cécile; Frigui, Wafa; Simeone, Roxane; Boritsch, Eva C.; Debrie, Anne-Sophie; Willery, Eve; Walker, Danielle; Quail, Michael A.; Ma, Laurence; Bouchier, Christiane; Salvignol, Grégory; Sayes, Fadel; Cascioferro, Alessandro; Seemann, Torsten; Barbe, Valérie; Locht, Camille; Gutierrez, Maria-Cristina; Leclerc, Claude; Bentley, Stephen; Stinear, Timothy P.; Brisse, Sylvain; Médigue, Claudine; Parkhill, Julian; Cruveiller, Stéphane; Brosch, Roland

2013-01-01

Global spread and genetic monomorphism are hallmarks of Mycobacterium tuberculosis, the agent of human tuberculosis. In contrast, Mycobacterium canettii, and related tubercle bacilli that also cause human tuberculosis and exhibit unusual smooth colony morphology, are restricted to East-Africa. Here, we sequenced and analyzed the genomes of five representative strains of smooth tubercle bacilli (STB) using Sanger (4-5x coverage), 454/Roche (13-18x coverage) and/or Illumina DNA sequencing (45-105x coverage). We show that STB are highly recombinogenic and evolutionary early-branching, with larger genome sizes, 25-fold more SNPs, fewer molecular scars and distinct CRISPR-Cas systems relative to M. tuberculosis. Despite the differences, all tuberculosis-causing mycobacteria share a highly conserved core genome. Mouse-infection experiments revealed that STB are less persistent and virulent than M. tuberculosis. We conclude that M. tuberculosis emerged from an ancestral, STB-like pool of mycobacteria by gain of persistence and virulence mechanisms and we provide genome-wide insights into the molecular events involved. PMID:23291586

Are moxifloxacin and levofloxacin equally effective to treat XDR tuberculosis?

PubMed

Maitre, Thomas; Petitjean, Grégoire; Chauffour, Aurélie; Bernard, Christine; El Helali, Najoua; Jarlier, Vincent; Reibel, Florence; Chavanet, Pascal; Aubry, Alexandra; Veziris, Nicolas

2017-08-01

Moxifloxacin retains partial activity against some fluoroquinolone-resistant mutants of Mycobacterium tuberculosis . Levofloxacin is presumed to be as active as moxifloxacin against drug-susceptible tuberculosis and to have a better safety profile. To compare the in vivo activity of levofloxacin and moxifloxacin against M. tuberculosis strains with various levels of fluoroquinolone resistance. BALB/c mice were intravenously infected with 10 6 M. tuberculosis H37Rv and three isogenic mutants: GyrA A90V, GyrB E540A and GyrB A543V. Treatment with 50 or 100 mg/kg levofloxacin and 60 or 66 mg/kg moxifloxacin was given orally every 6 h, for 4 weeks. Levofloxacin 50 and 100 mg/kg q6h and moxifloxacin 60 and 66 mg/kg q6h generated AUCs in mice equivalent to those of levofloxacin 750 and 1000 mg/day and moxifloxacin 400 and 800 mg/day, respectively, in humans. Moxifloxacin 60 and 66 mg/kg q6h had bactericidal activity against strain H37Rv (MIC ≤ 0.25 mg/L) and mutants GyrB E540A and GyrB A543V (MIC = 0.5 mg/L). Against mutant GyrA A90V (MIC = 2 mg/L), moxifloxacin 60 mg/kg q6h did not prevent bacillary growth, whereas 66 mg/kg q6h had bacteriostatic activity. Levofloxacin 50 mg/kg q6h had bactericidal activity against H37Rv (MIC ≤ 0.25 mg/L) but not against the mutant strains. Levofloxacin 100 mg/kg q6h had bactericidal activity against H37Rv and mutants GyrB E540A (MIC = 0.5 mg/L) and GyrB A543V (MIC= 1 mg/L) but not against mutant GyrA A90V (MIC = 4 mg/L). All mutations reduced fluoroquinolone activity, even those classified as susceptible according to phenotypic tests. High-dose levofloxacin is less effective than high-dose moxifloxacin against both fluoroquinolone-resistant and -susceptible M. tuberculosis strains in mice. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Locked Nucleic Acid Probe-Based Real-Time PCR Assay for the Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis

PubMed Central

Sun, Chongyun; Li, Chao; Wang, Xiaochen; Liu, Haican; Zhang, Pingping; Zhao, Xiuqin; Wang, Xinrui; Jiang, Yi; Yang, Ruifu; Wan, Kanglin; Zhou, Lei

2015-01-01

Drug-resistant Mycobacterium tuberculosis can be rapidly diagnosed through nucleic acid amplification techniques by analyzing the variations in the associated gene sequences. In the present study, a locked nucleic acid (LNA) probe-based real-time PCR assay was developed to identify the mutations in the rpoB gene associated with rifampin (RFP) resistance in M. tuberculosis. Six LNA probes with the discrimination capability of one-base mismatch were designed to monitor the 23 most frequent rpoB mutations. The target mutations were identified using the probes in a “probe dropout” manner (quantification cycle = 0); thus, the proposed technique exhibited superiority in mutation detection. The LNA probe-based real-time PCR assay was developed in a two-tube format with three LNA probes and one internal amplification control probe in each tube. The assay showed excellent specificity to M. tuberculosis with or without RFP resistance by evaluating 12 strains of common non-tuberculosis mycobacteria. The limit of detection of M. tuberculosis was 10 genomic equivalents (GE)/reaction by further introducing a nested PCR method. In a blind validation of 154 clinical mycobacterium isolates, 142/142 (100%) were correctly detected through the assay. Of these isolates, 88/88 (100%) were determined as RFP susceptible and 52/54 (96.3%) were characterized as RFP resistant. Two unrecognized RFP-resistant strains were sequenced and were found to contain mutations outside the range of the 23 mutation targets. In conclusion, this study established a sensitive, accurate, and low-cost LNA probe-based assay suitable for a four-multiplexing real-time PCR instrument. The proposed method can be used to diagnose RFP-resistant tuberculosis in clinical laboratories. PMID:26599667

Global Phylogeny of Mycobacterium tuberculosis Based on Single Nucleotide Polymorphism (SNP) Analysis: Insights into Tuberculosis Evolution, Phylogenetic Accuracy of Other DNA Fingerprinting Systems, and Recommendations for a Minimal Standard SNP Set†

PubMed Central

Filliol, Ingrid; Motiwala, Alifiya S.; Cavatore, Magali; Qi, Weihong; Hazbón, Manzour Hernando; Bobadilla del Valle, Miriam; Fyfe, Janet; García-García, Lourdes; Rastogi, Nalin; Sola, Christophe; Zozio, Thierry; Guerrero, Marta Inírida; León, Clara Inés; Crabtree, Jonathan; Angiuoli, Sam; Eisenach, Kathleen D.; Durmaz, Riza; Joloba, Moses L.; Rendón, Adrian; Sifuentes-Osornio, José; Ponce de León, Alfredo; Cave, M. Donald; Fleischmann, Robert; Whittam, Thomas S.; Alland, David

2006-01-01

We analyzed a global collection of Mycobacterium tuberculosis strains using 212 single nucleotide polymorphism (SNP) markers. SNP nucleotide diversity was high (average across all SNPs, 0.19), and 96% of the SNP locus pairs were in complete linkage disequilibrium. Cluster analyses identified six deeply branching, phylogenetically distinct SNP cluster groups (SCGs) and five subgroups. The SCGs were strongly associated with the geographical origin of the M. tuberculosis samples and the birthplace of the human hosts. The most ancestral cluster (SCG-1) predominated in patients from the Indian subcontinent, while SCG-1 and another ancestral cluster (SCG-2) predominated in patients from East Asia, suggesting that M. tuberculosis first arose in the Indian subcontinent and spread worldwide through East Asia. Restricted SCG diversity and the prevalence of less ancestral SCGs in indigenous populations in Uganda and Mexico suggested a more recent introduction of M. tuberculosis into these regions. The East African Indian and Beijing spoligotypes were concordant with SCG-1 and SCG-2, respectively; X and Central Asian spoligotypes were also associated with one SCG or subgroup combination. Other clades had less consistent associations with SCGs. Mycobacterial interspersed repetitive unit (MIRU) analysis provided less robust phylogenetic information, and only 6 of the 12 MIRU microsatellite loci were highly differentiated between SCGs as measured by GST. Finally, an algorithm was devised to identify two minimal sets of either 45 or 6 SNPs that could be used in future investigations to enable global collaborations for studies on evolution, strain differentiation, and biological differences of M. tuberculosis. PMID:16385065

Towards earlier inclusion of Children in Tuberculosis (TB) drugs trials: Consensus statements from an Expert Panel

PubMed Central

Nachman, Sharon; Ahmed, Amina; Amanullah, Farhana; Becerra, Mercedes C; Botgros, Radu; Brigden, Grania; Browning, Renee; Gardiner, Elizabeth; Hafner, Richard; Hesseling, Anneke; How, Cleotilde; Jean-Philippe, Patrick; Lessem, Erica; Makhene, Mamodikoe; Mbelle, Nontombi; Marais, Ben; McIlleron, Helen; Mc Neeley, David F; Mendel, Carl; Murray, Stephen; Navarro, Eileen; Oramasionwu, Gloria E; Porcalla, Ariel R; Powell, Clydette; Powell, Mair; Rigaud, Mona; Rouzier, Vanessa; Samson, Pearl; Schaaf, H. Simon; Shah, Seema; Starke, Jeff; Swaminathan, Soumya; Wobudeya, Eric; Worrell, Carol

2015-01-01

Children represent a significant proportion of the global tuberculosis (TB) burden, and may be disproportionately more affected by its most severe clinical manifestations. Currently available treatments for pediatric drug-susceptible (DS) and drug-resistant (DR) TB, albeit generally effective, are hampered by high pill burden, long duration of treatment, coexistent toxicities, and an overall lack of suitable, child-friendly formulations. The complex and burdensome nature of administering the existing regimens to treat DS TB also contributes to the rise of DR TB strains. Despite the availability and use of these therapies for decades, a dearth of dosing evidence in children underscores the importance of sustained efforts for TB drug development to better meet the treatment needs of children with TB. Several new TB drugs and regimens with promising activity against both DS and DR TB strains have recently entered clinical development and are in various phases of clinical evaluation in adults or have received marketing authorization for adults. However, initiation of clinical trials to evaluate these drugs in children is often deferred, pending the availability of complete safety and efficacy data in adults or after drug approval. This document summarizes consensus statements from an international panel of childhood TB opinion leaders which support the initiation of evaluation of new TB drugs and regimens in children at earlier phases of the TB Drug development cycle. PMID:25957923

[Determination of in vitro synergy by a checkerboard method when 3 core antimicrobial agents of the retreatment new scheme combined against MDR-MTB and XDR-MTB].

PubMed

Zhang, L L; Yang, H; Xiao, H P; Lu, J M; Sha, W; Zhang, Q

2016-06-01

In order to detect the in vitro synergistic effect of 4 drugs-pasiniazid (PA), moxifloxacin, rifabutin and rifapentini on multidrug-resistant mycobacterium tuberculosis (MDR-MTB) and extensively drug-resistant mycobacterium tuberculosis(XDR-MTB), which were core drugs of"The program of retreatment research of tuberculosis". The checkerboard method was used to detect the minimum inhibitory concentration (MIC) of antituberculosis drug combination schemes (moxifloxacin-PA, moxifloxacin-PA-rifabutin and moxifloxacin-PA-rifapentini) to 40 strains of clinical drug resistant MTB(20 strains of MDR-MTB and 20 XDR-MTB) and the standard strain H37Rv, by calculating the fractional inhibitory concentration index of joint action in vitro to judge the combined effect, with fractional inhibitory concentration index(FICI)≤0.5 and FICI≤0.75 as the basis of 2 drugs and 3 drugs showing synergy. The FICI of moxifloxacin-PA scheme for DR-MTB was 0.125 to 1.000, only 5 strains with a FICI ≤0.5, showing synergistic effect. The FICI of moxifloxacin-Pa-rifabutin scheme with 20 strains of MDR-MTB ranged from 0.310 to 1.260, 10 strains with a FICI≤0.75, showing synergistic effect. The FICI of moxifloxacin-PA-rifabutin scheme with 20 strains of XDR-MTB ranged from 0.215 to 1.250, 11 strains with a FICI≤0.75, showing synergistic effect. The FICI of moxifloxacin-PA-rifapentini scheme with 20 strains of MDR-MTB ranged from 0.150 to 0.780, 19 strains with a FICI≤0.75, showing synergistic effect. The FICI of moxifloxacin-PA-rifapentini scheme with 20 strains of XDR-MTB ranged from 0.200 to 1.280, 16 strains with a FICI≤0.75, showing synergistic effect. The synergistic effect of moxifloxacin-PA scheme was poor, but showing better synergy when further combined with rifabutin or rifapentini. Rifabutin showed better effect than rifapentini, but the synergistic effect of moxifloxacin-PA-rifabutin combination scheme was poor than that of moxifloxacin-PA-rifapentini combination scheme.

Florida's tuberculosis epidemic. Public health response.

PubMed

Witte, J J; Bigler, W J

1994-03-01

Florida ranked fourth in the nation with 1,707 tuberculosis cases reported in 1992 for a rate of 12.7 per 100,000 population. Thirteen percent of these patients had AIDS. Recent cases in prisons, shelters, hospitals and schools have stimulated interest and media coverage. Resurgence of strains of multiple-drug resistant tuberculosis is a serious concern. The Florida Department of Health and Rehabilitative Services, in collaboration with allied agencies, has utilized several initiatives in response. The most significant, Tuberculosis Epidemic Containment Plan, details intervention strategies needed to eliminate TB in the state by the year 2010. Successful implementation depends upon local TB prevention and control coalitions that include private and public sector providers.

The use of mutant mycobacteria as new vaccines to prevent tuberculosis.

PubMed

Hernàndez Pando, R; Aguilar, L D; Infante, E; Cataldi, A; Bigi, F; Martin, C; Gicquel, B

2006-01-01

Given the variable protective efficacy generated by Mycobacterium bovis BCG (Bacillus Calmette-Guérin), there is a concerted effort worldwide to develop better vaccines that could be used to reduce the burden of tuberculosis. Rational attenuated mutants of Mycobacterium tuberculosis are vaccine candidates that offer some potential in this area. In this paper, we will discuss the molecular methods used to generate mutant mycobacteria, as well as the results obtained with some of these strains, in terms of attenuation, immunogenicity and level of protection, when compared with the conventional BCG vaccine in diverse animal models. Tuberculosis vaccine candidates based on safe and live mycobacterial mutants could be promising candidates.

Antituberculous effect of silver nanoparticles

NASA Astrophysics Data System (ADS)

Kreytsberg, G. N.; Gracheva, I. E.; Kibrik, B. S.; Golikov, I. V.

2011-04-01

The in vitro experiment, involving 1164 strains of the tuberculosis mycobacteria, exhibited a potentiating effect of silver nanoparticles on known antituberculous preparations in respect of overcoming drug-resistance of the causative agent. The in vitro experiment, based on the model of resistant tuberculosis, was performed on 65 white mice. An evident antituberculous effect of the nanocomposite on the basis of silver nanoparticles and isoniazid was proved. Toxicological assessment of the of nanopreparations was carried out. The performed research scientifically establishes efficacy and safety of the nanocomposite application in combination therapy of patients suffering from drug-resistant tuberculosis.

A New Screen for Tuberculosis Drug Candidates Utilizing a Luciferase-Expressing Recombinant Mycobacterium bovis Bacillus Calmette-Guéren.

PubMed

Ozeki, Yuriko; Igarashi, Masayuki; Doe, Matsumi; Tamaru, Aki; Kinoshita, Naoko; Ogura, Yoshitoshi; Iwamoto, Tomotada; Sawa, Ryuichi; Umekita, Maya; Enany, Shymaa; Nishiuchi, Yukiko; Osada-Oka, Mayuko; Hayashi, Tetsuya; Niki, Mamiko; Tateishi, Yoshitaka; Hatano, Masaki; Matsumoto, Sohkichi

2015-01-01

Tuberculosis (TB) is a serious infectious disease caused by a bacterial pathogen. Mortality from tuberculosis was estimated at 1.5 million deaths worldwide in 2013. Development of new TB drugs is needed to not only to shorten the medication period but also to treat multi-drug resistant and extensively drug-resistant TB. Mycobacterium tuberculosis (Mtb) grows slowly and only multiplies once or twice per day. Therefore, conventional drug screening takes more than 3 weeks. Additionally, a biosafety level-3 (BSL-3) facility is required. Thus, we developed a new screening method to identify TB drug candidates by utilizing luciferase-expressing recombinant Mycobacterium bovis bacillus Calmette-Guéren (rBCG). Using this method, we identified several candidates in 4 days in a non-BSL-3 facility. We screened 10,080 individual crude extracts derived from Actinomyces and Streptomyces and identified 137 extracts which possessed suppressive activity to the luciferase of rBCG. Among them, 41 compounds inhibited the growth of both Mtb H37Rv and the extensively drug-resistant Mtb (XDR-Mtb) strains. We purified the active substance of the 1904-1 extract, which possessed strong activity toward rBCG, Mtb H37Rv, and XDR-Mtb but was harmless to the host eukaryotic cells. The MIC of this substance was 0.13 μg/ml, 0.5 μg/ml, and 2.0-7.5 μg/ml against rBCG, H37Rv, and 2 XDR-strains, respectively. Its efficacy was specific to acid-fast bacterium except for the Mycobacterium avium intracellular complex. Mass spectrometry and nuclear magnetic resonance analyses revealed that the active substance of 1904-1 was cyclomarin A. To confirm the mode of action of the 1904-1-derived compound, resistant BCG clones were used. Whole genome DNA sequence analysis showed that these clones contained a mutation in the clpc gene which encodes caseinolytic protein, an essential component of an ATP-dependent proteinase, and the likely target of the active substance of 1904-1. Our method provides a rapid and convenient screen to identify an anti-mycobacterial drug.

Hit Generation in TB Drug Discovery: From Genome to Granuloma

PubMed Central

2018-01-01

Current tuberculosis (TB) drug development efforts are not sufficient to end the global TB epidemic. Recent efforts have focused on the development of whole-cell screening assays because biochemical, target-based inhibitor screens during the last two decades have not delivered new TB drugs. Mycobacterium tuberculosis (Mtb), the causative agent of TB, encounters diverse microenvironments and can be found in a variety of metabolic states in the human host. Due to the complexity and heterogeneity of Mtb infection, no single model can fully recapitulate the in vivo conditions in which Mtb is found in TB patients, and there is no single “standard” screening condition to generate hit compounds for TB drug development. However, current screening assays have become more sophisticated as researchers attempt to mirror the complexity of TB disease in the laboratory. In this review, we describe efforts using surrogates and engineered strains of Mtb to focus screens on specific targets. We explain model culture systems ranging from carbon starvation to hypoxia, and combinations thereof, designed to represent the microenvironment which Mtb encounters in the human body. We outline ongoing efforts to model Mtb infection in the lung granuloma. We assess these different models, their ability to generate hit compounds, and needs for further TB drug development, to provide direction for future TB drug discovery. PMID:29384369

Induction of β-defensins by l-isoleucine as novel immunotherapy in experimental murine tuberculosis.

PubMed

Rivas-Santiago, C E; Rivas-Santiago, B; León, D A; Castañeda-Delgado, J; Hernández Pando, R

2011-04-01

Tuberculosis is a worldwide health problem, and multidrug-resistant (MDR) and extensively multidrug-resistant (XMDR) strains are rapidly emerging and threatening the control of this disease. These problems motivate the search for new treatment strategies. One potential strategy is immunotherapy using cationic anti-microbial peptides. The capacity of l-isoleucine to induce beta-defensin expression and its potential therapeutic efficiency were studied in a mouse model of progressive pulmonary tuberculosis. BALB/c mice were infected with Mycobacterium tuberculosis strain H37Rv or with a MDR clinical isolate by the intratracheal route. After 60 days of infection, when disease was in its progressive phase, mice were treated with 250 µg of intratracheal l-isoleucine every 48 h. Bacillary loads were determined by colony-forming units, protein and cytokine gene expression were determined by immunohistochemistry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR), respectively, and tissue damage was quantified by automated morphometry. Administration of l-isoleucine induced a significant increase of beta-defensins 3 and 4 which was associated with decreased bacillary loads and tissue damage. This was seen in animals infected with the antibiotic-sensitive strain H37Rv and with the MDR clinical isolate. Thus, induction of beta-defensins might be a potential therapy that can aid in the control of this significant infectious disease. © 2011 The Authors. Clinical and Experimental Immunology © 2011 British Society for Immunology.

Genetic markers, genotyping methods & next generation sequencing in Mycobacterium tuberculosis

PubMed Central

Desikan, Srinidhi; Narayanan, Sujatha

2015-01-01

Molecular epidemiology (ME) is one of the main areas in tuberculosis research which is widely used to study the transmission epidemics and outbreaks of tubercle bacilli. It exploits the presence of various polymorphisms in the genome of the bacteria that can be widely used as genetic markers. Many DNA typing methods apply these genetic markers to differentiate various strains and to study the evolutionary relationships between them. The three widely used genotyping tools to differentiate Mycobacterium tuberculosis strains are IS6110 restriction fragment length polymorphism (RFLP), spacer oligotyping (Spoligotyping), and mycobacterial interspersed repeat units - variable number of tandem repeats (MIRU-VNTR). A new prospect towards ME was introduced with the development of whole genome sequencing (WGS) and the next generation sequencing (NGS) methods, where the entire genome is sequenced that not only helps in pointing out minute differences between the various sequences but also saves time and the cost. NGS is also found to be useful in identifying single nucleotide polymorphisms (SNPs), comparative genomics and also various aspects about transmission dynamics. These techniques enable the identification of mycobacterial strains and also facilitate the study of their phylogenetic and evolutionary traits. PMID:26205019

Antimycobacterial, anti-inflammatory and genotoxicity evaluation of plants used for the treatment of tuberculosis and related symptoms in South Africa.

PubMed

Madikizela, B; Ndhlala, A R; Finnie, J F; Van Staden, J

2014-04-28

Emergence of drug-resistant tuberculosis strains and long duration of treatment has established an urgent need to search for new effective agents. The great floral diversity of South Africa has potential for producing new bioactive compounds, therefore pharmacological screening of plant extracts within this region offers much potential. To assess the in vitro antimycobacterial, anti-inflammatory and genotoxicity activity of selected plants that are used for the treatment of TB and related symptoms in South Africa. Ground plant materials from 10 plants were extracted sequentially with four solvents (petroleum ether, dichloromethane, 80% ethanol and water) and a total of 68 extracts were produced. A broth microdilution method was used to screen extracts against Mycobacterium tuberculosis H37Ra. The cyclooxygenase-2 (COX-2) enzyme was used to evaluate the anti-inflammatory activity of the extracts and the Salmonella microsome assay using two Salmonella typhimurium strains (TA98 and TA100) to establish genotoxicity. Six out of 68 extracts showed good antimycobacterial activity. Three extracts showed good inhibition (>70%) of COX-2 enzyme. All the extracts tested were non-genotoxic against the tested Salmonella strains. The results observed in this study indicate that some of the plants such as Abrus precatorius subsp. africanus, Ficus sur, Pentanisia prunelloides and Terminalia phanerophlebia could be investigated further against drug-resistant TB strains. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.