Autism-like behavioral phenotypes in BTBR T+tf/J mice.

PubMed

McFarlane, H G; Kusek, G K; Yang, M; Phoenix, J L; Bolivar, V J; Crawley, J N

2008-03-01

Autism is a behaviorally defined neurodevelopmental disorder of unknown etiology. Mouse models with face validity to the core symptoms offer an experimental approach to test hypotheses about the causes of autism and translational tools to evaluate potential treatments. We discovered that the inbred mouse strain BTBR T+tf/J (BTBR) incorporates multiple behavioral phenotypes relevant to all three diagnostic symptoms of autism. BTBR displayed selectively reduced social approach, low reciprocal social interactions and impaired juvenile play, as compared with C57BL/6J (B6) controls. Impaired social transmission of food preference in BTBR suggests communication deficits. Repetitive behaviors appeared as high levels of self-grooming by juvenile and adult BTBR mice. Comprehensive analyses of procedural abilities confirmed that social recognition and olfactory abilities were normal in BTBR, with no evidence for high anxiety-like traits or motor impairments, supporting an interpretation of highly specific social deficits. Database comparisons between BTBR and B6 on 124 putative autism candidate genes showed several interesting single nucleotide polymorphisms (SNPs) in the BTBR genetic background, including a nonsynonymous coding region polymorphism in Kmo. The Kmo gene encodes kynurenine 3-hydroxylase, an enzyme-regulating metabolism of kynurenic acid, a glutamate antagonist with neuroprotective actions. Sequencing confirmed this coding SNP in Kmo, supporting further investigation into the contribution of this polymorphism to autism-like behavioral phenotypes. Robust and selective social deficits, repetitive self-grooming, genetic stability and commercial availability of the BTBR inbred strain encourage its use as a research tool to search for background genes relevant to the etiology of autism, and to explore therapeutics to treat the core symptoms.

Method: a single nucleotide polymorphism genotyping method for Wheat streak mosaic virus.

PubMed

Rogers, Stephanie M; Payton, Mark; Allen, Robert W; Melcher, Ulrich; Carver, Jesse; Fletcher, Jacqueline

2012-05-17

The September 11, 2001 attacks on the World Trade Center and the Pentagon increased the concern about the potential for terrorist attacks on many vulnerable sectors of the US, including agriculture. The concentrated nature of crops, easily obtainable biological agents, and highly detrimental impacts make agroterrorism a potential threat. Although procedures for an effective criminal investigation and attribution following such an attack are available, important enhancements are still needed, one of which is the capability for fine discrimination among pathogen strains. The purpose of this study was to develop a molecular typing assay for use in a forensic investigation, using Wheat streak mosaic virus (WSMV) as a model plant virus. This genotyping technique utilizes single base primer extension to generate a genetic fingerprint. Fifteen single nucleotide polymorphisms (SNPs) within the coat protein and helper component-protease genes were selected as the genetic markers for this assay. Assay optimization and sensitivity testing was conducted using synthetic targets. WSMV strains and field isolates were collected from regions around the world and used to evaluate the assay for discrimination. The assay specificity was tested against a panel of near-neighbors consisting of genetic and environmental near-neighbors. Each WSMV strain or field isolate tested produced a unique SNP fingerprint, with the exception of three isolates collected within the same geographic location that produced indistinguishable fingerprints. The results were consistent among replicates, demonstrating the reproducibility of the assay. No SNP fingerprints were generated from organisms included in the near-neighbor panel, suggesting the assay is specific for WSMV. Using synthetic targets, a complete profile could be generated from as low as 7.15 fmoles of cDNA. The molecular typing method presented is one tool that could be incorporated into the forensic science tool box after a thorough validation study. This method incorporates molecular biology techniques that are already well established in research and diagnostic laboratories, allowing for an easy introduction of this method into existing laboratories. single nucleotide polymorphisms, genotyping, plant pathology, viruses, microbial forensics, Single base primer extension, SNaPshot Multiplex Kit.

Method: a single nucleotide polymorphism genotyping method for Wheat streak mosaic virus

PubMed Central

2012-01-01

Background The September 11, 2001 attacks on the World Trade Center and the Pentagon increased the concern about the potential for terrorist attacks on many vulnerable sectors of the US, including agriculture. The concentrated nature of crops, easily obtainable biological agents, and highly detrimental impacts make agroterrorism a potential threat. Although procedures for an effective criminal investigation and attribution following such an attack are available, important enhancements are still needed, one of which is the capability for fine discrimination among pathogen strains. The purpose of this study was to develop a molecular typing assay for use in a forensic investigation, using Wheat streak mosaic virus (WSMV) as a model plant virus. Method This genotyping technique utilizes single base primer extension to generate a genetic fingerprint. Fifteen single nucleotide polymorphisms (SNPs) within the coat protein and helper component-protease genes were selected as the genetic markers for this assay. Assay optimization and sensitivity testing was conducted using synthetic targets. WSMV strains and field isolates were collected from regions around the world and used to evaluate the assay for discrimination. The assay specificity was tested against a panel of near-neighbors consisting of genetic and environmental near-neighbors. Result Each WSMV strain or field isolate tested produced a unique SNP fingerprint, with the exception of three isolates collected within the same geographic location that produced indistinguishable fingerprints. The results were consistent among replicates, demonstrating the reproducibility of the assay. No SNP fingerprints were generated from organisms included in the near-neighbor panel, suggesting the assay is specific for WSMV. Using synthetic targets, a complete profile could be generated from as low as 7.15 fmoles of cDNA. Conclusion The molecular typing method presented is one tool that could be incorporated into the forensic science tool box after a thorough validation study. This method incorporates molecular biology techniques that are already well established in research and diagnostic laboratories, allowing for an easy introduction of this method into existing laboratories. Keywords: single nucleotide polymorphisms, genotyping, plant pathology, viruses, microbial forensics, Single base primer extension, SNaPshot Multiplex Kit PMID:22594601

Identification and characterization of a RAPD-PCR marker for distinguishing Asian and North American gypsy moths

Treesearch

K.J. Garner; J.M. Slavicek

1996-01-01

The recent introduction of the Asian gypsy moth (Lymantria dispar L.) into North America has necessitated the development of genetic markers to distinguish Asian moths from the established North American population, which originated in Europe. We used RAPD-PCR to identify a DNA length polymorphism that is diagnostic for the two moth strains. The...

Use of PCR-Based Methods for Rapid Differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis

PubMed Central

Torriani, Sandra; Zapparoli, Giacomo; Dellaglio, Franco

1999-01-01

Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy products were used. A numerical analysis of the RAPD-PCR patterns obtained with primer M13 gave results that were consistent with the results of specific PCR for all strains except L. delbrueckii subsp. delbrueckii LMG 6412T, which clustered with L. delbrueckii subsp. lactis strains. In addition, RAPD-PCR performed with primer 1254 provided highly polymorphic profiles and thus was superior for distinguishing individual L. delbrueckii strains. PMID:10508059

Use of PCR-based methods for rapid differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis.

PubMed

Torriani, S; Zapparoli, G; Dellaglio, F

1999-10-01

Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy products were used. A numerical analysis of the RAPD-PCR patterns obtained with primer M13 gave results that were consistent with the results of specific PCR for all strains except L. delbrueckii subsp. delbrueckii LMG 6412(T), which clustered with L. delbrueckii subsp. lactis strains. In addition, RAPD-PCR performed with primer 1254 provided highly polymorphic profiles and thus was superior for distinguishing individual L. delbrueckii strains.

Distinct prion-like strains of amyloid beta implicated in phenotypic diversity of Alzheimer's disease.

PubMed

Cohen, Mark; Appleby, Brian; Safar, Jiri G

2016-01-01

Vast evidence on human prions demonstrates that variable disease phenotypes, rates of propagation, and targeting of distinct brain structures are determined by unique conformers (strains) of pathogenic prion protein (PrP(Sc)). Recent progress in the development of advanced biophysical tools that inventory structural characteristics of amyloid beta (Aβ) in the brain cortex of phenotypically diverse Alzheimer's disease (AD) patients, revealed unique spectrum of oligomeric particles in the cortex of rapidly progressive cases, implicating these structures in variable rates of propagation in the brain, and in distict disease manifestation. Since only ∼30% of phenotypic diversity of AD can be explained by polymorphisms in risk genes, these and transgenic bioassay data argue that structurally distinct Aβ particles play a major role in the diverse pathogenesis of AD, and may behave as distinct prion-like strains encoding diverse phenotypes. From these observations and our growing understanding of prions, there is a critical need for new strain-specific diagnostic strategies for misfolded proteins causing these elusive disorders. Since targeted drug therapy can induce mutation and evolution of prions into new strains, effective treatments of AD will require drugs that enhance clearance of pathogenic conformers, reduce the precursor protein, or inhibit the conversion of precursors into prion-like states.

Characterization of Erwinia chrysanthemi by pectinolytic isozyme polymorphism and restriction fragment length polymorphism analysis of PCR-amplified fragments of pel genes.

PubMed Central

Nassar, A; Darrasse, A; Lemattre, M; Kotoujansky, A; Dervin, C; Vedel, R; Bertheau, Y

1996-01-01

Conserved regions about 420 bp long of the pelADE cluster specific to Erwinia chrysanthemi were amplified by PCR and used to differentiate 78 strains of E. chrysanthemi that were obtained from different hosts and geographical areas. No PCR products were obtained from DNA samples extracted from other pectinolytic and nonpectinolytic species and genera. The pel fragments amplified from the E. chrysanthemi strains studied were compared by performing a restriction fragment length polymorphism (RFLP) analysis. On the basis of similarity coefficients derived from the RFLP analysis, the strains were separated into 16 PCR RFLP patterns grouped in six clusters, These clusters appeared to be correlated with other infraspecific levels of E. chrysanthemi classification, such as pathovar and biovar, and occasionally with geographical origin. Moreover, the clusters correlated well with the polymorphism of pectate lyase and pectin methylesterase isoenzymes. While the pectin methylesterase profiles correlated with host monocot-dicot classification, the pectate lyase polymorphism might reflect the cell wall microdomains of the plants belonging to these classes. PMID:8779560

Outbreak Investigation Using High-Throughput Genome Sequencing within a Diagnostic Microbiology Laboratory

PubMed Central

Sherry, Norelle L.; Porter, Jessica L.; Seemann, Torsten; Watkins, Andrew; Stinear, Timothy P.

2013-01-01

Next-generation sequencing (NGS) of bacterial genomes has recently become more accessible and is now available to the routine diagnostic microbiology laboratory. However, questions remain regarding its feasibility, particularly with respect to data analysis in nonspecialist centers. To test the applicability of NGS to outbreak investigations, Ion Torrent sequencing was used to investigate a putative multidrug-resistant Escherichia coli outbreak in the neonatal unit of the Mercy Hospital for Women, Melbourne, Australia. Four suspected outbreak strains and a comparator strain were sequenced. Genome-wide single nucleotide polymorphism (SNP) analysis demonstrated that the four neonatal intensive care unit (NICU) strains were identical and easily differentiated from the comparator strain. Genome sequence data also determined that the NICU strains belonged to multilocus sequence type 131 and carried the blaCTX-M-15 extended-spectrum beta-lactamase. Comparison of the outbreak strains to all publicly available complete E. coli genome sequences showed that they clustered with neonatal meningitis and uropathogenic isolates. The turnaround time from a positive culture to the completion of sequencing (prior to data analysis) was 5 days, and the cost was approximately $300 per strain (for the reagents only). The main obstacles to a mainstream adoption of NGS technologies in diagnostic microbiology laboratories are currently cost (although this is decreasing), a paucity of user-friendly and clinically focused bioinformatics platforms, and a lack of genomics expertise outside the research environment. Despite these hurdles, NGS technologies provide unparalleled high-resolution genotyping in a short time frame and are likely to be widely implemented in the field of diagnostic microbiology in the next few years, particularly for epidemiological investigations (replacing current typing methods) and the characterization of resistance determinants. Clinical microbiologists need to familiarize themselves with these technologies and their applications. PMID:23408689

Polymorphism of Paramecium pentaurelia (Ciliophora, Oligohymenophorea) strains revealed by rDNA and mtDNA sequences.

PubMed

Przyboś, Ewa; Tarcz, Sebastian; Greczek-Stachura, Magdalena; Surmacz, Marta

2011-05-01

Paramecium pentaurelia is one of 15 known sibling species of the Paramecium aurelia complex. It is recognized as a species showing no intra-specific differentiation on the basis of molecular fingerprint analyses, whereas the majority of other species are polymorphic. This study aimed at assessing genetic polymorphism within P. pentaurelia including new strains recently found in Poland (originating from two water bodies, different years, seasons, and clones of one strain) as well as strains collected from distant habitats (USA, Europe, Asia), and strains representing other species of the complex. We compared two DNA fragments: partial sequences (349 bp) of the LSU rDNA and partial sequences (618 bp) of cytochrome B gene. A correlation between the geographical origin of the strains and the genetic characteristics of their genotypes was not observed. Different genotypes were found in Kraków in two types of water bodies (Opatkowice-natural pond; Jordan's Park-artificial pond). Haplotype diversity within a single water body was not recorded. Likewise, seasonal haplotype differences between the strains within the artificial water body, as well as differences between clones originating from one strain, were not detected. The clustering of some strains belonging to different species was observed in the phylogenies. Copyright © 2010 Elsevier GmbH. All rights reserved.

Bacillus "next generation" diagnostics: moving from detection toward subtyping and risk-related strain profiling.

PubMed

Ehling-Schulz, Monika; Messelhäusser, Ute

2013-01-01

The highly heterogeneous genus Bacillus comprises the largest species group of endospore forming bacteria. Because of their ubiquitous nature, Bacillus spores can enter food production at several stages resulting in significant economic losses and posing a potential risk to consumers due the capacity of certain Bacillus strains for toxin production. In the past, food microbiological diagnostics was focused on the determination of species using conventional culture-based methods, which are still widely used. However, due to the extreme intra-species diversity found in the genus Bacillus, DNA-based identification and typing methods are gaining increasing importance in routine diagnostics. Several studies showed that certain characteristics are rather strain-dependent than species-specific. Therefore, the challenge for current and future Bacillus diagnostics is not only the efficient and accurate identification on species level but also the development of rapid methods to identify strains with specific characteristics (such as stress resistance or spoilage potential), trace contamination sources, and last but not least discriminate potential hazardous strains from non-toxic strains.

The reporting of a Bacillus anthracis B-clade strain in South Africa after more than 20 years.

PubMed

Lekota, K E; Hassim, A; Rogers, P; Dekker, E H; Last, R; de Klerk-Lorist, L; van Heerden, H

2018-05-02

Anthrax is a disease with an age old history in Africa caused by the Gram-positive endospore forming soil bacterium Bacillus anthracis. Epizootics of wild ungulates occur annually in the enzootic region of Pafuri, Kruger National Park (KNP) in the Limpopo Province of South Africa. Rigorous routine surveillance and diagnostics in KNP, has not revealed these rare isolates since the 1990s, despite unabated annual outbreaks. In 2011 a cheetah was diagnosed as anthrax positive from a private game reserve in Limpopo Province and reported to State Veterinary Services for further investigation. Isolation, molecular diagnostics, whole genome sequencing and comparative genomics were carried out for B. anthracis KC2011. Bacteriological and molecular diagnostics confirmed the isolate as B. anthracis. Subsequent typing and whole genome single nucleotide polymorphisms analysis indicated it clustered alongside B. anthracis SA A0091 in the B.Br.010 SNP branch. Unlike B. anthracis KrugerB strain, KC2011 strain has unique SNPs and represents a new branch in the B-clade. The isolation and genotypic characterisation of KC2011 demonstrates a gap in the reporting of anthrax outbreaks in the greater Limpopo province area. The identification of vulnerable and susceptible cheetah mortalities due to this strain has implications for conservation measures and disease control.

Strains of Sarcocystis neurona exhibit differences in their surface antigens, including the absence of the major surface antigen SnSAG1.

PubMed

Howe, Daniel K; Gaji, Rajshekhar Y; Marsh, Antoinette E; Patil, Bhagyashree A; Saville, William J; Lindsay, David S; Dubey, J P; Granstrom, David E

2008-05-01

A gene family of surface antigens is expressed by merozoites of Sarcocystis neurona, the primary cause of equine protozoal myeloencephalitis (EPM). These surface proteins, designated SnSAGs, are immunodominant and therefore excellent candidates for development of EPM diagnostics or vaccines. Prior work had identified an EPM isolate lacking the major surface antigen SnSAG1, thus suggesting there may be some diversity in the SnSAGs expressed by different S. neurona isolates. Therefore, a bioinformatic, molecular and immunological study was conducted to assess conservation of the SnSAGs. Examination of an expressed sequence tag (EST) database revealed several notable SnSAG polymorphisms. In particular, the EST information implied that the EPM strain SN4 lacked the major surface antigen SnSAG1. The absence of this surface antigen from the SN4 strain was confirmed by both Western blot and Southern blot. To evaluate SnSAG polymorphisms in the S. neurona population, 14 strains were examined by Western blots using monospecific polyclonal antibodies against the four described SnSAGs. The results of these analyses demonstrated that SnSAG2, SnSAG3, and SnSAG4 are present in all 14 S. neurona strains tested, although some variance in SnSAG4 was observed. Importantly, SnSAG1 was not detected in seven of the strains, which included isolates from four cases of EPM and a case of fatal meningoencephalitis in a sea otter. Genetic analyses by PCR using gene-specific primers confirmed the absence of the SnSAG1 locus in six of these seven strains. Collectively, the data indicated that there is heterogeneity in the surface antigen composition of different S. neurona isolates, which is an important consideration for development of serological tests and prospective vaccines for EPM. Furthermore, the diversity reported herein likely extends to other phenotypes, such as strain virulence, and may have implications for the phylogeny of the various Sarcocystis spp. that undergo sexual stages of their life cycle in opossums.

Molecular diagnostic development for begomovirus-betasatellite complexes undergoing diversification: A case study.

PubMed

Brown, Judith K; Ur-Rehman, Muhammad Zia; Avelar, Sofia; Chingandu, N; Hameed, Usman; Haider, Saleem; Ilyas, Muhammad

2017-09-15

At least five begomoviral species that cause leaf curl disease of cotton have emerged recently in Asia and Africa, reducing fiber quality and yield. The potential for the spread of these viruses to other cotton-vegetable growing regions throughout the world is extensive, owing to routine, global transport of alternative hosts of the leaf curl viruses, especially ornamentals. The research reported here describes the design and validation of polymerase chain reaction (PCR) primers undertaken to facilitate molecular detection of the two most-prevalent leaf curl-associated begomovirus-betasatellite complexes in the Indian Subcontinent and Africa, the Cotton leaf curl Kokhran virus-Burewala strain and Cotton leaf curl Gezira virus, endemic to Asia and Africa, respectively. Ongoing genomic diversification of these begomoviral-satellite complexes was evident based on nucleotide sequence alignments, and analysis of single nucleotide polymorphisms, both factors that created new challenges for primer design. The additional requirement for species and strain-specific, and betasatellite-specific primer design, imposes further constraints on primer design and validation due to the large number of related species and strains extant in 'core leaf curl virus complex', now with expanded distribution in south Asia, the Pacific region, and Africa-Arabian Peninsula that have relatively highly conserved coding and non-coding regions, which precludes much of the genome-betasatellite sequence when selecting primer 'targets'. Here, PCR primers were successfully designed and validated for detection of cloned viral genomes and betasatellites for representative 'core leaf curl' strains and species, distant relatives, and total DNA isolated from selected plant species. The application of molecular diagnostics to screen plant imports prior to export or release from ports of entry is expected to greatly reduce the likelihood of exotic leaf curl virus introductions that could dramatically affect the production of cotton as well as vegetable and ornamental crop hosts. Copyright © 2017 Elsevier B.V. All rights reserved.

MULTIPLE ENZYME RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS FOR HIGH RESOLUTION DISTINCTION OF PSEUDOMONAS (SENSU STRICTO) 16S RRNA GENES

EPA Science Inventory

Pseudomonas specific 16S rDNA PCR amplification and multiple enzyme restriction fragment length polymorphism (MERFLP) analysis using a single digestion mixture of Alu I, Hinf I, Rsa I, and Tru 9I distinguished 150 published sequences and reference strains of authentic Pseudomonas...

Genomic survey of pathogenicity determinants and VNTR markers in the cassava bacterial pathogen Xanthomonas axonopodis pv. Manihotis strain CIO151.

PubMed

Arrieta-Ortiz, Mario L; Rodríguez-R, Luis M; Pérez-Quintero, Álvaro L; Poulin, Lucie; Díaz, Ana C; Arias Rojas, Nathalia; Trujillo, Cesar; Restrepo Benavides, Mariana; Bart, Rebecca; Boch, Jens; Boureau, Tristan; Darrasse, Armelle; David, Perrine; Dugé de Bernonville, Thomas; Fontanilla, Paula; Gagnevin, Lionel; Guérin, Fabien; Jacques, Marie-Agnès; Lauber, Emmanuelle; Lefeuvre, Pierre; Medina, Cesar; Medina, Edgar; Montenegro, Nathaly; Muñoz Bodnar, Alejandra; Noël, Laurent D; Ortiz Quiñones, Juan F; Osorio, Daniela; Pardo, Carolina; Patil, Prabhu B; Poussier, Stéphane; Pruvost, Olivier; Robène-Soustrade, Isabelle; Ryan, Robert P; Tabima, Javier; Urrego Morales, Oscar G; Vernière, Christian; Carrere, Sébastien; Verdier, Valérie; Szurek, Boris; Restrepo, Silvia; López, Camilo; Koebnik, Ralf; Bernal, Adriana

2013-01-01

Xanthomonas axonopodis pv. manihotis (Xam) is the causal agent of bacterial blight of cassava, which is among the main components of human diet in Africa and South America. Current information about the molecular pathogenicity factors involved in the infection process of this organism is limited. Previous studies in other bacteria in this genus suggest that advanced draft genome sequences are valuable resources for molecular studies on their interaction with plants and could provide valuable tools for diagnostics and detection. Here we have generated the first manually annotated high-quality draft genome sequence of Xam strain CIO151. Its genomic structure is similar to that of other xanthomonads, especially Xanthomonas euvesicatoria and Xanthomonas citri pv. citri species. Several putative pathogenicity factors were identified, including type III effectors, cell wall-degrading enzymes and clusters encoding protein secretion systems. Specific characteristics in this genome include changes in the xanthomonadin cluster that could explain the lack of typical yellow color in all strains of this pathovar and the presence of 50 regions in the genome with atypical nucleotide composition. The genome sequence was used to predict and evaluate 22 variable number of tandem repeat (VNTR) loci that were subsequently demonstrated as polymorphic in representative Xam strains. Our results demonstrate that Xanthomonas axonopodis pv. manihotis strain CIO151 possesses ten clusters of pathogenicity factors conserved within the genus Xanthomonas. We report 126 genes that are potentially unique to Xam, as well as potential horizontal transfer events in the history of the genome. The relation of these regions with virulence and pathogenicity could explain several aspects of the biology of this pathogen, including its ability to colonize both vascular and non-vascular tissues of cassava plants. A set of 16 robust, polymorphic VNTR loci will be useful to develop a multi-locus VNTR analysis scheme for epidemiological surveillance of this disease.

Genomic Survey of Pathogenicity Determinants and VNTR Markers in the Cassava Bacterial Pathogen Xanthomonas axonopodis pv. Manihotis Strain CIO151

PubMed Central

Arrieta-Ortiz, Mario L.; Rodríguez-R, Luis M.; Pérez-Quintero, Álvaro L.; Poulin, Lucie; Díaz, Ana C.; Arias Rojas, Nathalia; Trujillo, Cesar; Restrepo Benavides, Mariana; Bart, Rebecca; Boch, Jens; Boureau, Tristan; Darrasse, Armelle; David, Perrine; Dugé de Bernonville, Thomas; Fontanilla, Paula; Gagnevin, Lionel; Guérin, Fabien; Jacques, Marie-Agnès; Lauber, Emmanuelle; Lefeuvre, Pierre; Medina, Cesar; Medina, Edgar; Montenegro, Nathaly; Muñoz Bodnar, Alejandra; Noël, Laurent D.; Ortiz Quiñones, Juan F.; Osorio, Daniela; Pardo, Carolina; Patil, Prabhu B.; Poussier, Stéphane; Pruvost, Olivier; Robène-Soustrade, Isabelle; Ryan, Robert P.; Tabima, Javier; Urrego Morales, Oscar G.; Vernière, Christian; Carrere, Sébastien; Verdier, Valérie; Szurek, Boris; Restrepo, Silvia; López, Camilo

2013-01-01

Xanthomonas axonopodis pv. manihotis (Xam) is the causal agent of bacterial blight of cassava, which is among the main components of human diet in Africa and South America. Current information about the molecular pathogenicity factors involved in the infection process of this organism is limited. Previous studies in other bacteria in this genus suggest that advanced draft genome sequences are valuable resources for molecular studies on their interaction with plants and could provide valuable tools for diagnostics and detection. Here we have generated the first manually annotated high-quality draft genome sequence of Xam strain CIO151. Its genomic structure is similar to that of other xanthomonads, especially Xanthomonas euvesicatoria and Xanthomonas citri pv. citri species. Several putative pathogenicity factors were identified, including type III effectors, cell wall-degrading enzymes and clusters encoding protein secretion systems. Specific characteristics in this genome include changes in the xanthomonadin cluster that could explain the lack of typical yellow color in all strains of this pathovar and the presence of 50 regions in the genome with atypical nucleotide composition. The genome sequence was used to predict and evaluate 22 variable number of tandem repeat (VNTR) loci that were subsequently demonstrated as polymorphic in representative Xam strains. Our results demonstrate that Xanthomonas axonopodis pv. manihotis strain CIO151 possesses ten clusters of pathogenicity factors conserved within the genus Xanthomonas. We report 126 genes that are potentially unique to Xam, as well as potential horizontal transfer events in the history of the genome. The relation of these regions with virulence and pathogenicity could explain several aspects of the biology of this pathogen, including its ability to colonize both vascular and non-vascular tissues of cassava plants. A set of 16 robust, polymorphic VNTR loci will be useful to develop a multi-locus VNTR analysis scheme for epidemiological surveillance of this disease. PMID:24278159

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.

Bacillus “next generation” diagnostics: moving from detection toward subtyping and risk-related strain profiling

PubMed Central

Ehling-Schulz, Monika; Messelhäusser, Ute

2013-01-01

The highly heterogeneous genus Bacillus comprises the largest species group of endospore forming bacteria. Because of their ubiquitous nature, Bacillus spores can enter food production at several stages resulting in significant economic losses and posing a potential risk to consumers due the capacity of certain Bacillus strains for toxin production. In the past, food microbiological diagnostics was focused on the determination of species using conventional culture-based methods, which are still widely used. However, due to the extreme intra-species diversity found in the genus Bacillus, DNA-based identification and typing methods are gaining increasing importance in routine diagnostics. Several studies showed that certain characteristics are rather strain-dependent than species-specific. Therefore, the challenge for current and future Bacillus diagnostics is not only the efficient and accurate identification on species level but also the development of rapid methods to identify strains with specific characteristics (such as stress resistance or spoilage potential), trace contamination sources, and last but not least discriminate potential hazardous strains from non-toxic strains. PMID:23440299

Identification of verotoxin type 2 variant B subunit genes in Escherichia coli by the polymerase chain reaction and restriction fragment length polymorphism analysis.

PubMed Central

Tyler, S D; Johnson, W M; Lior, H; Wang, G; Rozee, K R

1991-01-01

A set of synthetic oligonucleotide primers was designed for use in a polymerase chain reaction protocol to specifically detect the B subunit genes in vtx2ha and vtx2hb, which code for the production of the VT2 (Shiga-like toxin II) variant cytotoxins VT2v-a and VT2v-b, respectively. An additional set of primers amplified a fragment common to the B subunits of the VT2 and the VT2 variant genes. Subsequent restriction endonuclease digestion of this amplicon permitted prediction of specific VT2 and variant genotypes on the basis of predetermined restriction fragment length polymorphisms. Genotypes of 21 VT2-producing strains of Escherichia coli were determined using this polymerase chain reaction-restriction fragment length polymorphism procedure. Four strains contained B subunit target sequences only for VT2 genes, 9 strains contained sequences only for VT2v-a genes, and 3 strains contained sequences only for VT2v-b. For genes in combination, one strain contained B subunit genes for both VT2 and VT2v-a and two strains contained B subunit genes for VT2 and VT2v-b. Two strains of E. coli O91:H21 contained both VT2v-a and VT2v-b B subunit genes. The VT2 reference strain of E. coli, E32511, was found to contain the targeted sequences from both VT2 and VT2v-a genes, whereas the recombinant E. coli, pEB1, possessed only that of the VT2 gene. The specific activities of extracellular VT2 determined in HeLa cells ranged from 0.3 to 41.7 TCD50 per microgram of protein in strains carrying the VT2 gene target and from 0 to 50.0 TCD50 per microgram of protein in strains carrying only the VT2 variant target (TCD50 is the tissue culture dose by which 50% of the cells were affected), suggesting that phenotypic expression does not correlate with genotype. Images PMID:1679436

Systematics of gastrointestinal nematodes of domestic ruminants: advances between 1992 and 1995 and proposals for future research.

PubMed

Lichtenfels, J R; Hoberg, E P; Zarlenga, D S

1997-11-01

The systematics of trichostrongyloid nematodes of ruminants provides a foundation for diagnostics and responds to the need to identify eggs in feces, free-living larvae from pastures or fecal cultures and larval or adult nematodes collected from hosts. These needs are associated with diagnostic problems or research projects. Difficulties in identifying all developmental stages of trichostrongyloid nematodes of domestic ruminants still severely limit the effective diagnosis and control of these parasites. Phylogenetic hypotheses as the basis for predictive classifications have been developed only for the subfamilies of the Trichostrongylidae. This report briefly describes recent progress in the development of improved tools for identification, phylogenetic analyses and predictive classifications. It also describes future research needed on the identification and classification of trichostrongyloid nematode parasites of domestic ruminants. Nematodes included are species of the super-family Trichostrongyloidea known to be important pathogens of domestic ruminants. The information summarized is presented by nematode developmental stage and by taxonomic groups. Eggs: While eggs of some trichostrongyloid nematode parasites of ruminants can be readily identified to their genus (Nematodirus), and some to species (e.g. Nematodirus battus), most of the important pathogens (including the Ostertagiinae and Haemonchinae) cannot be identified morphologically or morphometrically even to family level. However, DNA technology has been developed for determining not only the presence of specific pathogens in eggs from fecal samples, but also for estimating the percentage of the total eggs that each pathogen comprises. This new method will make possible a rapid determination of which individual animals in a herd should be treated. Larvae: The most commonly-used method for identifying infective larvae is time-consuming (several weeks), unreliable for estimating intensities of individual species as components of mixed populations and requires highly trained specialists. Available identification keys for larvae are not well illustrated and need to be augmented. Adults: Recent advances in the identification of adult trichostrongyloids and their systematics are organized by taxonomic group. Genera included are Ostertagia, Haemonchus, Cooperia, Trichostrongylus and Nematodirus. Recently, the first phylogenetic analysis of the Trichostrongylidae family established monophyly for the family. A similar analysis of the Molineidae is needed. Ostertagia: Several studies of polymorphism summarized the phenomenon and listed 19 polymorphic species in five genera. Two studies of DNA differences within and among polymorphic species of Ostertagiinae supported earlier hypotheses that the species pairs represent polymorphic species. A phylogenetic analysis of the Ostertagiinae and generic concepts are needed. Haemonchus: A key to three species of Haemonchus provides, for the first time, morphological characteristics for the microscopical identification to species of individual adult nematodes of either sex. The Food and Drug Administration is now requiring that results of drug trials include identification of Haemonchus to species. Cooperia: Studies using random amplified polymorphic DNA methods showed a high degree of variation within and among C. oncophora/C. surnabada, but supported a polymorphic relationship for the species pair. A phylogenetic analysis of the Cooperiinae is needed. Trichostrongylus: Restriction Fragment Length Polymorphisms (RFLPs) of genomic DNA of two strains of T. colubriformis indicated a high degree of intra- and inter-strain DNA polymorphism. However, other studies demonstrated expected species level differences between T. colubriformis and T. vitrinus using Random Amplified Polymorphic DNA (RAPD) methods. Sequences of the second Internal Transcribed Spacer Region (ITS-2) ribosomal repeat showed sequence differences of 1.3-7.6% among five

Phylogenetic reconstruction and polymorphism analysis of BK virus VP2 gene isolated from renal transplant recipients in China

PubMed Central

WANG, ZHANG-YANG; HONG, WEI-LONG; ZHU, ZHE-HUI; CHEN, YUN-HAO; YE, WEN-LE; CHU, GUANG-YU; LI, JIA-LIN; CHEN, BI-CHENG; XIA, PENG

2015-01-01

BK polyomavirus (BKV) is important pathogen for kidney transplant recipients, as it is frequently re-activated, leading to nephropathy. The aim of this study was to investigate the phylogenetic reconstruction and polymorphism of the VP2 gene in BKV isolated from Chinese kidney transplant recipients. Phylogenetic analysis was carried out in the VP2 region from 135 BKV-positive samples and 28 reference strains retrieved from GenBank. The unweighted pair-group method with arithmetic mean (UPGMA) grouped all strains into subtypes, but failed to subdivide strains into subgroups. Among the plasma and urine samples, all plasma (23/23) and 82 urine samples (82/95) were identified to contain subtype I; the other 10 urine samples contained subtype IV. A 86-bp fragment was identified as a highly conserved sequence. Following alignment with 36 published BKV sequences from China, 92 sites of polymorphism were identified, including 11 single nucleotide polymorphisms (SNPs) prevalent in Chinese individuals and 30 SNPs that were specific to the two predominant subtypes I and IV. The limitations of the VP2 gene segment in subgrouping were confirmed by phylogenetic analysis. The conserved sequence and polymorphism identified in this study may be helpful in the detection and genotyping of BKV. PMID:26640547

Importance of whole genome sequencing for the assessment of outbreaks in diagnostic laboratories: analysis of a case series of invasive Streptococcus pyogenes infections.

PubMed

Tagini, F; Aubert, B; Troillet, N; Pillonel, T; Praz, G; Crisinel, P A; Prod'hom, G; Asner, S; Greub, G

2017-07-01

Outbreaks of Streptococcus pyogenes hypervirulent clones are constant public health threats. In western Switzerland, an increase of severe cases of S. pyogenes invasive infections was observed between December 2015 and March 2016. Our aim was (i) to investigate these cases by the use of Whole Genome Sequencing (WGS) and (ii) to determine the specific virulome and resistome of each isolate in order to undertake adequate public health measures. Eleven Streptococcus pyogenes strains isolated from 11 patients with severe invasive infections between December 13, 2015 and March 12, 2016 were included in our study. Practically, emm-typing, MLST and WGS were used to investigate the relatedness between the isolates. The presence of virulence and antibiotic resistance genes as well as mutations in transcriptional regulators of virulence and in genes encoding for antibiotic targets were assessed. Three and two groups of isolates shared the same emm-type and ST type, respectively. Single Nucleotide Polymorphism (SNP) analysis revealed 14 to 32 SNPs between the strains of the same emm-type group, ruling out the possibility of a clonal outbreak. Mutations found in covS and rocA could partially explain an increased virulence. As these reassuring results were obtained in less than 10 days, no specific hospital hygiene and no dedicated public health measures had to be undertaken. WGS is a powerful technique to discriminate between closely related strains, excluding an outbreak in less than 10 days. Moreover, WGS provided extensive data on the virulome and resistome of all these strains.

Single nucleotide polymorphism analysis using different colored dye dimer probes

NASA Astrophysics Data System (ADS)

Marmé, Nicole; Friedrich, Achim; Denapaite, Dalia; Hakenbeck, Regine; Knemeyer, Jens-Peter

2006-09-01

Fluorescence quenching by dye dimer formation has been utilized to develop hairpin-structured DNA probes for the detection of a single nucleotide polymorphism (SNP) in the penicillin target gene pbp2x, which is implicated in the penicillin resistance of Streptococcus pneumoniae. We designed two specific DNA probes for the identification of the pbp2x genes from a penicillin susceptible strain R6 and a resistant strain Streptococcus mitis 661 using green-fluorescent tetramethylrhodamine (TMR) and red-fluorescent DY-636, respectively. Hybridization of each of the probes to its respective target DNA sequence opened the DNA hairpin probes, consequently breaking the nonfluorescent dye dimers into fluorescent species. This hybridization of the target with the hairpin probe achieved single nucleotide specific detection at nanomolar concentrations via increased fluorescence.

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.

Molecular Characterization of Herpes Simplex Virus 2 Strains by Analysis of Microsatellite Polymorphism

PubMed Central

Ait-Arkoub, Zaïna; Voujon, Delphine; Deback, Claire; Abrao, Emiliana P.; Agut, Henri; Boutolleau, David

2013-01-01

The complete 154-kbp linear double-stranded genomic DNA sequence of herpes simplex virus 2 (HSV-2), consisting of two extended regions of unique sequences bounded by a pair of inverted repeat elements, was published in 1998 and since then has been widely employed in a wide range of studies. Throughout the HSV-2 genome are scattered 150 microsatellites (also referred to as short tandem repeats) of 1- to 6-nucleotide motifs, mainly distributed in noncoding regions. Microsatellites are considered reliable markers for genetic mapping to differentiate herpesvirus strains, as shown for cytomegalovirus and HSV-1. The aim of this work was to characterize 12 polymorphic microsatellites within the HSV-2 genome by use of 3 multiplex PCR assays in combination with length polymorphism analysis for the rapid genetic differentiation of 56 HSV-2 clinical isolates and 2 HSV-2 laboratory strains (gHSV-2 and MS). This new system was applied to a specific new HSV-2 variant recently identified in HIV-1-infected patients originating from West Africa. Our results confirm that microsatellite polymorphism analysis is an accurate tool for studying the epidemiology of HSV-2 infections. PMID:23966512

The Feasibility of Classifying Breast Masses Using a Computer-Assisted Diagnosis (CAD) System Based on Ultrasound Elastography and BI-RADS Lexicon.

PubMed

Fleury, Eduardo F C; Gianini, Ana Claudia; Marcomini, Karem; Oliveira, Vilmar

2018-01-01

To determine the applicability of a computer-aided diagnostic system strain elastography system for the classification of breast masses diagnosed by ultrasound and scored using the criteria proposed by the breast imaging and reporting data system ultrasound lexicon and to determine the diagnostic accuracy and interobserver variability. This prospective study was conducted between March 1, 2016, and May 30, 2016. A total of 83 breast masses subjected to percutaneous biopsy were included. Ultrasound elastography images before biopsy were interpreted by 3 radiologists with and without the aid of computer-aided diagnostic system for strain elastography. The parameters evaluated by each radiologist results were sensitivity, specificity, and diagnostic accuracy, with and without computer-aided diagnostic system for strain elastography. Interobserver variability was assessed using a weighted κ test and an intraclass correlation coefficient. The areas under the receiver operating characteristic curves were also calculated. The areas under the receiver operating characteristic curve were 0.835, 0.801, and 0.765 for readers 1, 2, and 3, respectively, without computer-aided diagnostic system for strain elastography, and 0.900, 0.926, and 0.868, respectively, with computer-aided diagnostic system for strain elastography. The intraclass correlation coefficient between the 3 readers was 0.6713 without computer-aided diagnostic system for strain elastography and 0.811 with computer-aided diagnostic system for strain elastography. The proposed computer-aided diagnostic system for strain elastography system has the potential to improve the diagnostic performance of radiologists in breast examination using ultrasound associated with elastography.

Differential diagnosis of the honey bee trypanosomatids Crithidia mellificae and Lotmaria passim.

PubMed

Ravoet, Jorgen; Schwarz, Ryan S; Descamps, Tine; Yañez, Orlando; Tozkar, Cansu Ozge; Martin-Hernandez, Raquel; Bartolomé, Carolina; De Smet, Lina; Higes, Mariano; Wenseleers, Tom; Schmid-Hempel, Regula; Neumann, Peter; Kadowaki, Tatsuhiko; Evans, Jay D; de Graaf, Dirk C

2015-09-01

Trypanosomatids infecting honey bees have been poorly studied with molecular methods until recently. After the description of Crithidia mellificae (Langridge and McGhee, 1967) it took about forty years until molecular data for honey bee trypanosomatids became available and were used to identify and describe a new trypanosomatid species from honey bees, Lotmaria passim (Evans and Schwarz, 2014). However, an easy method to distinguish them without sequencing is not yet available. Research on the related bumble bee parasites Crithidia bombi and Crithidia expoeki revealed a fragment length polymorphism in the internal transcribed spacer 1 (ITS1), which enabled species discrimination. In search of fragment length polymorphisms for differential diagnostics in honey bee trypanosomatids, we studied honey bee trypanosomatid cell cultures of C. mellificae and L. passim. This research resulted in the identification of fragment length polymorphisms in ITS1 and ITS1-2 markers, which enabled us to develop a diagnostic method to differentiate both honey bee trypanosomatid species without the need for sequencing. However, the amplification success of the ITS1 marker depends probably on the trypanosomatid infection level. Further investigation confirmed that L. passim is the dominant species in Belgium, Japan and Switzerland. We found C. mellificae only rarely in Belgian honey bee samples, but not in honey bee samples from other countries. C. mellificae was also detected in mason bees (Osmia bicornis and Osmia cornuta) besides in honey bees. Further, the characterization and comparison of additional markers from L. passim strain SF (published as C. mellificae strain SF) and a Belgian honey bee sample revealed very low divergence in the 18S rRNA, ITS1-2, 28S rRNA and cytochrome b sequences. Nevertheless, a variable stretch was observed in the gp63 virulence factor. Copyright © 2015 Elsevier Inc. All rights reserved.

Deep comparative genomics among Chlamydia trachomatis lymphogranuloma venereum isolates highlights genes potentially involved in pathoadaptation.

PubMed

Borges, Vítor; Gomes, João Paulo

2015-06-01

Lymphogranuloma venereum (LGV) is a human sexually transmitted disease caused by the obligate intracellular bacterium Chlamydia trachomatis (serovars L1-L3). LGV clinical manifestations range from severe ulcerative proctitis (anorectal syndrome), primarily caused by the epidemic L2b strains, to painful inguinal lymphadenopathy (the typical LGV bubonic form). Besides potential host-related factors, the differential disease severity and tissue tropism among LGV strains is likely a function of the genetic backbone of the strains. We aimed to characterize the genetic variability among LGV strains as strain- or serovar-specific mutations may underlie phenotypic signatures, and to investigate the mutational events that occurred throughout the pathoadaptation of the epidemic L2b lineage. By analyzing 20 previously published genomes from L1, L2, L2b and L3 strains and two new genomes from L2b strains, we detected 1497 variant sites and about 100 indels, affecting 453 genes and 144 intergenic regions, with 34 genes displaying a clear overrepresentation of nonsynonymous mutations. Effectors and/or type III secretion substrates (almost all of those described in the literature) and inclusion membrane proteins showed amino acid changes that were about fivefold more frequent than silent changes. More than 120 variant sites occurred in plasmid-regulated virulence genes, and 66% yielded amino acid changes. The identified serovar-specific variant sites revealed that the L2b-specific mutations are likely associated with higher fitness and pointed out potential targets for future highly discriminatory diagnostic/typing tests. By evaluating the evolutionary pathway beyond the L2b clonal radiation, we observed that 90.2% of the intra-L2b variant sites occurring in coding regions involve nonsynonymous mutations, where CT456/tarp has been the main target. Considering the progress on C. trachomatis genetic manipulation, this study may constitute an important contribution for prioritizing study targets for functional genomics aiming to dissect the impact of the identified intra-LGV polymorphisms on virulence or tropism dissimilarities among LGV strains. Copyright © 2015 Elsevier B.V. All rights reserved.

Identification of three randomly amplified polymorphic DNA-polymerase chain reaction markers for distinguishing Asian and North American Gypsy Moths (Lepidoptera: Lymantriidae)

Treesearch

David E. Schreiber; Karen J. Garner; James M. Slavicek

1997-01-01

Gypsy moths originating in Asia have recently been introduced into North America, making it necessary to develop markers for distinguishing the Asian strain from the established North American population. We have identified 3 randomly amplified polymorphic DNA-polymerase chain reaction generated (RAPD-PCR) markers which are specific for either Asian or North American...

[Detection of rubella virus RNA in clinical material by real time polymerase chain reaction method].

PubMed

Domonova, É A; Shipulina, O Iu; Kuevda, D A; Larichev, V F; Safonova, A P; Burchik, M A; Butenko, A M; Shipulin, G A

2012-01-01

Development of a reagent kit for detection of rubella virus RNA in clinical material by PCR-RT. During development and determination of analytical specificity and sensitivity DNA and RNA of 33 different microorganisms including 4 rubella strains were used. Comparison of analytical sensitivity of virological and molecular-biological methods was performed by using rubella virus strains Wistar RA 27/3, M-33, "Orlov", Judith. Evaluation of diagnostic informativity of rubella virus RNAisolation in various clinical material by PCR-RT method was performed in comparison with determination of virus specific serum antibodies by enzyme immunoassay. A reagent kit for the detection of rubella virus RNA in clinical material by PCR-RT was developed. Analytical specificity was 100%, analytical sensitivity - 400 virus RNA copies per ml. Analytical sensitivity of the developed technique exceeds analytical sensitivity of the Vero E6 cell culture infection method in studies of rubella virus strains Wistar RA 27/3 and "Orlov" by 11g and 31g, and for M-33 and Judith strains is analogous. Diagnostic specificity is 100%. Diagnostic specificity for testing samples obtained within 5 days of rash onset: for peripheral blood sera - 20.9%, saliva - 92.5%, nasopharyngeal swabs - 70.1%, saliva and nasopharyngeal swabs - 97%. Positive and negative predictive values of the results were shown depending on the type of clinical material tested. Application of reagent kit will allow to increase rubella diagnostics effectiveness at the early stages of infectious process development, timely and qualitatively perform differential diagnostics of exanthema diseases, support tactics of anti-epidemic regime.

Specific Detection and Identification of Herpes B Virus by a PCR-Microplate Hybridization Assay

PubMed Central

Oya, Chika; Ochiai, Yoshitsugu; Taniuchi, Yojiro; Takano, Takashi; Ueda, Fukiko; Yoshikawa, Yasuhiro; Hondo, Ryo

2004-01-01

Herpes B virus DNA was specifically amplified by PCR, targeting the regions that did not cross-react with herpes simplex virus (HSV). The amplified products, which were shown to be highly genetic polymorphisms among herpes B virus isolates, were identified by microplate hybridization with probes generated by PCR. The products immobilized in microplate wells were hybridized with the biotin-labeled probes derived from the SMHV strain of herpes B virus. The amplified products derived from the SMHV and E2490 strains of herpes B virus were identified by microplate hybridization. PCR products amplified from the trigeminal ganglia of seropositive cynomolgus macaques were identified as herpes B virus DNA. The utility of the PCR-microplate hybridization assay for genetic detection and identification of the polymorphic region of herpes B virus was determined. PMID:15131142

A new double digestion ligation mediated suppression PCR method for simultaneous bacteria DNA-typing and confirmation of species: an Acinetobacter sp. model.

PubMed

Stojowska, Karolina; Krawczyk, Beata

2014-01-01

We have designed a new ddLMS PCR (double digestion Ligation Mediated Suppression PCR) method based on restriction site polymorphism upstream from the specific target sequence for the simultaneous identification and differentiation of bacterial strains. The ddLMS PCR combines a simple PCR used for species or genus identification and the LM PCR strategy for strain differentiation. The bacterial identification is confirmed in the form of the PCR product(s), while the length of the PCR product makes it possible to differentiate between bacterial strains. If there is a single copy of the target sequence within genomic DNA, one specific PCR product is created (simplex ddLMS PCR), whereas for multiple copies of the gene the fingerprinting patterns can be obtained (multiplex ddLMS PCR). The described ddLMS PCR method is designed for rapid and specific strain differentiation in medical and microbiological studies. In comparison to other LM PCR it has substantial advantages: enables specific species' DNA-typing without the need for pure bacterial culture selection, is not sensitive to contamination with other cells or genomic DNA, and gives univocal "band-based" results, which are easy to interpret. The utility of ddLMS PCR was shown for Acinetobacter calcoaceticus-baumannii (Acb) complex, the genetically closely related and phenotypically similar species and also important nosocomial pathogens, for which currently, there are no recommended methods for screening, typing and identification. In this article two models are proposed: 3' recA-ddLMS PCR-MaeII/RsaI for Acb complex interspecific typing and 5' rrn-ddLMS PCR-HindIII/ApaI for Acinetobacter baumannii intraspecific typing. ddLMS PCR allows not only for DNA-typing but also for confirmation of species in one reaction. Also, practical guidelines for designing a diagnostic test based on ddLMS PCR for genotyping different species of bacteria are provided.

A New Double Digestion Ligation Mediated Suppression PCR Method for Simultaneous Bacteria DNA-Typing and Confirmation of Species: An Acinetobacter sp. Model

PubMed Central

Stojowska, Karolina; Krawczyk, Beata

2014-01-01

We have designed a new ddLMS PCR (double digestion Ligation Mediated Suppression PCR) method based on restriction site polymorphism upstream from the specific target sequence for the simultaneous identification and differentiation of bacterial strains. The ddLMS PCR combines a simple PCR used for species or genus identification and the LM PCR strategy for strain differentiation. The bacterial identification is confirmed in the form of the PCR product(s), while the length of the PCR product makes it possible to differentiate between bacterial strains. If there is a single copy of the target sequence within genomic DNA, one specific PCR product is created (simplex ddLMS PCR), whereas for multiple copies of the gene the fingerprinting patterns can be obtained (multiplex ddLMS PCR). The described ddLMS PCR method is designed for rapid and specific strain differentiation in medical and microbiological studies. In comparison to other LM PCR it has substantial advantages: enables specific species' DNA-typing without the need for pure bacterial culture selection, is not sensitive to contamination with other cells or genomic DNA, and gives univocal “band-based” results, which are easy to interpret. The utility of ddLMS PCR was shown for Acinetobacter calcoaceticus-baumannii (Acb) complex, the genetically closely related and phenotypically similar species and also important nosocomial pathogens, for which currently, there are no recommended methods for screening, typing and identification. In this article two models are proposed: 3′ recA-ddLMS PCR-MaeII/RsaI for Acb complex interspecific typing and 5′ rrn-ddLMS PCR-HindIII/ApaI for Acinetobacter baumannii intraspecific typing. ddLMS PCR allows not only for DNA-typing but also for confirmation of species in one reaction. Also, practical guidelines for designing a diagnostic test based on ddLMS PCR for genotyping different species of bacteria are provided. PMID:25522278

Strain ratio ultrasound elastography increases the accuracy of colour-Doppler ultrasound in the evaluation of Thy-3 nodules. A bi-centre university experience.

PubMed

Cantisani, Vito; Maceroni, Piero; D'Andrea, Vito; Patrizi, Gregorio; Di Segni, Mattia; De Vito, Corrado; Grazhdani, Hektor; Isidori, Andrea M; Giannetta, Elisa; Redler, Adriano; Frattaroli, Fabrizio; Giacomelli, Laura; Di Rocco, Giorgio; Catalano, Carlo; D'Ambrosio, Ferdinando

2016-05-01

To assess whether ultrasound elastography (USE) with strain ratio increases diagnostic accuracy of Doppler ultrasound in further characterisation of cytologically Thy3 thyroid nodules. In two different university diagnostic centres, 315 patients with indeterminate cytology (Thy3) in thyroid nodules aspirates were prospectively evaluated with Doppler ultrasound and strain ratio USE before surgery. Ultrasonographic features were analysed separately and together as ultrasound score, to assess sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). Receiver operating characteristic (ROC) curves to identify optimal cut-off value of the strain ratio were also provided. Diagnosis on a surgical specimen was considered the standard of reference. Higher strain ratio values were found in malignant nodules, with an optimum strain ratio cut-off of 2.09 at ROC analysis. USE with strain ratio showed 90.6% sensitivity, 93% specificity, 82.8% PPV, 96.4% NPV, while US score yielded a sensitivity of 52.9%, specificity of 84.3%, PPV 55.6% and NPV 82.9%. The diagnostic gain with strain ratio was statistically significant as proved by ROC areas, which was 0.9182 for strain ratio and 0.6864 for US score. USE with strain ratio should be considered a useful additional tool to colour-Doppler US, since it improves characterisation of thyroid nodules with indeterminate cytology. • Strain ratio measurements improve differentiation of thyroid nodules with indeterminate cytology • Elastography with strain ratio is more reliable than ultrasound features and ultrasound score • Strain ratio may help to better select patients with Thy 3 nodules candidate for surgery.

PCR and restriction fragment length polymorphism of a pel gene as a tool to identify Erwinia carotovora in relation to potato diseases.

PubMed Central

Darrasse, A; Priou, S; Kotoujansky, A; Bertheau, Y

1994-01-01

Using a sequenced pectate lyase-encoding gene (pel gene), we developed a PCR test for Erwinia carotovora. A set of primers allowed the amplification of a 434-bp fragment in E. carotovora strains. Among the 89 E. carotovora strains tested, only the Erwinia carotovora subsp. betavasculorum strains were not detected. A restriction fragment length polymorphism (RFLP) study was undertaken on the amplified fragment with seven endonucleases. The Sau3AI digestion pattern specifically identified the Erwinia carotovora subsp. atroseptica strains, and the whole set of data identified the Erwinia carotovora subsp. wasabiae strains. However, Erwinia carotovora subsp. carotovora and Erwinia carotovora subsp. odorifera could not be separated. Phenetic and phylogenic analyses of RFLP results showed E. carotovora subsp. atroseptica as a homogeneous group while E. carotovora subsp. carotovora and E. carotovora subsp. odorifera strains exhibited a genetic diversity that may result from a nonmonophyletic origin. The use of RFLP on amplified fragments in epidemiology and for diagnosis is discussed. Images PMID:7912502

16S-23S rDNA intergenic spacer region polymorphism of Lactococcus garvieae, Lactococcus raffinolactis and Lactococcus lactis as revealed by PCR and nucleotide sequence analysis.

PubMed

Blaiotta, Giuseppe; Pepe, Olimpia; Mauriello, Gianluigi; Villani, Francesco; Andolfi, Rosamaria; Moschetti, Giancarlo

2002-12-01

The intergenic spacer region (ISR) between the 16S and 23S rRNA genes was tested as a tool for differentiating lactococci commonly isolated in a dairy environment. 17 reference strains, representing 11 different species belonging to the genera Lactococcus, Streptococcus, Lactobacillus, Enterococcus and Leuconostoc, and 127 wild streptococcal strains isolated during the whole fermentation process of "Fior di Latte" cheese were analyzed. After 16S-23S rDNA ISR amplification by PCR, species or genus-specific patterns were obtained for most of the reference strains tested. Moreover, results obtained after nucleotide analysis show that the 16S-23S rDNA ISR sequences vary greatly, in size and sequence, among Lactococcus garvieae, Lactococcus raffinolactis, Lactococcus lactis as well as other streptococci from dairy environments. Because of the high degree of inter-specific polymorphism observed, 16S-23S rDNA ISR can be considered a good potential target for selecting species-specific molecular assays, such as PCR primer or probes, for a rapid and extremely reliable differentiation of dairy lactococcal isolates.

Whole genome sequencing options for bacterial strain typing and epidemiologic analysis based on single nucleotide polymorphism versus gene-by-gene-based approaches.

PubMed

Schürch, A C; Arredondo-Alonso, S; Willems, R J L; Goering, R V

2018-04-01

Whole genome sequence (WGS)-based strain typing finds increasing use in the epidemiologic analysis of bacterial pathogens in both public health as well as more localized infection control settings. This minireview describes methodologic approaches that have been explored for WGS-based epidemiologic analysis and considers the challenges and pitfalls of data interpretation. Personal collection of relevant publications. When applying WGS to study the molecular epidemiology of bacterial pathogens, genomic variability between strains is translated into measures of distance by determining single nucleotide polymorphisms in core genome alignments or by indexing allelic variation in hundreds to thousands of core genes, assigning types to unique allelic profiles. Interpreting isolate relatedness from these distances is highly organism specific, and attempts to establish species-specific cutoffs are unlikely to be generally applicable. In cases where single nucleotide polymorphism or core gene typing do not provide the resolution necessary for accurate assessment of the epidemiology of bacterial pathogens, inclusion of accessory gene or plasmid sequences may provide the additional required discrimination. As with all epidemiologic analysis, realizing the full potential of the revolutionary advances in WGS-based approaches requires understanding and dealing with issues related to the fundamental steps of data generation and interpretation. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Polymorphisms in Inc Proteins and Differential Expression of inc Genes among Chlamydia trachomatis Strains Correlate with Invasiveness and Tropism of Lymphogranuloma Venereum Isolates

PubMed Central

Almeida, Filipe; Borges, Vítor; Ferreira, Rita; Borrego, Maria José; Gomes, João Paulo

2012-01-01

Chlamydia trachomatis is a human bacterial pathogen that multiplies only within an intracellular membrane-bound vacuole, the inclusion. C. trachomatis includes ocular and urogenital strains, usually causing infections restricted to epithelial cells of the conjunctiva and genital mucosa, respectively, and lymphogranuloma venereum (LGV) strains, which can infect macrophages and spread into lymph nodes. However, C. trachomatis genomes display >98% identity at the DNA level. In this work, we studied whether C. trachomatis Inc proteins, which have a bilobed hydrophobic domain that may mediate their insertion in the inclusion membrane, could be a factor determining these different types of infection and tropisms. Analyses of polymorphisms and phylogeny of 48 Inc proteins from 51 strains encompassing the three disease groups showed significant amino acid differences that were mainly due to variations between Inc proteins from LGV and ocular or urogenital isolates. Studies of the evolutionary dynamics of inc genes suggested that 10 of them are likely under positive selection and indicated that most nonsilent mutations are LGV specific. Additionally, real-time quantitative PCR analyses in prototype and clinical strains covering the three disease groups identified three inc genes with LGV-specific expression. We determined the transcriptional start sites of these genes and found LGV-specific nucleotides within their promoters. Thus, subtle variations in the amino acids of a subset of Inc proteins and in the expression of inc genes may contribute to the unique tropism and invasiveness of C. trachomatis LGV strains. PMID:23042990

Polymerase chain reaction: A molecular diagnostic tool in periodontology

PubMed Central

Maheaswari, Rajendran; Kshirsagar, Jaishree Tukaram; Lavanya, Nallasivam

2016-01-01

This review discusses the principles of polymerase chain reaction (PCR) and its application as a diagnostic tool in periodontology. The relevant MEDLINE and PubMed indexed journals were searched manually and electronically by typing PCR, applications of PCR, PCR in periodontics, polymorphism studies in periodontitis, and molecular techniques in periodontology. The searches were limited to articles in English language and the articles describing PCR process and its relation to periodontology were collected and used to prepare a concise review. PCR has now become a standard diagnostic and research tool in periodontology. Various studies reveal that its sensitivity and specificity allow it as a rapid, efficient method of detecting, identifying, and quantifying organism. Different immune and inflammatory markers can be identified at the mRNA expression level, and also the determination of genetic polymorphisms, thus providing the deeper insight into the mechanisms underlying the periodontal disease. PMID:27143822

Polymerase chain reaction: A molecular diagnostic tool in periodontology.

PubMed

Maheaswari, Rajendran; Kshirsagar, Jaishree Tukaram; Lavanya, Nallasivam

2016-01-01

This review discusses the principles of polymerase chain reaction (PCR) and its application as a diagnostic tool in periodontology. The relevant MEDLINE and PubMed indexed journals were searched manually and electronically by typing PCR, applications of PCR, PCR in periodontics, polymorphism studies in periodontitis, and molecular techniques in periodontology. The searches were limited to articles in English language and the articles describing PCR process and its relation to periodontology were collected and used to prepare a concise review. PCR has now become a standard diagnostic and research tool in periodontology. Various studies reveal that its sensitivity and specificity allow it as a rapid, efficient method of detecting, identifying, and quantifying organism. Different immune and inflammatory markers can be identified at the mRNA expression level, and also the determination of genetic polymorphisms, thus providing the deeper insight into the mechanisms underlying the periodontal disease.

Contrasting Genomic Diversity in Two Closely Related Postharvest Pathogens: Penicillium digitatum and Penicillium expansum.

PubMed

Julca, Irene; Droby, Samir; Sela, Noa; Marcet-Houben, Marina; Gabaldón, Toni

2015-12-14

Penicillium digitatum and Penicillium expansum are two closely related fungal plant pathogens causing green and blue mold in harvested fruit, respectively. The two species differ in their host specificity, being P. digitatum restricted to citrus fruits and P. expansum able to infect a wide range of fruits after harvest. Although host-specific Penicillium species have been found to have a smaller gene content, it is so far unclear whether these different host specificities impact genome variation at the intraspecific level. Here we assessed genome variation across four P. digitatum and seven P. expansum isolates from geographically distant regions. Our results show very high similarity (average 0.06 SNPs [single nucleotide polymorphism] per kb) between globally distributed isolates of P. digitatum pointing to a recent expansion of a single lineage. This low level of genetic variation found in our samples contrasts with the higher genetic variability observed in the similarly distributed P. expansum isolates (2.44 SNPs per kb). Patterns of polymorphism in P. expansum indicate that recombination exists between genetically diverged strains. Consistent with the existence of sexual recombination and heterothallism, which was unknown for this species, we identified the two alternative mating types in different P. expansum isolates. Patterns of polymorphism in P. digitatum indicate a recent clonal population expansion of a single lineage that has reached worldwide distribution. We suggest that the contrasting patterns of genomic variation between the two species reflect underlying differences in population dynamics related with host specificities and related agricultural practices. It should be noted, however, that this results should be confirmed with a larger sampling of strains, as new strains may broaden the diversity so far found in P. digitatum. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Phase variation and microevolution at homopolymeric tracts in Bordetella pertussis

PubMed Central

Gogol, Emily B; Cummings, Craig A; Burns, Ryan C; Relman, David A

2007-01-01

Background Bordetella pertussis, the causative agent of whooping cough, is a highly clonal pathogen of the respiratory tract. Its lack of genetic diversity, relative to many bacterial pathogens, could limit its ability to adapt to a hostile and changing host environment. This limitation might be overcome by phase variation, as observed for other mucosal pathogens. One of the most common mechanisms of phase variation is reversible expansion or contraction of homopolymeric tracts (HPTs). Results The genomes of B. pertussis and the two closely related species, B. bronchiseptica and B. parapertussis, were screened for homopolymeric tracts longer than expected on the basis of chance, given their nucleotide compositions. Sixty-nine such HPTs were found in total among the three genomes, 74% of which were polymorphic among the three species. Nine HPTs were genotyped in a collection of 90 geographically and temporally diverse B. pertussis strains using the polymerase chain reaction/ligase detection reaction (PCR/LDR) assay. Six HPTs were polymorphic in this collection of B. pertussis strains. Of note, one of these polymorphic HPTs was found in the fimX promoter, where a single base insertion variant was present in seven strains, all of which were isolated prior to introduction of the pertussis vaccine. Transcript abundance of fimX was found to be 3.8-fold lower in strains carrying the longer allele. HPTs in three other genes, tcfA, bapC, and BP3651, varied widely in composition across the strain collection and displayed allelic polymorphism within single cultures. Conclusion Allelic polymorphism at homopolymeric tracts is common within the B. pertussis genome. Phase variability may be an important mechanism in B. pertussis for evasion of the immune system and adaptation to different niches in the human host. High sensitivity and specificity make the PCR/LDR assay a powerful tool for investigating allelic variation at HPTs. Using this method, allelic diversity and phase variation were demonstrated at several B. pertussis loci. PMID:17509142

Phosphatidylinositol-specific phospholipase C activity in Lactobacillus rhamnosus with capacity to translocate.

PubMed

Rodriguez, A V; Baigorí, M D; Alvarez, S; Castro, G R; Oliver, G

2001-10-16

Phosphatidylinositol-specific phospholipase C (PI-PLC) activity was investigated in 25 different lactic acid bacteria (LAB) strains belonging to the genera Lactobacillus, Weisella, and Enterococcus. PI-PLC activity was detected in 44% of the strains studied in culture medium without carbon source. From the PI-PLC positive strains, Lactobacillus rhamnosus ATCC 7469 was selected for translocation studies. Healthy mice were orally administered with a daily dose of 2.0 x 10(9) of viable L. rhamnosus suspension. Viable bacteria were detected in liver and spleen of mice fed with LAB for 7 days. Bacterial colonies isolated from liver were biochemically characterized, and further subjected to randomly amplified polymorphic DNA. Amplification patterns of five strains displayed identical profiles to L. rhamnosus. PI-PLC activity was determined in the strains recovered from liver.

Genetic Interrelatedness among Clover Proliferation Mycoplasmalike Organisms (MLOs) and Other MLOs Investigated by Nucleic Acid Hybridization and Restriction Fragment Length Polymorphism Analyses

PubMed Central

Lee, Ing-Ming; Davis, Robert E.; Hiruki, Chuji

1991-01-01

DNA was isolated from clover proliferation (CP) mycoplasmalike organism (MLO)-diseased periwinkle plants (Catharanthus roseus (L.) G. Don.) and cloned into pSP6 plasmid vectors. CP MLO-specific recombinant DNA clones were biotin labeled and used as probes in dot hybridization and restriction fragment length polymorphism analyses to study the genetic interrelatedness among CP MLO and other MLOs, including potato witches'-broom (PWB) MLO. Results from dot hybridization analyses indicated that both a Maryland strain of aster yellows and a California strain of aster yellows are distantly related to CP MLO. Elm yellows, paulownia witches'-broom, peanut witches'-broom, loofah witches'-broom, and sweet potato witches'-broom may be very distantly related, if at all, to CP MLO. A new Jersey strain of aster yellows MLO, tomato big bud MLO, clover phyllody MLO, beet leafhopper-transmitted virescence MLO, and ash yellows MLO are related to CP MLO, but PWB MLO is the most closely related. Similarity coefficients derived from restriction fragment length polymorphism analyses revealed that PWB and CP MLOs are closely related strains and thus provided direct evidence of their relatedness in contrast to reliance solely on biological characterization. Images PMID:16348604

Molecular Basis of Allele-Specific Efficacy of a Blood-Stage Malaria Vaccine: Vaccine Development Implications

PubMed Central

Ouattara, Amed; Takala-Harrison, Shannon; Thera, Mahamadou A.; Coulibaly, Drissa; Niangaly, Amadou; Saye, Renion; Tolo, Youssouf; Dutta, Sheetij; Heppner, D. Gray; Soisson, Lorraine; Diggs, Carter L.; Vekemans, Johan; Cohen, Joe; Blackwelder, William C.; Dube, Tina; Laurens, Matthew B.; Doumbo, Ogobara K.; Plowe, Christopher V.

2013-01-01

The disappointing efficacy of blood-stage malaria vaccines may be explained in part by allele-specific immune responses that are directed against polymorphic epitopes on blood-stage antigens. FMP2.1/AS02A, a blood-stage candidate vaccine based on apical membrane antigen 1 (AMA1) from the 3D7 strain of Plasmodium falciparum, had allele-specific efficacy against clinical malaria in a phase II trial in Malian children. We assessed the cross-protective efficacy of the malaria vaccine and inferred which polymorphic amino acid positions in AMA1 were the targets of protective allele-specific immune responses. FMP2.1/AS02A had the highest efficacy against AMA1 alleles that were identical to the 3D7 vaccine-type allele at 8 highly polymorphic amino acid positions in the cluster 1 loop (c1L) but differed from 3D7 elsewhere in the molecule. Comparison of the incidence of vaccine-type alleles before and after vaccination in the malaria vaccine and control groups and examination of the patterns of allele change at polymorphic positions in consecutive malaria episodes suggest that the highly polymorphic amino acid position 197 in c1L was the most critical determinant of allele-specific efficacy. These results indicate that a multivalent AMA1 vaccine with broad efficacy could include only a limited set of key alleles of this extremely polymorphic antigen. PMID:23204168

[POSSIBILITIES OF APPLICATION OF MALDI-TOF MASS-SPECTROMETRY FOR STUDY OF CARBOHYDRATE-SPECIFIC RECEPTORS FOR DIAGNOSTIC BACTERIOPHAGE EL TOR].

PubMed

Telesmanich, N R; Goncharenko, E V; Chaika, S O; Chaika, I A; Telicheva, V O

2016-01-01

Study mechanisms of interaction of diagnostic bacteriophage El Tor with sensitive strain Vibrio cholerae El Tor 18507 using direct protein profiling, identification of constant and variable proteins, taking part in interaction of the phage and cell, as well as carbohydrate-specific phage receptors. . A commercial preparation of cholera diagnostic bacteriophage El Tor, strain V. cholerae El Tor 18507 were used. Effect of carbohydrates on bacteriophage activity was determined in experiments with phage by a classic and modified by us method. Protein profiles of the studied objects were studied using MSP-analysis method. Sucrose was shown to inhibit lytic activity of bacteriophage. Proteome profiles of El Tor bacteriophage and sensitive indicator strains were studied, identification of constant and variable proteins of the studied objects by MSP Peak-list program was carried out. Analysis of changes of profiles of phage and microbial cell during interaction with sucrose gave a basis for assuming, that sucrose in the mixture of culture-phage enters interaction namely with phage protein receptors, blocking receptors specific for cholera vibrio, that subsequently manifests in a sharp decrease of phage activity against the sensitive strain.

CRISPR/Cas9-mediated conversion of human platelet alloantigen allotypes.

PubMed

Zhang, Nanyan; Zhi, Huiying; Curtis, Brian R; Rao, Sridhar; Jobaliya, Chintan; Poncz, Mortimer; French, Deborah L; Newman, Peter J

2016-02-11

Human platelet alloantigens (HPAs) reside on functionally important platelet membrane glycoproteins and are caused by single nucleotide polymorphisms in the genes that encode them. Antibodies that form against HPAs are responsible for several clinically important alloimmune bleeding disorders, including fetal and neonatal alloimmune thrombocytopenia and posttransfusion purpura. The HPA-1a/HPA-1b alloantigen system, also known as the Pl(A1)/Pl(A2) polymorphism, is the most frequently implicated HPA among whites, and a single Leu33Pro amino acid polymorphism within the integrin β3 subunit is responsible for generating the HPA-1a/HPA-1b alloantigenic epitopes. HPA-1b/b platelets, like those bearing other low-frequency platelet-specific alloantigens, are relatively rare in the population and difficult to obtain for purposes of transfusion therapy and diagnostic testing. We used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) gene-editing technology to transform Leu33 (+) megakaryocytelike DAMI cells and induced pluripotent stem cells (iPSCs) to the Pro33 allotype. CD41(+) megakaryocyte progenitors derived from these cells expressed the HPA-1b (Pl(A2)) alloantigenic epitope, as reported by diagnostic NciI restriction enzyme digestion, DNA sequencing, and western blot analysis using HPA-1b-specific human maternal alloantisera. Application of CRISPR/Cas9 technology to genetically edit this and other clinically-important HPAs holds great potential for production of designer platelets for diagnostic, investigative, and, ultimately, therapeutic use. © 2016 by The American Society of Hematology.

CRISPR/Cas9-mediated conversion of human platelet alloantigen allotypes

PubMed Central

Zhang, Nanyan; Zhi, Huiying; Curtis, Brian R.; Rao, Sridhar; Jobaliya, Chintan; Poncz, Mortimer; French, Deborah L.

2016-01-01

Human platelet alloantigens (HPAs) reside on functionally important platelet membrane glycoproteins and are caused by single nucleotide polymorphisms in the genes that encode them. Antibodies that form against HPAs are responsible for several clinically important alloimmune bleeding disorders, including fetal and neonatal alloimmune thrombocytopenia and posttransfusion purpura. The HPA-1a/HPA-1b alloantigen system, also known as the PlA1/PlA2 polymorphism, is the most frequently implicated HPA among whites, and a single Leu33Pro amino acid polymorphism within the integrin β3 subunit is responsible for generating the HPA-1a/HPA-1b alloantigenic epitopes. HPA-1b/b platelets, like those bearing other low-frequency platelet-specific alloantigens, are relatively rare in the population and difficult to obtain for purposes of transfusion therapy and diagnostic testing. We used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) gene-editing technology to transform Leu33+ megakaryocytelike DAMI cells and induced pluripotent stem cells (iPSCs) to the Pro33 allotype. CD41+ megakaryocyte progenitors derived from these cells expressed the HPA-1b (PlA2) alloantigenic epitope, as reported by diagnostic NciI restriction enzyme digestion, DNA sequencing, and western blot analysis using HPA-1b–specific human maternal alloantisera. Application of CRISPR/Cas9 technology to genetically edit this and other clinically-important HPAs holds great potential for production of designer platelets for diagnostic, investigative, and, ultimately, therapeutic use. PMID:26634302

Using surface-enhanced Raman spectroscopy and electrochemically driven melting to discriminate Yersinia pestis from Y. pseudotuberculosis based on single nucleotide polymorphisms within unpurified polymerase chain reaction amplicons.

PubMed

Papadopoulou, Evanthia; Goodchild, Sarah A; Cleary, David W; Weller, Simon A; Gale, Nittaya; Stubberfield, Michael R; Brown, Tom; Bartlett, Philip N

2015-02-03

The development of sensors for the detection of pathogen-specific DNA, including relevant species/strain level discrimination, is critical in molecular diagnostics with major impacts in areas such as bioterrorism and food safety. Herein, we use electrochemically driven denaturation assays monitored by surface-enhanced Raman spectroscopy (SERS) to target single nucleotide polymorphisms (SNPs) that distinguish DNA amplicons generated from Yersinia pestis, the causative agent of plague, from the closely related species Y. pseudotuberculosis. Two assays targeting SNPs within the groEL and metH genes of these two species have been successfully designed. Polymerase chain reaction (PCR) was used to produce Texas Red labeled single-stranded DNA (ssDNA) amplicons of 262 and 251 bases for the groEL and metH targets, respectively. These amplicons were used in an unpurified form to hybridize to immobilized probes then subjected to electrochemically driven melting. In all cases electrochemically driven melting was able to discriminate between fully homologous DNA and that containing SNPs. The metH assay was particularly challenging due to the presence of only a single base mismatch in the middle of the 251 base long PCR amplicon. However, manipulation of assay conditions (conducting the electrochemical experiments at 10 °C) resulted in greater discrimination between the complementary and mismatched DNA. Replicate data were collected and analyzed for each duplex on different days, using different batches of PCR product and different sphere segment void (SSV) substrates. Despite the variability introduced by these differences, the assays are shown to be reliable and robust providing a new platform for strain discrimination using unpurified PCR samples.

Genetic polymorphism of natural Epstein-Barr virus isolates from infectious mononucleosis patients and healthy carriers.

PubMed Central

Lung, M L; Chang, R S; Jones, J H

1988-01-01

We analyzed Epstein-Barr virus (EBV) genomes from lymphoblastoid cell lines isolated from patients with infectious mononucleosis and from healthy subjects from California, Hawaii, and Hong Kong between 1970 and 1987. Using genetic polymorphism as epidemiological markers, we found that several genotypes of EBV cocirculate in a community and that although most EBV strains isolated from California and Southern China may be differentiated genotypically, there was no specific association between genotype and disease or time of isolation. Images PMID:2901499

Development of strain-specific PCR primers for quantitative detection of Bacillus mesentericus strain TO-A in human feces.

PubMed

Sato, Naoki; Seo, Genichiro; Benno, Yoshimi

2014-01-01

Strain-specific polymerase chain reaction (PCR) primers for detection of Bacillus mesentericus strain TO-A (BM TO-A) were developed. The randomly amplified polymorphic DNA (RAPD) technique was used to produce potential strain-specific markers. A 991-bp RAPD marker found to be strain-specific was sequenced, and two primer pairs specific to BM TO-A were constructed based on this sequence. In addition, we explored a more specific DNA region using inverse PCR, and designed a strain-specific primer set for use in real-time quantitative PCR (qPCR). These primer pairs were tested against 25 Bacillus subtilis strains and were found to be strain-specific. After examination of the detection limit and linearity of detection of BM TO-A in feces, the qPCR method and strain-specific primers were used to quantify BM TO-A in the feces of healthy volunteers who had ingested 3×10(8) colony forming unit (CFU) of BM TO-A per day in tablets. During the administration period, BM TO-A was detected in the feces of all 24 subjects, and the average number of BM TO-A detected using the culture method and qPCR was about 10(4.8) and 10(5.8) cells per gram of feces, respectively. Using the qPCR method, BM TO-A was detected in the feces of half of the subjects 3 d after withdrawal, and was detected in the feces of only one subject 1 week after withdrawal. These results suggest that the qPCR method using BM TO-A strain-specific primers is useful for the quantitative detection of this strain in feces.

Application of AFLP fingerprint analysis for studying the biodiversity of Streptococcus thermophilus.

PubMed

Lazzi, Camilla; Bove, Claudio Giorgio; Sgarbi, Elisa; Gatti, Monica; Monica, Gatti; La Gioia, Federica; Torriani, Sandra; Sandra, Torriani; Neviani, Erasmo

2009-10-01

Streptococcus thermophilus is a lactic acid bacteria (LAB) widely used in milk fermentation processes as a starter culture. In this work the genetic diversity of S. thermophilus isolates from different sources was analyzed using Amplified Fragment Length Polymorphism fingerprinting (AFLP). Since this is the first report that indicates the application of AFLP in order to study genotypic polymorphism in S. thermophilus species, an optimization of experimental conditions was carried out to decide the optimal AFLP analysis protocol. Furthermore the fingerprinting resolutions of AFLP and RAPD (Random Amplified Polymorphic DNA) were evaluated and compared. The overall data suggest that genotypic characterization performed by AFLP provide a better view of microbial diversity of S. thermophilus, indicating that RAPD is less discriminating than AFLP. The successful use of AFLP analysis in the characterization of S. thermophilus strains reported in this study suggests the potential uses for this technique to define the whole-genome diversity of each specific strain, as an alternative to the fingerprinting methods used till now.

Recurrent Reverse Evolution Maintains Polymorphism after Strong Bottlenecks in Commensal Gut Bacteria

PubMed Central

Ramiro, Ricardo S.; Barroso-Batista, João; Güleresi, Daniela; Lourenço, Marta; Gordo, Isabel

2017-01-01

Abstract The evolution of new strains within the gut ecosystem is poorly understood. We used a natural but controlled system to follow the emergence of intraspecies diversity of commensal Escherichia coli, during three rounds of adaptation to the mouse gut (∼1,300 generations). We previously showed that, in the first round, a strongly beneficial phenotype (loss-of-function for galactitol consumption; gat-negative) spread to >90% frequency in all colonized mice. Here, we show that this loss-of-function is repeatedly reversed when a gat-negative clone colonizes new mice. The regain of function occurs via compensatory mutation and reversion, the latter leaving no trace of past adaptation. We further show that loss-of-function adaptive mutants reevolve, after colonization with an evolved gat-positive clone. Thus, even under strong bottlenecks a regime of strong-mutation-strong-selection dominates adaptation. Coupling experiments and modeling, we establish that reverse evolution recurrently generates two coexisting phenotypes within the microbiota that can or not consume galactitol (gat-positive and gat-negative, respectively). Although the abundance of the dominant strain, the gat-negative, depends on the microbiota composition, gat-positive abundance is independent of the microbiota composition and can be precisely manipulated by supplementing the diet with galactitol. These results show that a specific diet is able to change the abundance of specific strains. Importantly, we find polymorphism for these phenotypes in indigenous Enterobacteria of mice and man. Our results demonstrate that natural selection can greatly overwhelm genetic drift at structuring the strain diversity of gut commensals and that competition for limiting resources may be a key mechanism for maintaining polymorphism in the gut. PMID:28961745

Development of diagnostic markers for use in breeding potatoes resistant to Globodera pallida pathotype Pa2/3 using germplasm derived from Solanum tuberosum ssp. andigena CPC 2802.

PubMed

Moloney, Claire; Griffin, Denis; Jones, Peter W; Bryan, Glenn J; McLean, Karen; Bradshaw, John E; Milbourne, Dan

2010-02-01

Quantitative resistance to Globodera pallida pathotype Pa2/3, originally derived from Solanum tuberosum ssp. andigena Commonwealth Potato Collection (CPC) accession 2802, is present in several potato cultivars and advanced breeding lines. One genetic component of this resistance, a large effect quantitative trait locus (QTL) on linkage group IV (which we have renamed GpaIV(adg)(s)) has previously been mapped in the tetraploid breeding line 12601ab1. In this study, we show that GpaIV(adg)(s) is also present in a breeding line called C1992/31 via genetic mapping in an F(1) population produced by crossing C1992/31 with the G. pallida susceptible cultivar Record. C1992/31 is relatively divergent from 12601ab1, confirming that GpaIV(adg)(s) is an ideal target for marker-assisted selection in currently available germplasm. To generate markers exhibiting diagnostic potential for GpaIV(adg)(s), three bacterial artificial chromosome clones were isolated from the QTL region, sequenced, and used to develop 15 primer sets generating single-copy amplicons, which were examined for polymorphisms exhibiting linkage to GpaIV(adg)(s) in C1992/31. Eight such polymorphisms were found. Subsequently, one insertion/deletion polymorphism, three single nucleotide polymorphisms and a specific allele of the microsatellite marker STM3016 were shown to exhibit diagnostic potential for the QTL in a panel of 37 potato genotypes, 12 with and 25 without accession CPC2082 in their pedigrees. STM3016 and one of the SNP polymorphisms, C237(119), were assayed in 178 potato genotypes, arising from crosses between C1992/31 and 16 G. pallida susceptible genotypes, undergoing selection in a commercial breeding programme. The results suggest that the diagnostic markers would most effectively be employed in MAS-based approaches to pyramid different resistance loci to develop cultivars exhibiting strong, durable resistance to G. pallida pathotype Pa2/3.

Identification of Erwinia species isolated from apples and pears by differential PCR.

PubMed

Gehring, I; Geider, K

2012-04-01

Many pathogenic and epiphytic bacteria isolated from apples and pears belong to the genus Erwinia; these include the species E. amylovora, E. pyrifoliae, E. billingiae, E. persicina, E. rhapontici and E. tasmaniensis. Identification and classification of freshly isolated bacterial species often requires tedious taxonomic procedures. To facilitate routine identification of Erwinia species, we have developed a PCR method based on species-specific oligonucleotides (SSOs) from the sequences of the housekeeping genes recA and gpd. Using species-specific primers that we report here, differentiation was done with conventional PCR (cPCR) and quantitative PCR (qPCR) applying two consecutive primer annealing temperatures. The specificity of the primers depends on terminal Single Nucleotide Polymorphisms (SNPs) that are characteristic for the target species. These PCR assays enabled us to distinguish eight Erwinia species, as well as to identify new Erwinia isolates from plant surfaces. When performed with mixed bacterial cultures, they only detected a single target species. This method is a novel approach to classify strains within the genus Erwinia by PCR and it can be used to confirm other diagnostic data, especially when specific PCR detection methods are not already available. The method may be applied to classify species within other bacterial genera. Copyright © 2012 Elsevier B.V. All rights reserved.

Diagnostic tools based on minor groove binder probe technology for rapid identification of vaccinal and field strains of canine parvovirus type 2b.

PubMed

Decaro, Nicola; Martella, Vito; Elia, Gabriella; Desario, Costantina; Campolo, Marco; Buonavoglia, Domenico; Bellacicco, Anna Lucia; Tempesta, Maria; Buonavoglia, Canio

2006-12-01

TaqMan-based diagnostic tests have been developed for the identification of canine parvovirus type 2 (CPV-2) strains in the faeces of dogs with diarrhoea, including a minor groove binder (MGB) probe assay for identification of type 2-based vaccines and field strains (types 2a, 2b and 2c). Since type 2b vaccines have been licensed recently in Europe, two novel MGB assays were developed for discrimination between type 2b vaccines and field strains of CPV. Such assays have been found to be highly sensitive, specific and reproducible, allowing for simultaneous detection of type 2b vaccinal and field strains present in the same specimens. These new assays will help resolution of the diagnostic problems related to the detection of a type 2b strain in the faeces of dogs shortly after the administration of a type 2b vaccine.

A Constitutive Relationship between Crack Propagation and Specific Damping Capacity in Steel

DTIC Science & Technology

1990-10-01

diagnostic tool for detecting crack growth in structures. The model must be simple to act as a tool, but it must be comprehensive to provide accuracy...strain for static fracture u ECritical strain above which plastic strain occursP EAverage value of the cyclic plastic-strain rangeP E t ln(Ao/AI), true

Primary hyperoxaluria type 1: diagnostic relevance of mutations and polymorphisms in the alanine:glyoxylate aminotransferase gene (AGXT).

PubMed

Tarn, A C; von Schnakenburg, C; Rumsby, G

1997-09-01

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive disorder of glyoxylate metabolism caused by deficiency of the hepatic peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT). The disease shows considerable phenotypic, enzymatic and genetic heterogeneity. To date, 7 polymorphisms and 11 point mutations have been described in the gene encoding AGT. We report on the prevalence of these polymorphisms and mutations in 79 patients with PH1 with the aim of assessing their diagnostic relevance. A strong association of the C154T, intron 1 insertion and C386T polymorphisms is confirmed and this linkage extends to include the type 1 variant of a polymorphic tandem repeat in intron 4. Only 64 of 158 (40%) PH1 alleles have one of the defined mutations, with the G630A mutation accounting for 39 of these and T853C for 14. Overall only 20 (25%) of the patients studied had the genetic basis of their disease fully explained: 7 were homozygous for the G630A mutation, 5 were homozygous for the T853C mutation, 1 was homozygous for the C819T mutation, and 7 had two different mutations identified and were presumed to be compound heterozygotes. Only the two more frequent G630A and T853C mutations are of general diagnostic relevance for mutation screening. It seems likely that there are a significant number of other mutations, perhaps family-specific, still to be described. There was no apparent difference in the types of mutations in patients presenting in the first year of life (36%), suggesting that other factors, such as periods of dehydration or urinary tract infections, might contribute more to the clinical manifestation than genotype.

Monitoring of antibiotic-induced alterations in the human intestinal microflora and detection of probiotic strains by use of terminal restriction fragment length polymorphism.

PubMed

Jernberg, Cecilia; Sullivan, Asa; Edlund, Charlotta; Jansson, Janet K

2005-01-01

Terminal restriction fragment length polymorphism (T-RFLP) was investigated as a tool for monitoring the human intestinal microflora during antibiotic treatment and during ingestion of a probiotic product. Fecal samples from eight healthy volunteers were taken before, during, and after administration of clindamycin. During treatment, four subjects were given a probiotic, and four subjects were given a placebo. Changes in the microbial intestinal community composition and relative abundance of specific microbial populations in each subject were monitored by using viable counts and T-RFLP fingerprints. T-RFLP was also used to monitor specific bacterial populations that were either positively or negatively affected by clindamycin. Some dominant bacterial groups, such as Eubacterium spp., were easily monitored by T-RFLP, while they were hard to recover by cultivation. Furthermore, the two probiotic Lactobacillus strains were easily tracked by T-RFLP and were shown to be the dominant Lactobacillus community members in the intestinal microflora of subjects who received the probiotic.

Monitoring of Antibiotic-Induced Alterations in the Human Intestinal Microflora and Detection of Probiotic Strains by Use of Terminal Restriction Fragment Length Polymorphism

PubMed Central

Jernberg, Cecilia; Sullivan, Åsa; Edlund, Charlotta; Jansson, Janet K.

2005-01-01

Terminal restriction fragment length polymorphism (T-RFLP) was investigated as a tool for monitoring the human intestinal microflora during antibiotic treatment and during ingestion of a probiotic product. Fecal samples from eight healthy volunteers were taken before, during, and after administration of clindamycin. During treatment, four subjects were given a probiotic, and four subjects were given a placebo. Changes in the microbial intestinal community composition and relative abundance of specific microbial populations in each subject were monitored by using viable counts and T-RFLP fingerprints. T-RFLP was also used to monitor specific bacterial populations that were either positively or negatively affected by clindamycin. Some dominant bacterial groups, such as Eubacterium spp., were easily monitored by T-RFLP, while they were hard to recover by cultivation. Furthermore, the two probiotic Lactobacillus strains were easily tracked by T-RFLP and were shown to be the dominant Lactobacillus community members in the intestinal microflora of subjects who received the probiotic. PMID:15640226

Morphological characterization and molecular fingerprinting of Nostoc strains by multiplex RAPD.

PubMed

Hillol, Chakdar; Pabbi, Sunil

2012-01-01

Morphological parameters studied for the twenty selected Nostoc strains were mostly found to be consistent with the earlier reports. But the shape of akinetes observed in this study was a little deviation from the existing descriptions and heterocyst frequency was also found to be different in different strains in spite of growing in the same nitrogen free media. Multiplex RAPD produced reproducible and completely polymorphic amplification profiles for all the strains including some strain specific unique bands which are intended to be useful for identification of those strains. At least one to a maximum of two unique bands was produced by different dual primer combinations. For ten strains out of twenty, strain specific bands were found to be generated. Cluster analysis revealed a vast heterogeneity among these Nostoc strains and no specific clustering based on geographical origin was found except a few strains. It was also observed that morphological data may not necessarily correspond to the genetic data in most of the cases. CCC92 (Nostoc muscorum) and CCC48 (Nostoc punctiforme) showed a high degree of similarity which was well supported by high bootstrap value. The level of similarity of the strains ranged from 0.15 to 0.94. Cluster analysis based on multiplex RAPD showed a good fit revealing the discriminatory power of this technique.

Identification of Medically Important Yeasts Using PCR-Based Detection of DNA Sequence Polymorphisms in the Internal Transcribed Spacer 2 Region of the rRNA Genes

PubMed Central

Chen, Y. C.; Eisner, J. D.; Kattar, M. M.; Rassoulian-Barrett, S. L.; LaFe, K.; Yarfitz, S. L.; Limaye, A. P.; Cookson, B. T.

2000-01-01

Identification of medically relevant yeasts can be time-consuming and inaccurate with current methods. We evaluated PCR-based detection of sequence polymorphisms in the internal transcribed spacer 2 (ITS2) region of the rRNA genes as a means of fungal identification. Clinical isolates (401), reference strains (6), and type strains (27), representing 34 species of yeasts were examined. The length of PCR-amplified ITS2 region DNA was determined with single-base precision in less than 30 min by using automated capillary electrophoresis. Unique, species-specific PCR products ranging from 237 to 429 bp were obtained from 92% of the clinical isolates. The remaining 8%, divided into groups with ITS2 regions which differed by ≤2 bp in mean length, all contained species-specific DNA sequences easily distinguishable by restriction enzyme analysis. These data, and the specificity of length polymorphisms for identifying yeasts, were confirmed by DNA sequence analysis of the ITS2 region from 93 isolates. Phenotypic and ITS2-based identification was concordant for 427 of 434 yeast isolates examined using sequence identity of ≥99%. Seven clinical isolates contained ITS2 sequences that did not agree with their phenotypic identification, and ITS2-based phylogenetic analyses indicate the possibility of new or clinically unusual species in the Rhodotorula and Candida genera. This work establishes an initial database, validated with over 400 clinical isolates, of ITS2 length and sequence polymorphisms for 34 species of yeasts. We conclude that size and restriction analysis of PCR-amplified ITS2 region DNA is a rapid and reliable method to identify clinically significant yeasts, including potentially new or emerging pathogenic species. PMID:10834993

Genotyping of Chromobacterium violaceum isolates by recA PCR-RFLP analysis.

PubMed

Scholz, Holger Christian; Witte, Angela; Tomaso, Herbert; Al Dahouk, Sascha; Neubauer, Heinrich

2005-03-15

Intraspecies variation of Chromobacterium violaceum was examined by comparative sequence - and by restriction fragment length polymorphism analysis of the recombinase A gene (recA-PCR-RFLP). Primers deduced from the known recA gene sequence of the type strain C. violaceum ATCC 12472(T) allowed the specific amplification of a 1040bp recA fragment from each of the 13 C. violaceum strains investigated, whereas other closely related organisms tested negative. HindII-PstI-recA RFLP analysis generated from 13 representative C. violaceum strains enabled us to identify at least three different genospecies. In conclusion, analysis of the recA gene provides a rapid and robust nucleotide sequence-based approach to specifically identify and classify C. violaceum on genospecies level.

Designing a SCAR molecular marker for monitoring Trichoderma cf. harzianum in experimental communities.

PubMed

Pérez, Gabriel; Verdejo, Valentina; Gondim-Porto, Clarissa; Orlando, Julieta; Carú, Margarita

2014-11-01

Several species of the fungal genus Trichoderma establish biological interactions with various micro- and macro-organisms. Some of these interactions are relevant in ecological terms and in biotechnological applications, such as biocontrol, where Trichoderma could be considered as an invasive species that colonizes a recipient community. The success of this invasion depends on multiple factors, which can be assayed using experimental communities as study models. Therefore, the aim of this work is to develop a species-specific sequence-characterized amplified region (SCAR) marker to monitor the colonization and growth of T. cf. harzianum when it invades experimental communities. For this study, 16 randomly amplified polymorphic DNA (RAPD) primers of 10-mer were used to generate polymorphic patterns, one of which generated a band present only in strains of T. cf. harzianum. This band was cloned, sequenced, and five primers of 20-23 mer were designed. Primer pairs 2F2/2R2 and 2F2/2R3 successfully and specifically amplified fragments of 278 and 448 bp from the T. cf. harzianum BpT10a strain DNA, respectively. Both primer pairs were also tested against the DNA from 14 strains of T. cf. harzianum and several strains of different fungal genera as specificity controls. Only the DNA from the strains of T. cf. harzianum was successfully amplified. Moreover, primer pair 2F2/2R2 was assessed by quantitative real-time polymerase chain reaction (PCR) using fungal DNA mixtures and DNA extracted from fungal experimental communities as templates. T. cf. harzianum was detectable even when as few as 100 copies of the SCAR marker were available or even when its population represented only 0.1% of the whole community.

Designing a SCAR molecular marker for monitoring Trichoderma cf. harzianum in experimental communities* #

PubMed Central

Pérez, Gabriel; Verdejo, Valentina; Gondim-Porto, Clarissa; Orlando, Julieta; Carú, Margarita

2014-01-01

Several species of the fungal genus Trichoderma establish biological interactions with various micro- and macro-organisms. Some of these interactions are relevant in ecological terms and in biotechnological applications, such as biocontrol, where Trichoderma could be considered as an invasive species that colonizes a recipient community. The success of this invasion depends on multiple factors, which can be assayed using experimental communities as study models. Therefore, the aim of this work is to develop a species-specific sequence-characterized amplified region (SCAR) marker to monitor the colonization and growth of T. cf. harzianum when it invades experimental communities. For this study, 16 randomly amplified polymorphic DNA (RAPD) primers of 10-mer were used to generate polymorphic patterns, one of which generated a band present only in strains of T. cf. harzianum. This band was cloned, sequenced, and five primers of 20–23 mer were designed. Primer pairs 2F2/2R2 and 2F2/2R3 successfully and specifically amplified fragments of 278 and 448 bp from the T. cf. harzianum BpT10a strain DNA, respectively. Both primer pairs were also tested against the DNA from 14 strains of T. cf. harzianum and several strains of different fungal genera as specificity controls. Only the DNA from the strains of T. cf. harzianum was successfully amplified. Moreover, primer pair 2F2/2R2 was assessed by quantitative real-time polymerase chain reaction (PCR) using fungal DNA mixtures and DNA extracted from fungal experimental communities as templates. T. cf. harzianum was detectable even when as few as 100 copies of the SCAR marker were available or even when its population represented only 0.1% of the whole community. PMID:25367789

Polymorphisms in the lcrV gene of Yersinia enterocolitica and their effect on plague protective immunity.

PubMed

Miller, Nathan C; Quenee, Lauriane E; Elli, Derek; Ciletti, Nancy A; Schneewind, Olaf

2012-04-01

Current efforts to develop plague vaccines focus on LcrV, a polypeptide that resides at the tip of type III secretion needles. LcrV-specific antibodies block Yersinia pestis type III injection of Yop effectors into host immune cells, thereby enabling phagocytes to kill the invading pathogen. Earlier work reported that antibodies against Y. pestis LcrV cannot block type III injection by Yersinia enterocolitica strains and suggested that lcrV polymorphisms may provide for escape from LcrV-mediated plague immunity. We show here that polyclonal or monoclonal antibodies raised against Y. pestis KIM D27 LcrV (LcrV(D27)) bind LcrV from Y. enterocolitica O:9 strain W22703 (LcrV(W22703)) or O:8 strain WA-314 (LcrV(WA-314)) but are otherwise unable to block type III injection by Y. enterocolitica strains. Replacing the lcrV gene on the pCD1 virulence plasmid of Y. pestis KIM D27 with either lcrV(W22703) or lcrV(WA-314) does not affect the ability of plague bacteria to secrete proteins via the type III pathway, to inject Yops into macrophages, or to cause lethal plague infections in mice. LcrV(D27)-specific antibodies blocked type III injection by Y. pestis expressing lcrV(W22703) or lcrV(WA-314) and protected mice against intravenous lethal plague challenge with these strains. Thus, although antibodies raised against LcrV(D27) are unable to block the type III injection of Y. enterocolitica strains, expression of lcrV(W22703) or lcrV(WA-314) in Y. pestis did not allow these strains to escape LcrV-mediated plague protective immunity in the intravenous challenge model.

Primary hypolactasia diagnosis: Comparison between the gaxilose test, shortened lactose tolerance test, and clinical parameters corresponding to the C/T-13910 polymorphism.

PubMed

Domínguez Jiménez, José Luis; Fernández Suárez, Antonio; Muñoz Colmenero, Aurora Úrsula; Fatela Cantillo, Daniel; López Pelayo, Iratxe

2017-04-01

There is no consensus on the most accurate method to diagnose primary hypolactasia. We aimed to compare the diagnostic accuracy of the new gaxilose test with 2 traditional tests (lactose tolerance test and clinical criteria) for the diagnosis of primary hypolactasia using the C/T-13910 polymorphism as a reference standard. Patients with a clinical suspicion of lactose intolerance were subjected to gaxilose tests, shortened lactose tolerance tests, and symptom questionnaires before and after overload with 50 g lactose and after a lactose-free diet. The diagnostic accuracy and degree of agreement and correlation were assessed using a genetic test (C/T-13910 polymorphism) as a reference standard and their respective 95% confidence intervals. Thirty consecutive patients (70% women) participated in the study. The genetic test confirmed the C/T-13910 polymorphism in 11 patients (36.8%). The presence of diarrhoea and the symptom score after lactose overload, along with the tolerance test, were the variables with the highest degree of agreement (κ > 0.60). Area under the ROC curve was >0.82 (p < 0.05), with sensitivity and specificity values of >0.80. However, the gaxilose test obtained lower values: κ, 0.47; area under curve, 0.75 (0.57-0.94); sensitivity, 0.82 (0.55-1); and specificity, 0.68 (0.45-0.92). The multivariate analysis showed an association between the post-overload symptom questionnaire and the results of the genetic test (odds ratio: 1.17; 1.04-1.31; p < 0.01). The presence of diarrhoea and the symptom score after overload with 50 g lactose showed a higher degree of agreement and diagnostic accuracy for primary hypolactasia than the gaxilose test when the genetic test is used as a reference standard. Copyright © 2016 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

[Effect of elastic strain rate ratio method and virtual touch tissue quantification on the diagnosis of breast masses].

PubMed

Gong, LiJie; He, Yan; Tian, Peng; Yan, Yan

2016-07-01

To determine the effect of elastic strain rate ratio method and virtual touch tissue quantification (VTQ) on the diagnosis of breast masses.
 Sixty female patients with breast cancer, who received surgical treatment in Daqing Oilfield General Hospital, were enrolled. All patients signed the informed consent paperwork and they were treated by routine ultrasound examination, compression elastography (CE) examination, and VTQ examination in turn. Strain ratio (SR) was checked by CE and shear wave velocity (SWV) value was measured by VTQ. The diagnostic values of different methods were evaluated by receiver operating characteristic (ROC) curves in the diagnosis of benign and malignant breast tumors.
 The maximum diameter and SWV value of the benign tumors were lower than those of the malignant tumors, and the SR ratio of benign masses was higher than that of malignant tumors (P<0.01). The AUC, sensitivity and specificity for elastic strain rate and VTQ for single or combined use were higher than those of conventional ultrasound (0.904, 97.5%, 69.2%; 0.946, 87.5%, 87.2%; 0.976, 90%, 97.4% vs 0.783, 85%, 61.5%). The AUC and specificity of VTQ were higher than those of the elastic strain rate (0.946, 87.2% vs 0.904, 69.2%), but the sensitivity of VTQ was higher than that of the latter (87.5% vs 97.5%). The AUC and specificity for combination of both methods were higher than those of single method, but the sensitivity was lower than that of the elastic strain rate. 
 Combination of elastic strain rate ratio method with VTQ possesses the best diagnostic value and the highest diagnostic accuracy in the diagnosis of breast mass than that used alone.

Use of bacteriophage cell wall-binding proteins for rapid diagnostics of Listeria.

PubMed

Schmelcher, Mathias; Loessner, Martin J

2014-01-01

Diagnostic protocols for food-borne bacterial pathogens such as Listeria need to be sensitive, specific, rapid, and inexpensive. Conventional culture methods are hampered by lengthy enrichment and incubation steps. Bacteriophage-derived high-affinity binding molecules (cell wall-binding domains, CBDs) specific for Listeria cells have recently been introduced as tools for detection and differentiation of this pathogen in foods. When coupled with magnetic separation, these proteins offer advantages in sensitivity and speed compared to the standard diagnostic methods. Furthermore, fusion of CBDs to differently colored fluorescent reporter proteins enables differentiation of Listeria strains in mixed cultures. This chapter provides protocols for detection of Listeria in food by CBD-based magnetic separation and subsequent multiplexed identification of strains of different serotypes with reporter-CBD fusion proteins.

Comparative Transcriptome of Wild Type and Selected Strains of the Microalgae Tisochrysis lutea Provides Insights into the Genetic Basis, Lipid Metabolism and the Life Cycle

PubMed Central

Carrier, Gregory; Garnier, Matthieu; Le Cunff, Loïc; Bougaran, Gaël; Probert, Ian; De Vargas, Colomban; Corre, Erwan; Cadoret, Jean-Paul; Saint-Jean, Bruno

2014-01-01

The applied exploitation of microalgae cultures has to date almost exclusively involved the use of wild type strains, deposited over decades in dedicated culture collections. Concomitantly, the concept of improving algae with selection programs for particular specific purposes is slowly emerging. Studying since a decade an economically and ecologically important haptophyte Tisochrysis lutea (Tiso), we took advantage of the availability of wild type (Tiso-Wt) and selected (Tiso-S2M2) strains to conduct a molecular variations study. This endeavour presented substantial challenges: the genome assembly was not yet available, the life cycle unknown and genetic diversity of Tiso-Wt poorly documented. This study brings the first molecular data in order to set up a selection strategy for that microalgae. Following high-throughput Illumina sequencing, transcriptomes of Tiso-Wt and Tiso-S2M2 were de novo assembled and annotated. Genetic diversity between both strains was analyzed and revealed a clear conservation, while a comparison of transcriptomes allowed identification of polymorphisms resulting from the selection program. Of 34,374 transcripts, 291 were differentially expressed and 165 contained positional polymorphisms (SNP, Indel). We focused on lipid over-accumulation of the Tiso-S2M2 strain and 8 candidate genes were identified by combining analysis of positional polymorphism, differential expression levels, selection signature and by study of putative gene function. Moreover, genetic analysis also suggests the existence of a sexual cycle and genetic recombination in Tisochrysis lutea. PMID:24489800

Comprehensive analysis of Salmonella sequence polymorphisms and development of a LDR-UA assay for the detection and characterization of selected serotypes.

PubMed

Lauri, Andrea; Castiglioni, Bianca; Mariani, Paola

2011-07-01

Salmonella is a major cause of food-borne disease, and Salmonella enterica subspecies I includes the most clinically relevant serotypes. Salmonella serotype determination is important for the disease etiology assessment and contamination source tracking. This task will be facilitated by the disclosure of Salmonella serotype sequence polymorphisms, here annotated in seven genes (sefA, safA, safC, bigA, invA, fimA, and phsB) from 139 S. enterica strains, of which 109 belonging to 44 serotypes of subsp. I. One hundred nineteen polymorphic sites were scored and associated to single serotypes or to serotype groups belonging to S. enterica subsp. I. A diagnostic tool was constructed based on the Ligation Detection Reaction-Universal Array (LDR-UA) for the detection of polymorphic sites uniquely associated to serotypes of primary interest (Salmonella Hadar, Salmonella Infantis, Salmonella Enteritidis, Salmonella Typhimurium, Salmonella Gallinarum, Salmonella Virchow, and Salmonella Paratyphi B). The implementation of promiscuous probes allowed the diagnosis of ten further serotypes that could be associated to a unique hybridization pattern. Finally, the sensitivity and applicability of the tool was tested on target DNA dilutions and with controlled meat contamination, allowing the detection of one Salmonella CFU in 25 g of meat.

Nuclear Ribosomal DNA Variation and Pathogenic Specialization in Alternaria Fungi Known To Produce Host-Specific Toxins †

PubMed Central

Kusaba, Motoaki; Tsuge, Takashi

1994-01-01

A total of 99 strains of 11 Alternaria species, including 68 strains of seven fungi known to produce host-specific toxins, were subjected to analysis of restriction fragment length polymorphism (RFLP) in nuclear ribosomal DNA (rDNA). Total DNA was digested with XbaI, and the Southern blots were probed with a nuclear rDNA clone of Alternaria kikuchiana. The hybridization gave 17 different RFLPs from the 99 strains. On the basis of these RFLPs, populations of host-specific toxin-producing fungi could not be differentiated from one another nor from nonpathogenic A. alternata. Each population of the toxin-producing fungi carried rDNA variants. Nine different types, named A1 to A6 and B1 to B3, were detected among the toxin-producing fungi and nonpathogenic A. alternata. All of the populations contained the type A4 variant, and the other rDNA types were also shared by different toxin-producing fungi and A. alternata. In contrast, Alternaria species that are morphologically distinguishable from A. alternata could be differentiated from A. alternata on the basis of the rDNA RFLPs. Polymorphisms in rDNA digested with HaeIII and MspI were also evaluated in 61 Alternaria strains. These restriction enzymes produced 31 variations among all of the samples. The seven toxin-producing fungi and nonpathogenic A. alternata could not be resolved by phylogenetic analysis based on the RFLPs, although they could be differentiated from the other Alternaria species studied. These results provide support for the hypothesis that Alternaria fungi known to produce host-specific toxins are intraspecific variants of A. alternata specialized in pathogenicity. Images PMID:16349367

Natural variation of piRNA expression affects immunity to transposable elements.

PubMed

Ryazansky, Sergei; Radion, Elizaveta; Mironova, Anastasia; Akulenko, Natalia; Abramov, Yuri; Morgunova, Valeriya; Kordyukova, Maria Y; Olovnikov, Ivan; Kalmykova, Alla

2017-04-01

In the Drosophila germline, transposable elements (TEs) are silenced by PIWI-interacting RNA (piRNA) that originate from distinct genomic regions termed piRNA clusters and are processed by PIWI-subfamily Argonaute proteins. Here, we explore the variation in the ability to restrain an alien TE in different Drosophila strains. The I-element is a retrotransposon involved in the phenomenon of I-R hybrid dysgenesis in Drosophila melanogaster. Genomes of R strains do not contain active I-elements, but harbour remnants of ancestral I-related elements. The permissivity to I-element activity of R females, called reactivity, varies considerably in natural R populations, indicating the existence of a strong natural polymorphism in defense systems targeting transposons. To reveal the nature of such polymorphisms, we compared ovarian small RNAs between R strains with low and high reactivity and show that reactivity negatively correlates with the ancestral I-element-specific piRNA content. Analysis of piRNA clusters containing remnants of I-elements shows increased expression of the piRNA precursors and enrichment by the Heterochromatin Protein 1 homolog, Rhino, in weak R strains, which is in accordance with stronger piRNA expression by these regions. To explore the nature of the differences in piRNA production, we focused on two R strains, weak and strong, and showed that the efficiency of maternal inheritance of piRNAs as well as the I-element copy number are very similar in both strains. At the same time, germline and somatic uni-strand piRNA clusters generate more piRNAs in strains with low reactivity, suggesting the relationship between the efficiency of primary piRNA production and variable response to TE invasions. The strength of adaptive genome defense is likely driven by naturally occurring polymorphisms in the rapidly evolving piRNA pathway proteins. We hypothesize that hyper-efficient piRNA production is contributing to elimination of a telomeric retrotransposon HeT-A, which we have observed in one particular transposon-resistant R strain.

Impact of gamma rays on the Phaffia rhodozyma genome revealed by RAPD-PCR.

PubMed

Najafi, N; Hosseini, Ramin; Ahmadi, Ar

2011-12-01

Phaffia rhodozyma is a red yeast which produces astaxanthin as the major carotenoid pigment. Astaxanthin is thought to reduce the incidence of cancer and degenerative diseases in man. It also enhances the immune response and acts as a free-radical quencher, a precursor of vitamin A, or a pigment involved in the visual attraction of animals as mating partners. The impact of gamma irradiation was studied on the Phaffia rhodozyma genome. Ten mutant strains, designated Gam1-Gam10, were obtained using gamma irradiation. Ten decamer random amplified polymorphic DNA (RAPD) primers were employed to assess genetic changes. Nine primers revealed scorable polymorphisms and a total of 95 band positions were scored; amongst which 38 bands (37.5%) were polymorphic. Primer F with 3 bands and primer J20 with 13 bands produced the lowest and the highest number of bands, respectively. Primer A16 produced the highest number of polymorphic bands (70% polymorphism) and primer F showed the lowest number of polymorphic bands (0% polymorphism). Genetic distances were calculated using Jaccard's coefficient and the UPGMA method. A dendrogram was created using SPSS (version 11.5) and the strains were clustered into four groups. RAPD markers could distinguish between the parental and the mutant strains of P. rhodozyma. RAPD technique showed that some changes had occurred in the genome of the mutated strains. This technique demonstrated the capability to differentiate between the parental and the mutant strains.

Identification of Legionella Species by Random Amplified Polymorphic DNA Profiles

PubMed Central

Lo Presti, François; Riffard, Serge; Vandenesch, François; Etienne, Jerome

1998-01-01

Random amplified polymorphic DNA (RAPD) was used for the identification of Legionella species. Primer SK2 (5′-CGGCGGCGGCGG-3′) and standardized RAPD conditions gave the technique a reproducibility of 93 to 100%, depending on the species tested. Species-specific patterns corresponding to the 42 Legionella species were consequently defined by this method; the patterns were dependent on the recognition of a core of common bands for each species. This specificity was demonstrated by testing 65 type strains and 265 environmental and clinical isolates. No serogroup-specific profiles were obtained. A number of unidentified Legionella isolates potentially corresponding to new species were clustered in four groups. RAPD analysis appears to be a rapid and reproducible technique for identification of Legionella isolates to the species level without further restriction or hybridization. PMID:9774564

Physiological typing of Pseudallescheria and Scedosporium strains using Taxa Profile, a semi-automated, 384-well microtitre system.

PubMed

Horré, R; Schaal, K P; Marklein, G; de Hoog, G S; Reiffert, S-M

2011-10-01

During the last few decades, Pseudallescheria and Scedosporium infections in humans are noted with increasing frequency. Multi-drug resistance commonly occurring in this species complex interferes with adequate therapy. Rapid and correct identification of clinical isolates is of paramount significance for optimal treatment in the early stages of infection, while strain typing is necessary for epidemiological purposes. In view of the development of physiological diagnostic parameters, 570 physiological reactions were evaluated using the Taxa Profile Micronaut system, a semi-automatic, computer-assisted, 384-well microtitre platform. Thirty two strains of the Pseudallescheria and Scedosporium complex were analysed after molecular verification of correct species attribution. Of the compounds tested, 254 proved to be polymorphic. Cluster analysis was performed with the Micronaut profile software, which is linked to the ntsypc® program. The systemic opportunist S. prolificans was unambiguously separated from the remaining species. Within the P. boydii/P. apiosperma complex differentiation was noted at the level of individual strains, but no unambiguous parameters for species recognition were revealed. © 2011 Blackwell Verlag GmbH.

Aspergillus collagen-like genes (acl): identification, sequence polymorphism, and assessment for PCR-based pathogen detection.

PubMed

Tuntevski, Kiril; Durney, Brandon C; Snyder, Anna K; Lasala, P Rocco; Nayak, Ajay P; Green, Brett J; Beezhold, Donald H; Rio, Rita V M; Holland, Lisa A; Lukomski, Slawomir

2013-12-01

The genus Aspergillus is a burden to public health due to its ubiquitous presence in the environment, its production of allergens, and wide demographic susceptibility among cystic fibrosis, asthmatic, and immunosuppressed patients. Current methods of detection of Aspergillus colonization and infection rely on lengthy morphological characterization or nonstandardized serological assays that are restricted to identifying a fungal etiology. Collagen-like genes have been shown to exhibit species-specific conservation across the noncollagenous regions as well as strain-specific polymorphism in the collagen-like regions. Here we assess the conserved region of the Aspergillus collagen-like (acl) genes and explore the application of PCR amplicon size-based discrimination among the five most common etiologic species of the Aspergillus genus, including Aspergillus fumigatus, A. flavus, A. nidulans, A. niger, and A. terreus. Genetic polymorphism and phylogenetic analysis of the aclF1 gene were additionally examined among the available strains. Furthermore, the applicability of the PCR-based assay to identification of these five species in cultures derived from sputum and bronchoalveolar fluid from 19 clinical samples was explored. Application of capillary electrophoresis on nanogels was additionally demonstrated to improve the discrimination between Aspergillus species. Overall, this study demonstrated that Aspergillus acl genes could be used as PCR targets to discriminate between clinically relevant Aspergillus species. Future studies aim to utilize the detection of Aspergillus acl genes in PCR and microfluidic applications to determine the sensitivity and specificity for the identification of Aspergillus colonization and invasive aspergillosis in immunocompromised subjects.

Diagnosis of Caprine Arthritis Encephalitis Virus infection in dairy goats by ELISA, PCR and Viral Culture.

PubMed

Panneum, S; Rukkwamsuk, T

2017-03-01

For preventive and control strategies of Caprine Arthritis Encephalitis Virus (CAEV) infection in dairy goats, performance of the available diagnostic tests was described as one of the most important and necessary aspects. The study aimed at evaluating the diagnostic test performance, including PCR, ELISA and viral culture, for CAEV infection in dairy goats in Thailand. Blood samples of 29 dairy goats from five low- to medium-prevalence herds and one very low-prevalence herd were collected for PCR and ELISA methods. The performance of these two diagnostic methods was evaluated by comparing with cytopathic effects (CPE) in the co-cultivation of CAEV and primary synovial cells. Results indicated that sensitivity, specificity were, respectively, 69.6%, 100%, for PCR; and 95.7%, 83.3% for ELISA. The PCR assay tended to have lower sensitivity and higher specificity than ELISA. When multiple tests were applied, parallel testing provided sensitivity and specificity of 98.7% and 83.3%, while series testing showed sensitivity and specificity of 66.6% and 100% respectively. These results indicated that combination of ELISA and PCR provided some advantages and possibly offered optimal methods to detect CAEV-infected goats. Kappa value of the agreement between PCR and ELISA test was 0.34, indicating fair agreement. Regarding the possibility of antigenic variation between CAEV strains used in both PCR and ELISA assays, the actual circulating CAEV strain should be reviewed in order to develop and enhance the diagnostic tests using the CAE viral antigens derived from specific local strains of Thailand.

Diversity of Survival Patterns among Escherichia coli O157:H7 Genotypes Subjected to Food-Related Stress Conditions.

PubMed

Elhadidy, Mohamed; Álvarez-Ordóñez, Avelino

2016-01-01

The purpose of this study was to evaluate the resistance patterns to food-related stresses of Shiga toxin producing Escherichia coli O157:H7 strains belonging to specific genotypes. A total of 33 E. coli O157:H7 strains were exposed to seven different stress conditions acting as potential selective pressures affecting the transmission of E. coli O157:H7 to humans through the food chain. These stress conditions included cold, oxidative, osmotic, acid, heat, freeze-thaw, and starvation stresses. The genotypes used for comparison included lineage-specific polymorphism, Shiga-toxin-encoding bacteriophage insertion sites, clade type, tir (A255T) polymorphism, Shiga toxin 2 subtype, and antiterminator Q gene allele. Bacterial resistance to different stressors was calculated by determining D-values (times required for inactivation of 90% of the bacterial population), which were then subjected to univariate and multivariate analyses. In addition, a relative stress resistance value, integrating resistance values to all tested stressors, was calculated for each bacterial strain and allowed for a ranking-type classification of E. coli O157:H7 strains according to their environmental robustness. Lineage I/II strains were found to be significantly more resistant to acid, cold, and starvation stress than lineage II strains. Similarly, tir (255T) and clade 8 encoding strains were significantly more resistant to acid, heat, cold, and starvation stress than tir (255A) and non-clade 8 strains. Principal component analysis, which allows grouping of strains with similar stress survival characteristics, separated strains of lineage I and I/II from strains of lineage II, which in general showed reduced survival abilities. Results obtained suggest that lineage I/II, tir (255T), and clade 8 strains, which have been previously reported to be more frequently associated with human disease cases, have greater multiple stress resistance than strains of other genotypes. The results from this study provide a better insight into how selective pressures encountered through the food chain may play a role in the epidemiology of STEC O157:H7 through controlling the transmission of highly adapted strains to humans.

Diversity of Survival Patterns among Escherichia coli O157:H7 Genotypes Subjected to Food-Related Stress Conditions

PubMed Central

Elhadidy, Mohamed; Álvarez-Ordóñez, Avelino

2016-01-01

The purpose of this study was to evaluate the resistance patterns to food-related stresses of Shiga toxin producing Escherichia coli O157:H7 strains belonging to specific genotypes. A total of 33 E. coli O157:H7 strains were exposed to seven different stress conditions acting as potential selective pressures affecting the transmission of E. coli O157:H7 to humans through the food chain. These stress conditions included cold, oxidative, osmotic, acid, heat, freeze-thaw, and starvation stresses. The genotypes used for comparison included lineage-specific polymorphism, Shiga-toxin-encoding bacteriophage insertion sites, clade type, tir (A255T) polymorphism, Shiga toxin 2 subtype, and antiterminator Q gene allele. Bacterial resistance to different stressors was calculated by determining D-values (times required for inactivation of 90% of the bacterial population), which were then subjected to univariate and multivariate analyses. In addition, a relative stress resistance value, integrating resistance values to all tested stressors, was calculated for each bacterial strain and allowed for a ranking-type classification of E. coli O157:H7 strains according to their environmental robustness. Lineage I/II strains were found to be significantly more resistant to acid, cold, and starvation stress than lineage II strains. Similarly, tir (255T) and clade 8 encoding strains were significantly more resistant to acid, heat, cold, and starvation stress than tir (255A) and non-clade 8 strains. Principal component analysis, which allows grouping of strains with similar stress survival characteristics, separated strains of lineage I and I/II from strains of lineage II, which in general showed reduced survival abilities. Results obtained suggest that lineage I/II, tir (255T), and clade 8 strains, which have been previously reported to be more frequently associated with human disease cases, have greater multiple stress resistance than strains of other genotypes. The results from this study provide a better insight into how selective pressures encountered through the food chain may play a role in the epidemiology of STEC O157:H7 through controlling the transmission of highly adapted strains to humans. PMID:27014242

Detection of Penicillin Susceptibility in Streptococcus pneumoniae by pbp2b PCR-Restriction Fragment Length Polymorphism Analysis

PubMed Central

O’Neill, A. M.; Gillespie, S. H.; Whiting, G. C.

1999-01-01

A PCR-restriction fragment length polymorphism strategy directed against the pbp2b gene was evaluated for identification of penicillin susceptibility. A total of 106 United Kingdom (U.K.), 30 Danish, and 11 Papua New Guinean strains were tested. Of the U.K. strains, all the susceptible and all but one of the resistant isolates were correctly assigned. By using conventional definitions of “not resistant” and “not susceptible,” the sensitivities were 97.5 and 94.4%, the specificities were 100 and 98.9%, the positive predictive values were 100 and 94.4%, and the negative predictive values were 93.1 and 98.9%, respectively. This technique may allow susceptible (MIC, <0.1 mg/liter) and resistant (MIC, >1 mg/liter) isolates to be distinguished in a single PCR. PMID:9854082

Comparative genome analysis identifies novel nucleic acid diagnostic targets for use in the specific detection of Haemophilus influenzae.

PubMed

Coughlan, Helena; Reddington, Kate; Tuite, Nina; Boo, Teck Wee; Cormican, Martin; Barrett, Louise; Smith, Terry J; Clancy, Eoin; Barry, Thomas

2015-10-01

Haemophilus influenzae is recognised as an important human pathogen associated with invasive infections, including bloodstream infection and meningitis. Currently used molecular-based diagnostic assays lack specificity in correctly detecting and identifying H. influenzae. As such, there is a need to develop novel diagnostic assays for the specific identification of H. influenzae. Whole genome comparative analysis was performed to identify putative diagnostic targets, which are unique in nucleotide sequence to H. influenzae. From this analysis, we identified 2H. influenzae putative diagnostic targets, phoB and pstA, for use in real-time PCR diagnostic assays. Real-time PCR diagnostic assays using these targets were designed and optimised to specifically detect and identify all 55H. influenzae strains tested. These novel rapid assays can be applied to the specific detection and identification of H. influenzae for use in epidemiological studies and could also enable improved monitoring of invasive disease caused by these bacteria. Copyright © 2015 Elsevier Inc. All rights reserved.

[Tuberculous cheilitis revealing pulmonary tuberculosis].

PubMed

Bricha, Myriem; Slimani, Hajar; Hammi, Sanae; Bourkadi, Jamal Eddine

2016-01-01

Tuberculosis of the oral cavity is rare. It is associated with clinical polymorphism and poses above all a diagnostic problem. We report the case of a 42-year-old male patient with tubercular cheilitis. This study aims to focus attention on tuberculosis that can be detected exceptionally in specific locations, such as the oral lip.

Characterization of Heterobasidion occidentale transcriptomes reveals candidate genes and DNA polymorphisms for virulence variations.

PubMed

Liu, Jun-Jun; Shamoun, Simon Francis; Leal, Isabel; Kowbel, Robert; Sumampong, Grace; Zamany, Arezoo

2018-05-01

Characterization of genes involved in differentiation of pathogen species and isolates with variations of virulence traits provides valuable information to control tree diseases for meeting the challenges of sustainable forest health and phytosanitary trade issues. Lack of genetic knowledge and genomic resources hinders novel gene discovery, molecular mechanism studies and development of diagnostic tools in the management of forest pathogens. Here, we report on transcriptome profiling of Heterobasidion occidentale isolates with contrasting virulence levels. Comparative transcriptomic analysis identified orthologous groups exclusive to H. occidentale and its isolates, revealing biological processes involved in the differentiation of isolates. Further bioinformatics analyses identified an H. occidentale secretome, CYPome and other candidate effectors, from which genes with species- and isolate-specific expression were characterized. A large proportion of differentially expressed genes were revealed to have putative activities as cell wall modification enzymes and transcription factors, suggesting their potential roles in virulence and fungal pathogenesis. Next, large numbers of simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) were detected, including more than 14 000 interisolate non-synonymous SNPs. These polymorphic loci and species/isolate-specific genes may contribute to virulence variations and provide ideal DNA markers for development of diagnostic tools and investigation of genetic diversity. © 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Variability in response to nicotine in the LSxSS RI strains: potential role of polymorphisms in alpha4 and alpha6 nicotinic receptor genes.

PubMed

Tritto, Theresa; Stitzel, Jerry A; Marks, Michael J; Romm, Elena; Collins, Allan C

2002-04-01

Several studies have shown that genetic factors influence the effects of nicotine on respiration, acoustic startle, Y-maze crosses and rears, heart rate and body temperature in the mouse. Recently, we identified restriction fragment length polymorphisms (RFLPs) associated with the alpha4 (Chrna4) and alpha6 (Chrna6) nicotinic cholinergic receptor genes in the recombinant inbred (RI) strains derived from the Long-Sleep (LS) and Short-Sleep (SS) mouse lines. The alpha4 polymorphism has been identified as a point-mutation at position 529 (threonine to alanine) and the alpha6 polymorphism has not yet been identified. The studies described here evaluated the potential role of these polymorphisms in regulating sensitivity to nicotine by constructing dose-response curves for the effects of nicotine on six responses in the LSxSS RI strains. The results obtained suggest that both of the polymorphisms may play a role in regulating variability in sensitivity to nicotine. Those RI strains carrying the LS-like alpha4 RFLP were significantly more sensitive to the effects of nicotine on Y-maze crosses and rears, temperature and respiration and were less sensitive to the effects of nicotine on acoustic startle than those strains carrying the SS-like alpha4 RFLP. Those RI strains carrying the LS-like alpha6 RFLP were more sensitive to the effects of nicotine on respiration and acoustic startle, and less sensitive to the effects of nicotine on Y-maze crosses than those strains carrying the SS-like alpha6 RFLP. These results suggest that genetically determined differences in sensitivity to nicotine may be explained, in part, by variability associated with at least two of the neuronal nicotinic receptor genes, alpha4 and alpha6.

Use of molecular techniques to evaluate the survival of a microorganism injected into an aquifer

USGS Publications Warehouse

Thiem, S.M.; Krumme, M.L.; Smith, R.L.; Tiedje, J.M.

1994-01-01

A PCR primer set and an internal probe that are specific for Pseudomonas sp. strain B13, a 3-chlorobenzoate-metabolizing strain, were developed. Using this primer set and probe, we were able to detect Pseudomonas sp. strain B13 DNA sequences in DNA extracted from aquifer samples 14.5 months after Pseudomonas sp. strain B13 had been injected into a sand and gravel aquifer. This primer set and probe were also used to analyze isolates from 3-chlorobenzoate enrichments of the aquifer samples by Southern blot analysis. Hybridization of Southern blots with the Pseudomonas sp. strain B13-specific probe and a catabolic probe in conjunction with restriction fragment length polymorphism (RFLP) analysis of ribosome genes was used to determine that viable Pseudomonas sp. strain B13 persisted in this environment. We isolated a new 3-chlorobenzoate-degrading strain from one of these enrichment cultures. The B13-specific probe does not hybridize to DNA from this isolate. The new strain could be the result of gene exchange between Pseudomonas sp. strain B13 and an indigenous bacterium. This speculation is based on an RFLP pattern of ribosome genes that differs from that of Pseudomonas sp. strain B13, the fact that identically sized restriction fragments hybridized to the catabolic gene probe, and the absence of any enrichable 3-chlorobenzoate-degrading strains in the aquifer prior to inoculation.

Characterization of early follicular cDNA library suggests evidence for genetic polymorphisms in the inbred strain C108 of Bombyx mori.

PubMed

Mills, D R; Goldsmith, M R

2000-04-01

Recent work towards the completion of a saturated molecular genetic linkage map for the lepidopteran silkworm, Bombyx mori (n = 28), has provided evidence for existing polymorphisms in the inbred strain C108. Two inbred parental strains, p50 and C108, were crossed to produce the F1 (P/C) hybrid offspring. The populations used in this project were comprised of a combination of 29 F2 (F1 x F1) and 31 reciprocal backcross (P/C x C/C, P/C x P/P) progeny. All restriction fragment length polymorphisms (RFLPs) for the initial analysis were hybridized with anonymous probes derived from a random early follicular cDNA (Rcf) library from Bombyx. A total of 19 Rcf probes were selected as showing scorable codominant polymorphic patterns when screened against F2 and backcross DNAs digested with the restriction enzymes EcoRI, HindIII, or PstI, and Southern blotted to nylon membranes for hybridization. Of the newly reported Rcf probes, 7 (37%) were characterized as producing 'simple' polymorphic patterns, while 12 (63%) were characterized as producing 'complex' polymorphic patterns. Further characterization of the complex patterns subdivided this group into two general classes: polymorphisms that contained an additional allele, and multiple bands that contained an easily scored two banded polymorphism. Because the extra allele class was limited to the (P/C x C/C) backcross progeny, it is suggested that the inbred parental strain C108 harbors polymorphic loci that are inherited in a simple Mendelian fashion. A genetic analysis discussing plausible origins and maintenance of these polymorphisms is presented.

The Rhoptry Proteins ROP18 and ROP5 Mediate Toxoplasma gondii Evasion of the Murine, But Not the Human, Interferon-Gamma Response

PubMed Central

Niedelman, Wendy; Gold, Daniel A.; Rosowski, Emily E.; Sprokholt, Joris K.; Lim, Daniel; Farid Arenas, Ailan; Melo, Mariane B.; Spooner, Eric; Yaffe, Michael B.; Saeij, Jeroen P. J.

2012-01-01

The obligate intracellular parasite Toxoplasma gondii secretes effector proteins into the host cell that manipulate the immune response allowing it to establish a chronic infection. Crosses between the types I, II and III strains, which are prevalent in North America and Europe, have identified several secreted effectors that determine strain differences in mouse virulence. The polymorphic rhoptry protein kinase ROP18 was recently shown to determine the difference in virulence between type I and III strains by phosphorylating and inactivating the interferon-γ (IFNγ)-induced immunity-related GTPases (IRGs) that promote killing by disrupting the parasitophorous vacuole membrane (PVM) in murine cells. The polymorphic pseudokinase ROP5 determines strain differences in virulence through an unknown mechanism. Here we report that ROP18 can only inhibit accumulation of the IRGs on the PVM of strains that also express virulent ROP5 alleles. In contrast, specific ROP5 alleles can reduce IRG coating even in the absence of ROP18 expression and can directly interact with one or more IRGs. We further show that the allelic combination of ROP18 and ROP5 also determines IRG evasion and virulence of strains belonging to other lineages besides types I, II and III. However, neither ROP18 nor ROP5 markedly affect survival in IFNγ-activated human cells, which lack the multitude of IRGs present in murine cells. These findings suggest that ROP18 and ROP5 have specifically evolved to block the IRGs and are unlikely to have effects in species that do not have the IRG system, such as humans. PMID:22761577

Polymorphic amplified typing sequences (PATS) and pulsed-field gel electrophoresis (PFGE) yield comparable results in the strain typing of a diverse set of bovine Escherichia coli O157 isolates

USDA-ARS?s Scientific Manuscript database

The PCR-based Escherichia coli O157 (O157) strain typing system, Polymorphic Amplified Typing Sequences (PATS), targets insertions-deletions (Indels) and single nucleotide polymorphisms (SNPs) at the XbaI and AvrII(BlnI) restriction enzyme sites, respectively, besides amplifying four known virulenc...

Epitaxially grown strained pentacene thin film on graphene membrane.

PubMed

Kim, Kwanpyo; Santos, Elton J G; Lee, Tae Hoon; Nishi, Yoshio; Bao, Zhenan

2015-05-06

Organic-graphene system has emerged as a new platform for various applications such as flexible organic photovoltaics and organic light emitting diodes. Due to its important implication in charge transport, the study and reliable control of molecular packing structures at the graphene-molecule interface are of great importance for successful incorporation of graphene in related organic devices. Here, an ideal membrane of suspended graphene as a molecular assembly template is utilized to investigate thin-film epitaxial behaviors. Using transmission electron microscopy, two distinct molecular packing structures of pentacene on graphene are found. One observed packing structure is similar to the well-known bulk-phase, which adapts a face-on molecular orientation on graphene substrate. On the other hand, a rare polymorph of pentacene crystal, which shows significant strain along the c-axis, is identified. In particular, the strained film exhibits a specific molecular orientation and a strong azimuthal correlation with underlying graphene. Through ab initio electronic structure calculations, including van der Waals interactions, the unusual polymorph is attributed to the strong graphene-pentacene interaction. The observed strained organic film growth on graphene demonstrates the possibility to tune molecular packing via graphene-molecule interactions. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cephalosporium maydis is a distinct species in the Gaeumannomyces-Harpophora species complex.

PubMed

Saleh, Amgad A; Leslie, John F

2004-01-01

Cephalosporium maydis is an important plant pathogen whose phylogenetic position relative to other fungi has not been established clearly. We compared strains of C. maydis, strains from several other plant-pathogenic Cephalosporium spp. and several possible relatives within the Gaeumannomyces-Harpophora species complex, to which C. maydis has been suggested to belong based on previous preliminary DNA sequence analyses. DNA sequences of the nuclear genes encoding the rDNA ITS region, β-tubulin, histone H3, and MAT-2 support the hypothesis that C. maydis is a distinct taxon within the Gaeumannomyces-Harpophora species complex. Based on amplified fragment length polymorphism (AFLP) profiles, C. maydis also is distinct from the other tested species of Cephalosporium, Phialophora sensu lato and members of Gaeumannomyces-Harpophora species complex, which supports its classification as Harpophora maydis. Oligonucleotide primers for H. maydis were developed that can be used in a PCR diagnostic protocol to rapidly and reliably detect and identify this pathogen. These diagnostic PCR primers will aid the detection of H. maydis in diseased maize because this fungus can be difficult to detect and isolate, and the movement of authentic cultures may be limited by quarantine restrictions.

Genetic Diversity of Toll-Like Receptors and Immunity to M. leprae Infection

PubMed Central

Hart, Bryan E.; Tapping, Richard I.

2012-01-01

Genetic association studies of leprosy cohorts across the world have identified numerous polymorphisms which alter susceptibility and outcome to infection with Mycobacterium leprae. As expected, many of the polymorphisms reside within genes that encode components of the innate and adaptive immune system. Despite the preponderance of these studies, our understanding of the mechanisms that underlie these genetic associations remains sparse. Toll-like receptors (TLRs) have emerged as an essential family of innate immune pattern recognition receptors which play a pivotal role in host defense against microbes, including pathogenic strains of mycobacteria. This paper will highlight studies which have uncovered the association of specific TLR gene polymorphisms with leprosy or tuberculosis: two important diseases resulting from mycobacterial infection. This analysis will focus on the potential influence these polymorphic variants have on TLR expression and function and how altered TLR recognition or signaling may contribute to successful antimycobacterial immunity. PMID:22529866

Photobacterium damselae ssp. piscicida: detection by direct amplification of 16S rRNA gene sequences and genotypic variation as determined by amplified fragment length polymorphism (AFLP).

PubMed

Kvitt, H; Ucko, M; Colorni, A; Batargias, C; Zlotkin, A; Knibb, W

2002-04-05

A PCR protocol for the rapid diagnosis of fish 'pasteurellosis' based on 16S rRNA gene sequences was developed. The procedure combines low annealing temperature that detects low titers of Photobacterium damselae but also related species, and high annealing temperature for the specific identification of P. damselae directly from infected fish. The PCR protocol was validated on 19 piscine isolates of P. damselae ssp. piscicida from different geographic regions (Japan, Italy, Spain, Greece and Israel), on spontaneously infected sea bream Sparus aurata and sea bass Dicentrarchus labrax, and on closely related American Type Culture Collection (ATCC) reference strains. PCR using high annealing temperature (64 degrees C) discriminated between P. damselae and closely related reference strains, including P. histaminum. Sixteen isolates of P. damselae ssp. piscicida, 2 P. damselae ssp. piscicida reference strains and 1 P. damselae ssp. damselae reference strain were subjected to Amplified Fragment Length Polymorphism (AFLP) analysis, and a similarity matrix was produced. Accordingly, the Japanese isolates of P. damselae ssp. piscicida were distinguished from the Mediterranean/European isolates at a cut-off value of 83% similarity. A further subclustering at a cut-off value of 97% allowed discrimination between the Israeli P. damselae ssp. piscicida isolates and the other Mediterranean/European isolates. The combination of PCR direct amplification and AFLP provides a 2-step procedure, where P. damselae is rapidly identified at genus level on the basis of its 16S rRNA gene sequence and then grouped into distinct clusters on the basis of AFLP polymorphisms. The first step of direct amplification is highly sensitive and has immediate practical consequences, offering fish farmers a rapid diagnosis, while the AFLP is more specific and detects intraspecific variation which, in our study, also reflected geographic correspondence. Because of its superior discriminative properties, AFLP can be an important tool for epidemiological and taxonomic studies of this highly homogeneous genus.

Identification on commercialized products of AFLP markers able to discriminate slow- from fast-growing chicken strains.

PubMed

Fumière, Olivier; Dubois, Marc; Grégoire, Dimitrie; Théwis, André; Berben, Gilbert

2003-02-26

The European chicken meat market is characterized by numerous quality marks: "Label de Qualité Wallon" in Belgium, "Label Rouge" in France, denominations of geographical origin, organic agriculture, etc. Most of those certified productions have specifications requiring the use of slow-growing chicken strains. The amplified fragment length polymorphism (AFLP) technique has been used to search molecular markers able to discriminate slow-growing chicken strains from fast-growing ones and to authenticate certified products. Two pairs of restriction enzymes (EcoRI/MseI and EcoRI/TaqI) and 121 selective primer combinations were tested on individual DNA samples from chicken products essentially in carcass form that were ascribed as belonging to either slow- or fast-growing strains. Within the resulting fingerprints, two fragments were identified as type-strains specific markers. One primer combination gives a band (333 bp) that is specific for slow-growing chickens, and another primer pair generates a band (372 bp) that was found to be characteristic of fast-growing chickens. The two markers were isolated, cloned, and sequenced. The effectiveness and the specificity of the two interesting determinants were assessed on individuals of two well-known strains (ISA 657 and Cobb 500) and on commercialized products coming from various origins.

Strain-specific Fibril Propagation by an Aβ Dodecamer

NASA Astrophysics Data System (ADS)

Dean, Dexter N.; Das, Pradipta K.; Rana, Pratip; Burg, Franklin; Levites, Yona; Morgan, Sarah E.; Ghosh, Preetam; Rangachari, Vijayaraghavan

2017-01-01

Low molecular weight oligomers of amyloid-β (Aβ) have emerged as the primary toxic agents in the etiology of Alzheimer disease (AD). Polymorphism observed within the aggregation end products of fibrils are known to arise due to microstructural differences among the oligomers. Diversity in aggregate morphology correlates with the differences in AD, cementing the idea that conformational strains of oligomers could be significant in phenotypic outcomes. Therefore, it is imperative to determine the ability of strains to faithfully propagate their structure. Here we report fibril propagation of an Aβ42 dodecamer called large fatty acid-derived oligomers (LFAOs). The LFAO oligomeric strain selectively induces acute cerebral amyloid angiopathy (CAA) in neonatally-injected transgenic CRND8 mice. Propagation in-vitro occurs as a three-step process involving the association of LFAO units. LFAO-seeded fibrils possess distinct morphology made of repeating LFAO units that could be regenerated upon sonication. Overall, these data bring forth an important mechanistic perspective into strain-specific propagation of oligomers that has remained elusive thus far.

Strain-Specific Protective Effect of the Immunity Induced by Live Malarial Sporozoites under Chloroquine Cover

PubMed Central

Wijayalath, Wathsala; Cheesman, Sandra; Tanabe, Kazuyuki; Handunnetti, Shiroma; Carter, Richard; Pathirana, Sisira

2012-01-01

The efficacy of a whole-sporozoite malaria vaccine would partly be determined by the strain-specificity of the protective responses against malarial sporozoites and liver-stage parasites. Evidence from previous reports were inconsistent, where some studies have shown that the protective immunity induced by irradiated or live sporozoites in rodents or humans were cross-protective and in others strain-specific. In the present work, we have studied the strain-specificity of live sporozoite-induced immunity using two genetically and immunologically different strains of Plasmodium cynomolgi, Pc746 and PcCeylon, in toque monkeys. Two groups of monkeys were immunized against live sporozoites of either the Pc746 (n = 5), or the PcCeylon (n = 4) strain, by the bites of 2–4 sporozoite-infected Anopheles tessellates mosquitoes per monkey under concurrent treatments with chloroquine and primaquine to abrogate detectable blood infections. Subsequently, a group of non-immunized monkeys (n = 4), and the two groups of immunized monkeys were challenged with a mixture of sporozoites of the two strains by the bites of 2–5 infective mosquitoes from each strain per monkey. In order to determine the strain-specificity of the protective immunity, the proportions of parasites of the two strains in the challenge infections were quantified using an allele quantification assay, Pyrosequencing™, based on a single nucleotide polymorphism (SNP) in the parasites’ circumsporozoite protein gene. The Pyrosequencing™ data showed that a significant reduction of parasites of the immunizing strain in each group of strain-specifically immunized monkeys had occurred, indicating a stronger killing effect on parasites of the immunizing strain. Thus, the protective immunity developed following a single, live sporozoite/chloroquine immunization, acted specifically against the immunizing strain and was, therefore, strain-specific. As our experiment does not allow us to determine the parasite stage at which the strain-specific protective immunity is directed, it is possible that the target of this immunity could be either the pre-erythrocytic stage, or the blood-stage, or both. PMID:23029282

Whole genome evaluation of tandem repeat polymorphisms between two pathogenically similar strains of Xylella fastidiosa isolated from almond and grape in California

USDA-ARS?s Scientific Manuscript database

Whole genome tandem repeat polymorphisms were evaluated between two closely related Xylella fastidiosa strains, M23 and Temecula1, both cause almond leaf scorch disease (ALSD) and grape Pierce’s disease (PD) in California. Strain M23 was isolated from almond and the genome was sequenced in this stu...

Immunological and molecular polymorphisms of OspC, an immunodominant major outer surface protein of Borrelia burgdorferi.

PubMed Central

Wilske, B; Preac-Mursic, V; Jauris, S; Hofmann, A; Pradel, I; Soutschek, E; Schwab, E; Will, G; Wanner, G

1993-01-01

The gene of the immunodominant major protein pC of Borrelia burgdorferi was previously cloned and sequenced (R. Fuchs, S. Jauris, F. Lottspeich, V. Preac-Mursic, B. Wilske, and E. Soutschek, Mol. Microbiol. 6:503-509, 1992). pC is abundantly expressed on the outer surface of B. burgdorferi, as demonstrated by immunoelectron microscopy with monoclonal antibody L22 1F8. Accordingly, pC is renamed OspC, by analogy to the outer surface proteins OspA and OspB. Western immunoblot analysis of 45 B. burgdorferi isolates with monoclonal antibodies revealed that OspC is immunologically heterogeneous. Partial sequence analysis of the ospC gene confirmed the protein heterogeneity at the genetic level. We found that the degree of identity between the ospC partial sequences of five strains representing different OspA serotypes was only 63.3 to 85.4%. Immunological heterogeneity was also observed among representatives of the three newly designated genospecies of B. burgdorferi sensu lato, B. burgdorferi sensu stricto, B. garinii, and group VS461. Heterogeneity was confirmed for B. garinii at the genetic level. The ospC gene was also cloned from strains that did not express OspC, and antibody-reactive OspC was expressed in Escherichia coli. In addition, OspC-expressing variants were obtained from a nonexpressing strain by plating single colonies on solid medium. These findings confirm that the ospC gene is also present in nonexpressing strains. Because OspC is an immunodominant protein for the early immune response in Lyme borreliosis and was effective as a vaccine in an animal model, the immunological and molecular polymorphisms of ospC and OspC have important implications for the development of diagnostic reagents and vaccines. Images PMID:8478108

Whole Genome Sequencing for Genomics-Guided Investigations of Escherichia coli O157:H7 Outbreaks.

PubMed

Rusconi, Brigida; Sanjar, Fatemeh; Koenig, Sara S K; Mammel, Mark K; Tarr, Phillip I; Eppinger, Mark

2016-01-01

Multi isolate whole genome sequencing (WGS) and typing for outbreak investigations has become a reality in the post-genomics era. We applied this technology to strains from Escherichia coli O157:H7 outbreaks. These include isolates from seven North America outbreaks, as well as multiple isolates from the same patient and from different infected individuals in the same household. Customized high-resolution bioinformatics sequence typing strategies were developed to assess the core genome and mobilome plasticity. Sequence typing was performed using an in-house single nucleotide polymorphism (SNP) discovery and validation pipeline. Discriminatory power becomes of particular importance for the investigation of isolates from outbreaks in which macrogenomic techniques such as pulse-field gel electrophoresis or multiple locus variable number tandem repeat analysis do not differentiate closely related organisms. We also characterized differences in the phage inventory, allowing us to identify plasticity among outbreak strains that is not detectable at the core genome level. Our comprehensive analysis of the mobilome identified multiple plasmids that have not previously been associated with this lineage. Applied phylogenomics approaches provide strong molecular evidence for exceptionally little heterogeneity of strains within outbreaks and demonstrate the value of intra-cluster comparisons, rather than basing the analysis on archetypal reference strains. Next generation sequencing and whole genome typing strategies provide the technological foundation for genomic epidemiology outbreak investigation utilizing its significantly higher sample throughput, cost efficiency, and phylogenetic relatedness accuracy. These phylogenomics approaches have major public health relevance in translating information from the sequence-based survey to support timely and informed countermeasures. Polymorphisms identified in this work offer robust phylogenetic signals that index both short- and long-term evolution and can complement currently employed typing schemes for outbreak ex- and inclusion, diagnostics, surveillance, and forensic studies.

Whole Genome Sequencing for Genomics-Guided Investigations of Escherichia coli O157:H7 Outbreaks

PubMed Central

Rusconi, Brigida; Sanjar, Fatemeh; Koenig, Sara S. K.; Mammel, Mark K.; Tarr, Phillip I.; Eppinger, Mark

2016-01-01

Multi isolate whole genome sequencing (WGS) and typing for outbreak investigations has become a reality in the post-genomics era. We applied this technology to strains from Escherichia coli O157:H7 outbreaks. These include isolates from seven North America outbreaks, as well as multiple isolates from the same patient and from different infected individuals in the same household. Customized high-resolution bioinformatics sequence typing strategies were developed to assess the core genome and mobilome plasticity. Sequence typing was performed using an in-house single nucleotide polymorphism (SNP) discovery and validation pipeline. Discriminatory power becomes of particular importance for the investigation of isolates from outbreaks in which macrogenomic techniques such as pulse-field gel electrophoresis or multiple locus variable number tandem repeat analysis do not differentiate closely related organisms. We also characterized differences in the phage inventory, allowing us to identify plasticity among outbreak strains that is not detectable at the core genome level. Our comprehensive analysis of the mobilome identified multiple plasmids that have not previously been associated with this lineage. Applied phylogenomics approaches provide strong molecular evidence for exceptionally little heterogeneity of strains within outbreaks and demonstrate the value of intra-cluster comparisons, rather than basing the analysis on archetypal reference strains. Next generation sequencing and whole genome typing strategies provide the technological foundation for genomic epidemiology outbreak investigation utilizing its significantly higher sample throughput, cost efficiency, and phylogenetic relatedness accuracy. These phylogenomics approaches have major public health relevance in translating information from the sequence-based survey to support timely and informed countermeasures. Polymorphisms identified in this work offer robust phylogenetic signals that index both short- and long-term evolution and can complement currently employed typing schemes for outbreak ex- and inclusion, diagnostics, surveillance, and forensic studies. PMID:27446025

Long-term colonization with single and multiple strains of Helicobacter pylori assessed by DNA fingerprinting.

PubMed Central

Taylor, N S; Fox, J G; Akopyants, N S; Berg, D E; Thompson, N; Shames, B; Yan, L; Fontham, E; Janney, F; Hunter, F M

1995-01-01

The gastric pathogen Helicobacter pylori establishes long-term chronic infections that can lead to gastritis, peptic ulcers, and cancer. The species is so diverse that distinctly different strains are generally recovered from each patient. To better understand the dynamics of long-term carriage, we characterized H. pylori isolates from initial and follow-up biopsy specimens from a patient population at high risk of H. pylori infection and gastric cancer. Eighty-five isolates were obtained from 23 patients and were analyzed by genomic restriction enzyme analysis, arbitrarily primed PCR fingerprinting, (random amplified polymorphic DNA analysis), and/or restriction of specific PCR-amplified genes (restriction fragment length polymorphism analysis). A single strain was found in sequential biopsy specimens from 12 of 15 patients (80%) receiving sucralfate. In the remaining three patients treated with sucralfate, two strains were identified in two patients and three strains were identified in the third patient. In contrast, a single strain was found in sequential biopsy specimens from only three of eight patients (37%) receiving bismuth, metronidazole, and nitrofurantoin. Two strains were identified in five other patients receiving bismuth-antibiotic (63%). Immunoglobulin G antibodies to H. pylori were present in the sera of all patients. Thus, H. pylori colonization can persist for long periods (up to at least 4 years), despite high titers of immunoglobulin G antibodies in serum. Resistance to metronidazole was noted in some strains before and/or after treatment, but all strains remained susceptible to amoxicillin, tetracycline, and nitrofurantoin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7790461

Rapid estimation of microbial populations in fish samples by using terminal restriction fragment length polymorphism analysis of 16S rDNA.

PubMed

Tanaka, Yuichiro; Takahashi, Hajime; Kitazawa, Nao; Kimura, Bon

2010-01-01

A rapid system using terminal restriction fragment length polymorphism (T-RFLP) analysis targeting 16S rDNA is described for microbial population analysis in edible fish samples. The defined terminal restriction fragment database was constructed by collecting 102 strains of bacteria representing 53 genera that are associated with fish. Digestion of these 102 strains with two restriction enzymes, HhaI and MspI, formed 54 pattern groups with discrimination to the genus level. This T-RFLP system produced results comparable to those from a culture-based method in six natural fish samples with a qualitative correspondence of 71.4 to 92.3%. Using the T-RFLP system allowed an estimation of the microbial population within 7 h. Rapid assay of the microbial population is advantageous for food manufacturers and testing laboratories; moreover, the strategy presented here allows adaptation to specific testing applications.

Sequencing Needs for Viral Diagnostics

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

Gardner, S N; Lam, M; Mulakken, N J

2004-01-26

We built a system to guide decisions regarding the amount of genomic sequencing required to develop diagnostic DNA signatures, which are short sequences that are sufficient to uniquely identify a viral species. We used our existing DNA diagnostic signature prediction pipeline, which selects regions of a target species genome that are conserved among strains of the target (for reliability, to prevent false negatives) and unique relative to other species (for specificity, to avoid false positives). We performed simulations, based on existing sequence data, to assess the number of genome sequences of a target species and of close phylogenetic relatives (''nearmore » neighbors'') that are required to predict diagnostic signature regions that are conserved among strains of the target species and unique relative to other bacterial and viral species. For DNA viruses such as variola (smallpox), three target genomes provide sufficient guidance for selecting species-wide signatures. Three near neighbor genomes are critical for species specificity. In contrast, most RNA viruses require four target genomes and no near neighbor genomes, since lack of conservation among strains is more limiting than uniqueness. SARS and Ebola Zaire are exceptional, as additional target genomes currently do not improve predictions, but near neighbor sequences are urgently needed. Our results also indicate that double stranded DNA viruses are more conserved among strains than are RNA viruses, since in most cases there was at least one conserved signature candidate for the DNA viruses and zero conserved signature candidates for the RNA viruses.« less

Structural and functional characterization of two alpha-synuclein strains

NASA Astrophysics Data System (ADS)

Bousset, Luc; Pieri, Laura; Ruiz-Arlandis, Gemma; Gath, Julia; Jensen, Poul Henning; Habenstein, Birgit; Madiona, Karine; Olieric, Vincent; Böckmann, Anja; Meier, Beat H.; Melki, Ronald

2013-10-01

α-synuclein aggregation is implicated in a variety of diseases including Parkinson’s disease, dementia with Lewy bodies, pure autonomic failure and multiple system atrophy. The association of protein aggregates made of a single protein with a variety of clinical phenotypes has been explained for prion diseases by the existence of different strains that propagate through the infection pathway. Here we structurally and functionally characterize two polymorphs of α-synuclein. We present evidence that the two forms indeed fulfil the molecular criteria to be identified as two strains of α-synuclein. Specifically, we show that the two strains have different structures, levels of toxicity, and in vitro and in vivo seeding and propagation properties. Such strain differences may account for differences in disease progression in different individuals/cell types and/or types of synucleinopathies.

HTR2A A-1438G/T102C polymorphisms predict negative symptoms performance upon aripiprazole treatment in schizophrenic patients.

PubMed

Chen, Shih-Fen; Shen, Yu-Chih; Chen, Chia-Hsiang

2009-08-01

Aripiprazole acts as a partial agonist at dopamine D2 and D3 and serotonin 1A receptors and as an antagonist at serotonin 2A receptors (HTR2A). Since aripiprazole acts as an antagonist at HTR2A, genetic variants of HTR2A may be important in explaining variability in response to aripiprazole. This study investigated whether the efficacy of aripiprazole can be predicted by functional HTR2A A-1438G/T102C polymorphisms (rs63311/rs6313) as modified by clinical factors in Han Chinese hospitalized patients with acutely exacerbated schizophrenia. After hospitalization, the patients (n = 128) were given a 4-week course of aripiprazole. Patients were genotyped for HTR2A A-1438G/T102C polymorphisms via the restriction fragment length polymorphism method. Clinical factors such as gender, age, duration of illness, education level, diagnostic subtype, and medication dosage were noted as well. The researchers measured psychopathology biweekly, using the Positive and Negative Syndrome Scale (PANSS). A mixed model regression approach (SAS Proc MIXED) was used to analyze the effects of genetic and clinical factors on PANSS performance after aripiprazole treatment. We found that the GG/CC genotype group of HTR2A A-1438G/T102C polymorphisms predicts poor aripiprazole response specifically for negative symptoms. In addition, the clinical factors, including dosage of aripiprazole, age, duration of illness, and diagnostic subtype, were found to influence PANSS performance after aripiprazole treatment. The data suggest HTR2A A-1438G/T102C polymorphisms may predict negative symptoms performance upon aripiprazole treatment in schizophrenic patients as modified by clinical factors.

Human immunodeficiency virus type 1 seroprevalence and antiretroviral drug resistance-associated mutations in miners in Gabon, central Africa.

PubMed

Caron, Mélanie; Makuwa, Maria; Souquière, Sandrine; Descamps, Diane; Brun-Vézinet, Françoise; Kazanji, Mirdad

2008-09-01

Miners in sub-Saharan African are known to have an extremely high prevalence of HIV-1 infection. We therefore evaluated the prevalence of HIV-1 infection among manganese miners in Gabon, central Africa and examined the diversity of HIV-1 strains by characterizing the polymorphism of the pol gene in order to observe drug resistance-associated mutations. In 857 samples tested, the HIV-1 prevalence was 2.9%. By pol sequence analysis, we showed that all the HIV-1 strains belonged to group M, with a majority of CRF02_AG (57%) followed by subtype A (9%) and CRF01_AE or subtype B (4%). The remaining HIV-1 strains demonstrated discordant genomic results and exhibited a mosaic pol genome (30%). Most of the mutations detected in pol coding regions corresponded to the subtype polymorphism, with no specific antiretroviral drug resistance. To avoid the rapid emergence of resistant viruses in this part of central Africa, continuous surveillance of the circulation of drug-resistant viruses must be maintained to guide treatment strategies.

Aspergillus Collagen-Like Genes (acl): Identification, Sequence Polymorphism, and Assessment for PCR-Based Pathogen Detection

PubMed Central

Tuntevski, Kiril; Durney, Brandon C.; Snyder, Anna K.; LaSala, P. Rocco; Nayak, Ajay P.; Green, Brett J.; Beezhold, Donald H.; Rio, Rita V. M.; Holland, Lisa A.

2013-01-01

The genus Aspergillus is a burden to public health due to its ubiquitous presence in the environment, its production of allergens, and wide demographic susceptibility among cystic fibrosis, asthmatic, and immunosuppressed patients. Current methods of detection of Aspergillus colonization and infection rely on lengthy morphological characterization or nonstandardized serological assays that are restricted to identifying a fungal etiology. Collagen-like genes have been shown to exhibit species-specific conservation across the noncollagenous regions as well as strain-specific polymorphism in the collagen-like regions. Here we assess the conserved region of the Aspergillus collagen-like (acl) genes and explore the application of PCR amplicon size-based discrimination among the five most common etiologic species of the Aspergillus genus, including Aspergillus fumigatus, A. flavus, A. nidulans, A. niger, and A. terreus. Genetic polymorphism and phylogenetic analysis of the aclF1 gene were additionally examined among the available strains. Furthermore, the applicability of the PCR-based assay to identification of these five species in cultures derived from sputum and bronchoalveolar fluid from 19 clinical samples was explored. Application of capillary electrophoresis on nanogels was additionally demonstrated to improve the discrimination between Aspergillus species. Overall, this study demonstrated that Aspergillus acl genes could be used as PCR targets to discriminate between clinically relevant Aspergillus species. Future studies aim to utilize the detection of Aspergillus acl genes in PCR and microfluidic applications to determine the sensitivity and specificity for the identification of Aspergillus colonization and invasive aspergillosis in immunocompromised subjects. PMID:24123732

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

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

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.

A new assay based on terminal restriction fragment length polymorphism of homocitrate synthase gene fragments for Candida species identification.

PubMed

Szemiako, Kasjan; Śledzińska, Anna; Krawczyk, Beata

2017-08-01

Candida sp. have been responsible for an increasing number of infections, especially in patients with immunodeficiency. Species-specific differentiation of Candida sp. is difficult in routine diagnosis. This identification can have a highly significant association in therapy and prophylaxis. This work has shown a new application of the terminal restriction fragment length polymorphism (t-RFLP) method in the molecular identification of six species of Candida, which are the most common causes of fungal infections. Specific for fungi homocitrate synthase gene was chosen as a molecular target for amplification. The use of three restriction enzymes, DraI, RsaI, and BglII, for amplicon digestion can generate species-specific fluorescence labeled DNA fragment profiles, which can be used to determine the diagnostic algorithm. The designed method can be a cost-efficient high-throughput molecular technique for the identification of six clinically important Candida species.

Single Assay for Simultaneous Detection and Differential Identification of Human and Avian Influenza Virus Types, Subtypes, and Emergent Variants

PubMed Central

Metzgar, David; Myers, Christopher A.; Russell, Kevin L.; Faix, Dennis; Blair, Patrick J.; Brown, Jason; Vo, Scott; Swayne, David E.; Thomas, Colleen; Stenger, David A.; Lin, Baochuan; Malanoski, Anthony P.; Wang, Zheng; Blaney, Kate M.; Long, Nina C.; Schnur, Joel M.; Saad, Magdi D.; Borsuk, Lisa A.; Lichanska, Agnieszka M.; Lorence, Matthew C.; Weslowski, Brian; Schafer, Klaus O.; Tibbetts, Clark

2010-01-01

For more than four decades the cause of most type A influenza virus infections of humans has been attributed to only two viral subtypes, A/H1N1 or A/H3N2. In contrast, avian and other vertebrate species are a reservoir of type A influenza virus genome diversity, hosting strains representing at least 120 of 144 combinations of 16 viral hemagglutinin and 9 viral neuraminidase subtypes. Viral genome segment reassortments and mutations emerging within this reservoir may spawn new influenza virus strains as imminent epidemic or pandemic threats to human health and poultry production. Traditional methods to detect and differentiate influenza virus subtypes are either time-consuming and labor-intensive (culture-based) or remarkably insensitive (antibody-based). Molecular diagnostic assays based upon reverse transcriptase-polymerase chain reaction (RT-PCR) have short assay cycle time, and high analytical sensitivity and specificity. However, none of these diagnostic tests determine viral gene nucleotide sequences to distinguish strains and variants of a detected pathogen from one specimen to the next. Decision-quality, strain- and variant-specific pathogen gene sequence information may be critical for public health, infection control, surveillance, epidemiology, or medical/veterinary treatment planning. The Resequencing Pathogen Microarray (RPM-Flu) is a robust, highly multiplexed and target gene sequencing-based alternative to both traditional culture- or biomarker-based diagnostic tests. RPM-Flu is a single, simultaneous differential diagnostic assay for all subtype combinations of type A influenza viruses and for 30 other viral and bacterial pathogens that may cause influenza-like illness. These other pathogen targets of RPM-Flu may co-infect and compound the morbidity and/or mortality of patients with influenza. The informative specificity of a single RPM-Flu test represents specimen-specific viral gene sequences as determinants of virus type, A/HN subtype, virulence, host-range, and resistance to antiviral agents. PMID:20140251

Single assay for simultaneous detection and differential identification of human and avian influenza virus types, subtypes, and emergent variants.

PubMed

Metzgar, David; Myers, Christopher A; Russell, Kevin L; Faix, Dennis; Blair, Patrick J; Brown, Jason; Vo, Scott; Swayne, David E; Thomas, Colleen; Stenger, David A; Lin, Baochuan; Malanoski, Anthony P; Wang, Zheng; Blaney, Kate M; Long, Nina C; Schnur, Joel M; Saad, Magdi D; Borsuk, Lisa A; Lichanska, Agnieszka M; Lorence, Matthew C; Weslowski, Brian; Schafer, Klaus O; Tibbetts, Clark

2010-02-03

For more than four decades the cause of most type A influenza virus infections of humans has been attributed to only two viral subtypes, A/H1N1 or A/H3N2. In contrast, avian and other vertebrate species are a reservoir of type A influenza virus genome diversity, hosting strains representing at least 120 of 144 combinations of 16 viral hemagglutinin and 9 viral neuraminidase subtypes. Viral genome segment reassortments and mutations emerging within this reservoir may spawn new influenza virus strains as imminent epidemic or pandemic threats to human health and poultry production. Traditional methods to detect and differentiate influenza virus subtypes are either time-consuming and labor-intensive (culture-based) or remarkably insensitive (antibody-based). Molecular diagnostic assays based upon reverse transcriptase-polymerase chain reaction (RT-PCR) have short assay cycle time, and high analytical sensitivity and specificity. However, none of these diagnostic tests determine viral gene nucleotide sequences to distinguish strains and variants of a detected pathogen from one specimen to the next. Decision-quality, strain- and variant-specific pathogen gene sequence information may be critical for public health, infection control, surveillance, epidemiology, or medical/veterinary treatment planning. The Resequencing Pathogen Microarray (RPM-Flu) is a robust, highly multiplexed and target gene sequencing-based alternative to both traditional culture- or biomarker-based diagnostic tests. RPM-Flu is a single, simultaneous differential diagnostic assay for all subtype combinations of type A influenza viruses and for 30 other viral and bacterial pathogens that may cause influenza-like illness. These other pathogen targets of RPM-Flu may co-infect and compound the morbidity and/or mortality of patients with influenza. The informative specificity of a single RPM-Flu test represents specimen-specific viral gene sequences as determinants of virus type, A/HN subtype, virulence, host-range, and resistance to antiviral agents.

Rapid screening of toxigenic vibrio cholerae O1 strains from south Iran by PCR-ELISA.

PubMed

Mousavi, Seyed Latif; Nazarian, Shahram; Amani, Jafar; Rahgerdi, Ahmad Karimi

2008-01-01

The ability to sensitively detect Vibrio cholera with PCR-ELISA method represents a considerable advancement over alternative more time-consuming methods for detection of this pathogen. The aim of this research is to evaluate the suitability of a PCR-enzyme-linked immunosorbent assay for sensitive and rapid detection of V. cholera O1. The 398-bp sequence of a gene that codes for the cholera toxin B subunit was amplified by PCR. The digoxigenin-labeled amplified products were coated on microplates and detected by ELISA. The PCR product was also hybridized with biotin labelled probe and detected by ELISA using streptavidin. The specificity of the PCR was determined using 10 bacterial strains and 50 samples from south Iran. The detection limit was 0.5 pg of the genomic DNA and five bacterial cells. Adaptation of PCR into PCR-ELISA assay format facilitates specific and sensitive detection and diagnosis of human cholera disease. We conclude that this PCR-ELISA is a diagnostic method that specifically detects toxin genes in V. cholera O1 strains. It is more rapid and less cumbersome than other diagnostic methods for detection of toxicity in these strains.

Single Nucleotide Polymorphisms (SNP)-specific Quantitative Real Time Polymerase Chain Reaction (PCR) Assay for Analyzing Competition and Emergence of the Military Hypersporulating Strains of Bacillus Atrophaeous var. Globigii

DTIC Science & Technology

2012-09-01

Emergence of the Military Hypersporulating Strains of Bacillus Atrophaeous var. Globigii by Doncho V. Zhelev, Christopher Dupuis , Suelynn Ren, Anna...Globigii Doncho V. Zhelev, Christopher Dupuis , Suelynn Ren, Anna Le, and Mia Hunt Sensors and Electron Devices Directorate, ARL Henry Gibbons...Zhelev, Christopher Dupuis , Suelynn Ren, Anna Le, Mia Hunt, and Henry Gibbons 5d. PROJECT NUMBER EC-SE-2011-05 5e. TASK NUMBER 5f. WORK UNIT

Ultrasound elastography-based assessment of the elasticity of the supraspinatus muscle in impingement syndrome: does elastography has any diagnostic value?

PubMed

Demirel, Adnan; Baykara, Murat; Koca, Tuba Tülay; Berk, Ejder

2018-06-01

Ultrasound elastography (UE) is a new ultrasound-based imaging technique that provides information about elasticity and stiffness of tissues. This cross-sectional study aimed to identify the diagnostic importance of UE in supraspinatus impingement syndrome. Forty-one subjects, aged 38-70 years, were included in the study. UE was used to determine the elasticity of the supraspinatus muscle. The strain ratio was calculated as the evaluation criteria to measure the elasticity of the muscle. High strain ratio indicated low elasticity. The measurements were made by the blinded radiologist while the patients sat with their shoulder in a neutral position. The diagnostic value of the strain ratio was evaluated using the receiver operating characteristic (ROC) analysis. The mean strain value of the supraspinatus muscle on the intact and pathological shoulders determined by UE was 0.74 ± 0.33 and 0.31 ± 0.24, respectively. A low strain ratio value in the supraspinatus muscle on the side with impingement syndrome was measured. When the test variable was evaluated as "strain ratio" according to ROC curve analysis, it was found to be above the reference line [0.849 (> 0.5)] (P = 0.00). When the cutoff value was selected as 0.495, the sensitivity and specificity were found to be 75.6 and 78% (the strain ratio value > 0.495), respectively. Measurement of strain ratio with UE can be used as a noninvasive, inexpensive, and practical diagnostic test for the shoulder impingement disease.

Naked-eye fingerprinting of single nucleotide polymorphisms on psoriasis patients

NASA Astrophysics Data System (ADS)

Valentini, Paola; Marsella, Alessandra; Tarantino, Paolo; Mauro, Salvatore; Baglietto, Silvia; Congedo, Maurizio; Paolo Pompa, Pier

2016-05-01

We report a low-cost test, based on gold nanoparticles, for the colorimetric (naked-eye) fingerprinting of a panel of single nucleotide polymorphisms (SNPs), relevant for the personalized therapy of psoriasis. Such pharmacogenomic tests are not routinely performed on psoriasis patients, due to the high cost of standard technologies. We demonstrated high sensitivity and specificity of our colorimetric test by validating it on a cohort of 30 patients, through a double-blind comparison with two state-of-the-art instrumental techniques, namely reverse dot blotting and sequencing, finding 100% agreement. This test offers high parallelization capabilities and can be easily generalized to other SNPs of clinical relevance, finding broad utility in diagnostics and pharmacogenomics.We report a low-cost test, based on gold nanoparticles, for the colorimetric (naked-eye) fingerprinting of a panel of single nucleotide polymorphisms (SNPs), relevant for the personalized therapy of psoriasis. Such pharmacogenomic tests are not routinely performed on psoriasis patients, due to the high cost of standard technologies. We demonstrated high sensitivity and specificity of our colorimetric test by validating it on a cohort of 30 patients, through a double-blind comparison with two state-of-the-art instrumental techniques, namely reverse dot blotting and sequencing, finding 100% agreement. This test offers high parallelization capabilities and can be easily generalized to other SNPs of clinical relevance, finding broad utility in diagnostics and pharmacogenomics. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr02200f

Temporal dynamics and population genetic structure of Fusarium graminearum in the upper Midwestern United States.

PubMed

Liang, J M; Xayamongkhon, H; Broz, K; Dong, Y; McCormick, S P; Abramova, S; Ward, T J; Ma, Z H; Kistler, H C

2014-12-01

Fusarium graminearum sensu stricto causes Fusarium head blight (FHB) in wheat and barley, and contaminates grains with several trichothecene mycotoxins, causing destructive yield losses and economic impact in the United States. Recently, a F. graminearum strain collected from Minnesota (MN) was determined to produce a novel trichothecene toxin, called NX-2. In order to determine the spatial and temporal dynamics of NX-2 producing strains in MN, North Dakota (ND) and South Dakota (SD), a total of 463 F. graminearum strains were collected from three sampling periods, 1999-2000, 2006-2007 and 2011-2013. A PCR-RFLP based diagnostic test was developed and validated for NX-2 producing strains based on polymorphisms in the Tri1 gene. Trichothecene biosynthesis gene (Tri gene)-based polymerase chain reaction (PCR) assays and ten PCR-restriction fragment length polymorphism (RFLP) markers were used to genotype all strains. NX-2 strains were detected in each sampling period but with a very low overall frequency (2.8%) and were mainly collected near the borders of MN, ND and SD. Strains with the 3ADON chemotype were relatively infrequent in 1999-2000 (4.5%) but increased to 29.4% in 2006-2007 and 17.2% in 2011-2013. The distribution of 3ADON producing strains also expanded from a few border counties between ND and MN in 1999-2000, southward toward the border between SD and MN in 2006-2007 and westward in 2011-2013. Genetic differentiation between 2006-2007 and 2011-2013 populations (3%) was much lower than that between 1999-2000 and 2006-2007 (22%) or 1999-2000 and 2011-2013 (20%) suggesting that most change to population genetic structure of F. graminearum occurred between 1999-2000 and 2006-2007. This change was associated with the emergence of a new population consisting largely of individuals with a 3ADON chemotype. A Bayesian clustering analysis suggested that NX-2 chemotype strains are part of a previously described Upper Midwestern population. However, these analyses also suggest that the NX-2 isolates could represent a distinct population, but that interpretations of population assignment are influenced by the small number of NX-2 strains available for analysis. Published by Elsevier Inc.

Natural variation in non-coding regions underlying phenotypic diversity in budding yeast

PubMed Central

Salinas, Francisco; de Boer, Carl G.; Abarca, Valentina; García, Verónica; Cuevas, Mara; Araos, Sebastian; Larrondo, Luis F.; Martínez, Claudio; Cubillos, Francisco A.

2016-01-01

Linkage mapping studies in model organisms have typically focused their efforts in polymorphisms within coding regions, ignoring those within regulatory regions that may contribute to gene expression variation. In this context, differences in transcript abundance are frequently proposed as a source of phenotypic diversity between individuals, however, until now, little molecular evidence has been provided. Here, we examined Allele Specific Expression (ASE) in six F1 hybrids from Saccharomyces cerevisiae derived from crosses between representative strains of the four main lineages described in yeast. ASE varied between crosses with levels ranging between 28% and 60%. Part of the variation in expression levels could be explained by differences in transcription factors binding to polymorphic cis-regulations and to differences in trans-activation depending on the allelic form of the TF. Analysis on highly expressed alleles on each background suggested ASN1 as a candidate transcript underlying nitrogen consumption differences between two strains. Further promoter allele swap analysis under fermentation conditions confirmed that coding and non-coding regions explained aspartic and glutamic acid consumption differences, likely due to a polymorphism affecting Uga3 binding. Together, we provide a new catalogue of variants to bridge the gap between genotype and phenotype. PMID:26898953

Genetic basis of allochronic differentiation in the fall armyworm.

PubMed

Hänniger, Sabine; Dumas, Pascaline; Schöfl, Gerhard; Gebauer-Jung, Steffi; Vogel, Heiko; Unbehend, Melanie; Heckel, David G; Groot, Astrid T

2017-03-06

Very little is known on how changes in circadian rhythms evolve. The noctuid moth Spodoptera frugiperda (Lepidoptera: Noctuidae) consists of two strains that exhibit allochronic differentiation in their mating time, which acts as a premating isolation barrier between the strains. We investigated the genetic basis of the strain-specific timing differences to identify the molecular mechanisms of differentiation in circadian rhythms. Through QTL analyses we identified one major Quantitative trait chromosome (QTC) underlying differentiation in circadian timing of mating activity. Using RADtags, we identified this QTC to be homologous to Bombyx mori C27, on which the clock gene vrille is located, which thus became the major candidate gene. In S. frugiperda, vrille showed strain-specific polymorphisms. Also, vrille expression differed significantly between the strains, with the rice-strain showing higher expression levels than the corn-strain. In addition, RT-qPCR experiments with the other main clock genes showed that pdp1, antagonist of vrille in the modulatory feedback loop of the circadian clock, showed higher expression levels in the rice-strain than in the corn-strain. Together, our results indicate that the allochronic differentiation in the two strains of S. frugiperda is associated with differential transcription of vrille or a cis-acting gene close to vrille, which contributes to the evolution of prezygotic isolation in S. frugiperda.

Chloroplast DNA polymorphism and evolutional relationships between Asian cultivated rice (Oryza sativa) and its wild relatives (O. rufipogon).

PubMed

Li, W J; Zhang, B; Huang, G W; Kang, G P; Liang, M Z; Chen, L B

2012-12-17

We analyzed chloroplast DNA (cpDNA) polymorphism and phylogenic relationships between 6 typical indica rice, 4 japonica rice, 8 javanica rice, and 12 Asian common wild rice (Oryza rufipogon) strains collected from different latitudes in China by comparing polymorphism at 9 highly variable regions. One hundred and forty-four polymorphic bases were detected. The O. rufipogon samples had 117 polymorphic bases, showing rich genetic diversity. One hundred and thirty-one bases at 13 sites were identified with indica/japonica characteristics; they showed differences between the indica and japonica subspecies at these sites. The javanica strains and japonica shared similar bases at these 131 polymorphic sites, suggesting that javanica is closely related to japonica. On the basis of length analyses of the open reading frame (ORF)100 and (ORF)29-tRNA-Cys(GCA) (TrnC(GCA)) fragments, the O. rufipogon strains were classified into indica/japonica subgroups, which was consistent with the results of the phylogenic tree assay based on concatenated datasets. These results indicated that differences in indica and japonica also exist in the cpDNA genome of the O. rufipogon strains. However, these differences demonstrated a certain degree of primitiveness and incompleteness, as an O. rufipogon line may show different indica/ japonica attributes at different sites. Consequently, O. rufipogon cannot be simply classified into the indica/japonica types as O. sativa. Our data support the hypothesis that Asian cultivated rice, O. indica and O. japonica, separately evolved from Asian common wild rice (O. rufipogon) strains, which have different indica-japonica differentiation trends.

Sequence-related amplified polymorphism (SRAP) marker as a new method for identification of endophytic fungi from Taxus.

PubMed

Ren, Na; Liu, Jiajia; Yang, Dongliang; Chen, Jianhua; Luan, Mingbao; Hong, Juan

2012-01-01

A total of 20 endophytic fungi stains were classified into four groups using traditional morphological identification method, and were studied for genetic diversity by sequence-related amplified polymorphism (SRAP) technique. Genomic DNA (deoxyribonucleic acid) of these strains was extracted with CTAB method. SRAP analysis was done with 24 pairs of primers. All strains could be uniquely distinguished with 584 bands and 446 polymorphism bands which generated 76.4% of polymorphic ratio. Unweighted pair-group method with arithmetical averages cluster analysis enabled construction of a dendrogram for estimating genetic distances between different strains. All strains, which were just divided into four groups by traditional morphology identification, were clustered into four major groups at GS = 0.603 and further separated into eight sub-groups at GS = 0.921. Dendrogram also revealed a large genetic variation in 20 strains; different primer combinations allowed them distinctly distinguished one from others with relatively low genetic similarity. The results show that the SRAP technology is more efficient than traditional morphology identification. It is found that SRAP markers could more really reflect the genetic diversity of endophytic fungi strains from Taxus, and also could be used as a method for identification of endophytic fungi from Taxus. It also suggests that SRAP can be used to establish foundation for further screening of taxol-producing endophytic fungi strains which can produce high levels of paclitaxel.

Genetic variability in isolates of Chromobacterium violaceum from pulmonary secretion, water, and soil.

PubMed

Santini, A C; Magalhães, J T; Cascardo, J C M; Corrêa, R X

2016-04-28

Chromobacterium violaceum is a free-living Gram-negative bacillus usually found in the water and soil in tropical regions, which causes infections in humans. Chromobacteriosis is characterized by rapid dissemination and high mortality. The aim of this study was to detect the genetic variability among C. violaceum type strain ATCC 12472, and seven isolates from the environment and one from a pulmonary secretion from a chromobacteriosis patient from Ilhéus, Bahia. The molecular characterization of all samples was performed by polymerase chain reaction (PCR) sequencing and 16S rDNA analysis. Primers specific for two ATCC 12472 pathogenicity genes, hilA and yscD, as well as random amplified polymorphic DNA (RAPD), were used for PCR amplification and comparative sequencing of the products. For a more specific approach, the PCR products of 16S rDNA were digested with restriction enzymes. Seven of the samples, including type-strain ATCC 12472, were amplified by the hilA primers; these were subsequently sequenced. Gene yscD was amplified only in type-strain ATCC 12472. MspI and AluI digestion revealed 16S rDNA polymorphisms. This data allowed the generation of a dendogram for each analysis. The isolates of C. violaceum have variability in random genomic regions demonstrated by RAPD. Also, these isolates have variability in pathogenicity genes, as demonstrated by sequencing and restriction enzyme digestion.

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.

Detection and Identification of Probiotic Lactobacillus plantarum Strains by Multiplex PCR Using RAPD-Derived Primers

PubMed Central

Galanis, Alex; Kourkoutas, Yiannis; Tassou, Chrysoula C.; Chorianopoulos, Nikos

2015-01-01

Lactobacillus plantarum 2035 and Lactobacillus plantarum ACA-DC 2640 are two lactic acid bacteria (LAB) strains that have been isolated from Feta cheese. Both display significant potential for the production of novel probiotic food products. The aim of the present study was the development of an accurate and efficient method for the molecular detection and identification of the above strains in a single reaction. A multiplex PCR assay was designed for each strain, based on specific primers derived from Random Amplified Polymorphic DNA (RAPD) Sequenced Characterized Amplified Region (SCAR) analysis. The specificity of the assay was tested with a total of 23 different LAB strains, for L. plantarum 2035 and L. plantarum ACA-DC 2640. The multiplex PCR assay was also successfully applied for the detection of the above cultures in yogurt samples prepared in our lab. The proposed methodology may be applied for monitoring the presence of these strains in food products, thus evaluating their probiotic character. Moreover, our strategy may be adapted for other novel LAB strains with probiotic potential, thus providing a powerful tool for molecular discrimination that could be invaluable to the food industry. PMID:26506345

Detection and Identification of Probiotic Lactobacillus plantarum Strains by Multiplex PCR Using RAPD-Derived Primers.

PubMed

Galanis, Alex; Kourkoutas, Yiannis; Tassou, Chrysoula C; Chorianopoulos, Nikos

2015-10-22

Lactobacillus plantarum 2035 and Lactobacillus plantarum ACA-DC 2640 are two lactic acid bacteria (LAB) strains that have been isolated from Feta cheese. Both display significant potential for the production of novel probiotic food products. The aim of the present study was the development of an accurate and efficient method for the molecular detection and identification of the above strains in a single reaction. A multiplex PCR assay was designed for each strain, based on specific primers derived from Random Amplified Polymorphic DNA (RAPD) Sequenced Characterized Amplified Region (SCAR) analysis. The specificity of the assay was tested with a total of 23 different LAB strains, for L. plantarum 2035 and L. plantarum ACA-DC 2640. The multiplex PCR assay was also successfully applied for the detection of the above cultures in yogurt samples prepared in our lab. The proposed methodology may be applied for monitoring the presence of these strains in food products, thus evaluating their probiotic character. Moreover, our strategy may be adapted for other novel LAB strains with probiotic potential, thus providing a powerful tool for molecular discrimination that could be invaluable to the food industry.

Complete Genome Sequence of Yersinia pestis Strains Antiqua andNepal516: Evidence of Gene Reduction in an Emerging Pathogen

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

Chain, Patrick S.G.; Hu, Ping; Malfatti, Stephanie A.

2006-01-16

Yersinia pestis, the causative agent of bubonic andpneumonicplague, has undergone detailed study at the molecular level. Tofurther investigate the genomic diversity among this group and to helpcharacterize lineages of the plague organism that have no sequencedmembers, we present here the genomes of two isolates of the "classical"Antiqua biovar, strains Antiqua and Nepal516. The genomes of Antiqua andNepal516 are 4.7 Mb and 4.5 Mb and encode 4,138 and 3,956 open readingframes respectively. Though both strains belong to one of the threeclassical biovars, they represent separate lineages defined by recentphylogenetic studies. We compare all five currently sequenced Y. pestisgenomes and the correspondingmore » features in Y. pseudotuberculosis. Thereare strain-specific rearrangements, insertions, deletions, singlenucleotide polymorphisms and a unique distribution of insertionsequences. We found 453 single nucleotide polymorphisms in protein codingregions, which were used to assess evolutionary relationships of these Y.pestis strains. Gene reduction analysis revealed that the gene deletionprocesses are under selective pressure and many of the inactivations areprobably related to the organism s interaction with its host environment.The results presented here clearly demonstrate the differences betweenthe two Antiqua lineages and support the notion that grouping Y. pestisstrains based strictly on the classical definition of biovars (predicatedupon two biochemical assays) does not accurately reflect the phylogeneticrelationships within this species. Comparison of four virulent Y. pestisstrains with the human-avirulent strain 91001 provides further insightinto the genetic basis of virulence to humans.« less

KDNA Genetic Signatures Obtained by LSSP-PCR Analysis of Leishmania (Leishmania) infantum Isolated from the New and the Old World

PubMed Central

Alvarenga, Janaína Sousa Campos; Ligeiro, Carla Maia; Gontijo, Célia Maria Ferreira; Cortes, Sofia; Campino, Lenea; Vago, Annamaria Ravara; Melo, Maria Norma

2012-01-01

Background Visceral Leishmaniasis (VL) caused by species from the Leishmania donovani complex is the most severe form of the disease, lethal if untreated. VL caused by Leishmania infantum is a zoonosis with an increasing number of human cases and millions of dogs infected in the Old and the New World. In this study, L. infantum (syn. L.chagasi) strains were isolated from human and canine VL cases. The strains were obtained from endemic areas from Brazil and Portugal and their genetic polymorphism was ascertained using the LSSP-PCR (Low-Stringency Single Specific Primer PCR) technique for analyzing the kinetoplastid DNA (kDNA) minicircles hypervariable region. Principal Findings KDNA genetic signatures obtained by minicircle LSSP-PCR analysis of forty L. infantum strains allowed the grouping of strains in several clades. Furthermore, LSSP-PCR profiles of L. infantum subpopulations were closely related to the host origin (human or canine). To our knowledge this is the first study which used this technique to compare genetic polymorphisms among strains of L. infantum originated from both the Old and the New World. Conclusions LSSP-PCR profiles obtained by analysis of L. infantum kDNA hypervariable region of parasites isolated from human cases and infected dogs from Brazil and Portugal exhibited a genetic correlation among isolates originated from the same reservoir, human or canine. However, no association has been detected among the kDNA signatures and the geographical origin of L. infantum strains. PMID:22912862

Comparative analysis of response to selection with three insecticides in the dengue mosquito Aedes aegypti using mRNA sequencing

PubMed Central

2014-01-01

Background Mosquito control programmes using chemical insecticides are increasingly threatened by the development of resistance. Such resistance can be the consequence of changes in proteins targeted by insecticides (target site mediated resistance), increased insecticide biodegradation (metabolic resistance), altered transport, sequestration or other mechanisms. As opposed to target site resistance, other mechanisms are far from being fully understood. Indeed, insecticide selection often affects a large number of genes and various biological processes can hypothetically confer resistance. In this context, the aim of the present study was to use RNA sequencing (RNA-seq) for comparing transcription level and polymorphism variations associated with adaptation to chemical insecticides in the mosquito Aedes aegypti. Biological materials consisted of a parental susceptible strain together with three child strains selected across multiple generations with three insecticides from different classes: the pyrethroid permethrin, the neonicotinoid imidacloprid and the carbamate propoxur. Results After ten generations, insecticide-selected strains showed elevated resistance levels to the insecticides used for selection. RNA-seq data allowed detecting over 13,000 transcripts, of which 413 were differentially transcribed in insecticide-selected strains as compared to the susceptible strain. Among them, a significant enrichment of transcripts encoding cuticle proteins, transporters and enzymes was observed. Polymorphism analysis revealed over 2500 SNPs showing > 50% allele frequency variations in insecticide-selected strains as compared to the susceptible strain, affecting over 1000 transcripts. Comparing gene transcription and polymorphism patterns revealed marked differences among strains. While imidacloprid selection was linked to the over transcription of many genes, permethrin selection was rather linked to polymorphism variations. Focusing on detoxification enzymes revealed that permethrin selection strongly affected the polymorphism of several transcripts encoding cytochrome P450 monooxygenases likely involved in insecticide biodegradation. Conclusions The present study confirmed the power of RNA-seq for identifying concomitantly quantitative and qualitative transcriptome changes associated with insecticide resistance in mosquitoes. Our results suggest that transcriptome modifications can be selected rapidly by insecticides and affect multiple biological functions. Previously neglected by molecular screenings, polymorphism variations of detoxification enzymes may play an important role in the adaptive response of mosquitoes to insecticides. PMID:24593293

Comparative analysis of response to selection with three insecticides in the dengue mosquito Aedes aegypti using mRNA sequencing.

PubMed

David, Jean-Philippe; Faucon, Frédéric; Chandor-Proust, Alexia; Poupardin, Rodolphe; Riaz, Muhammad Asam; Bonin, Aurélie; Navratil, Vincent; Reynaud, Stéphane

2014-03-05

Mosquito control programmes using chemical insecticides are increasingly threatened by the development of resistance. Such resistance can be the consequence of changes in proteins targeted by insecticides (target site mediated resistance), increased insecticide biodegradation (metabolic resistance), altered transport, sequestration or other mechanisms. As opposed to target site resistance, other mechanisms are far from being fully understood. Indeed, insecticide selection often affects a large number of genes and various biological processes can hypothetically confer resistance. In this context, the aim of the present study was to use RNA sequencing (RNA-seq) for comparing transcription level and polymorphism variations associated with adaptation to chemical insecticides in the mosquito Aedes aegypti. Biological materials consisted of a parental susceptible strain together with three child strains selected across multiple generations with three insecticides from different classes: the pyrethroid permethrin, the neonicotinoid imidacloprid and the carbamate propoxur. After ten generations, insecticide-selected strains showed elevated resistance levels to the insecticides used for selection. RNA-seq data allowed detecting over 13,000 transcripts, of which 413 were differentially transcribed in insecticide-selected strains as compared to the susceptible strain. Among them, a significant enrichment of transcripts encoding cuticle proteins, transporters and enzymes was observed. Polymorphism analysis revealed over 2500 SNPs showing > 50% allele frequency variations in insecticide-selected strains as compared to the susceptible strain, affecting over 1000 transcripts. Comparing gene transcription and polymorphism patterns revealed marked differences among strains. While imidacloprid selection was linked to the over transcription of many genes, permethrin selection was rather linked to polymorphism variations. Focusing on detoxification enzymes revealed that permethrin selection strongly affected the polymorphism of several transcripts encoding cytochrome P450 monooxygenases likely involved in insecticide biodegradation. The present study confirmed the power of RNA-seq for identifying concomitantly quantitative and qualitative transcriptome changes associated with insecticide resistance in mosquitoes. Our results suggest that transcriptome modifications can be selected rapidly by insecticides and affect multiple biological functions. Previously neglected by molecular screenings, polymorphism variations of detoxification enzymes may play an important role in the adaptive response of mosquitoes to insecticides.

Structural and functional characterization of two alpha-synuclein strains

PubMed Central

Bousset, Luc; Pieri, Laura; Ruiz-Arlandis, Gemma; Gath, Julia; Jensen, Poul Henning; Habenstein, Birgit; Madiona, Karine; Olieric, Vincent; Böckmann, Anja; Meier, Beat H.; Melki, Ronald

2013-01-01

α-synuclein aggregation is implicated in a variety of diseases including Parkinson’s disease, dementia with Lewy bodies, pure autonomic failure and multiple system atrophy. The association of protein aggregates made of a single protein with a variety of clinical phenotypes has been explained for prion diseases by the existence of different strains that propagate through the infection pathway. Here we structurally and functionally characterize two polymorphs of α-synuclein. We present evidence that the two forms indeed fulfil the molecular criteria to be identified as two strains of α-synuclein. Specifically, we show that the two strains have different structures, levels of toxicity, and in vitro and in vivo seeding and propagation properties. Such strain differences may account for differences in disease progression in different individuals/cell types and/or types of synucleinopathies. PMID:24108358

Development of a simple and practical method of discrimination between Vibrio furnissii and V. fluvialis based on single-nucleotide polymorphisms of 16S rRNA genes observed in V. furnissii but not in V. fluvialis.

PubMed

Takajo, Ichiro; Yamada, Akiteru; Umeki, Kazumi; Saeki, Yuji; Hashikura, Yuuki; Yamamoto, Ikuo; Umekita, Kunihiko; Urayama-Kawano, Midori; Yamasaki, Shogo; Taniguchi, Takako; Misawa, Naoaki; Okayama, Akihiko

2018-01-01

Vibrio furnissii and V. fluvialis are closely related, the discrimination of which by conventional biochemical assay remains a challenge. Investigation of the sequence of the 16S rRNA genes in a clinical isolate of V. furnissii by visual inspection of a sequencing electropherogram revealed two sites of single-nucleotide polymorphisms (SNPs; positions 460 A/G and 1261 A/G) in these genes. A test of 12 strains each of V. fluvialis and V. furnissii revealed these SNPs to be common in V. furnissii but not in V. fluvialis. Divergence of SNP frequency was observed among the strains of V. furnissii tested. Because the SNPs described in V. furnissii produce a difference in the target sequence of restriction enzymes, a combination of polymerase chain reaction (PCR) of the 16S rRNA genes using conventional primers and restriction fragment length polymorphism analysis using Eco RV and Eae I was shown to discriminate between V. fluvialis and V. furnissii. This method is simple and alleviates the need for expensive equipment or primer sets specific to these bacteria. Therefore, we believe that this method can be useful, alongside specific PCR and mass spectrometry, when there is a need to discriminate between V. fluvialis and V. furnissii. Copyright © 2017 Elsevier B.V. All rights reserved.

Epoxyalkane:Coenzyme M Transferase Gene Diversity and Distribution in Groundwater Samples from Chlorinated-Ethene-Contaminated Sites

PubMed Central

Liu, Xikun

2016-01-01

ABSTRACT Epoxyalkane:coenzyme M transferase (EaCoMT) plays a critical role in the aerobic biodegradation and assimilation of alkenes, including ethene, propene, and the toxic chloroethene vinyl chloride (VC). To improve our understanding of the diversity and distribution of EaCoMT genes in the environment, novel EaCoMT-specific terminal-restriction fragment length polymorphism (T-RFLP) and nested-PCR methods were developed and applied to groundwater samples from six different contaminated sites. T-RFLP analysis revealed 192 different EaCoMT T-RFs. Using clone libraries, we retrieved 139 EaCoMT gene sequences from these samples. Phylogenetic analysis revealed that a majority of the sequences (78.4%) grouped with EaCoMT genes found in VC- and ethene-assimilating Mycobacterium strains and Nocardioides sp. strain JS614. The four most-abundant T-RFs were also matched with EaCoMT clone sequences related to Mycobacterium and Nocardioides strains. The remaining EaCoMT sequences clustered within two emergent EaCoMT gene subgroups represented by sequences found in propene-assimilating Gordonia rubripertincta strain B-276 and Xanthobacter autotrophicus strain Py2. EaCoMT gene abundance was positively correlated with VC and ethene concentrations at the sites studied. IMPORTANCE The EaCoMT gene plays a critical role in assimilation of short-chain alkenes, such as ethene, VC, and propene. An improved understanding of EaCoMT gene diversity and distribution is significant to the field of bioremediation in several ways. The expansion of the EaCoMT gene database and identification of incorrectly annotated EaCoMT genes currently in the database will facilitate improved design of environmental molecular diagnostic tools and high-throughput sequencing approaches for future bioremediation studies. Our results further suggest that potentially significant aerobic VC degraders in the environment are not well represented in pure culture. Future research should aim to isolate and characterize aerobic VC-degrading bacteria from these underrepresented groups. PMID:27016563

Molecular genetic analysis of the V kappa Ser group associated with two mouse light chain genetic markers. Complementary DNA cloning and southern hybridization analysis

PubMed Central

1985-01-01

Previous studies (21) have shown that two mouse kappa light (L) chain variable (V) region polymorphisms, the IB-peptide and Efla markers, reflect expression of a characteristic group of V kappa regions, called V kappa Ser, by some inbred strains and not others. Expression of V kappa Ser is controlled by a locus on chromosome 6, the chromosome that contains the kappa locus. To further characterize this V kappa group and begin to analyze the basis for its strain-specific expression, full- length complementary DNA (cDNA) copies were produced of L chain mRNA from the M75 myeloma that had been induced in the C.C58 strain of mice, and which produces a V kappa Ser L chain. The C.C58 strain is congenic with BALB/cAn, differing in the region of chromosome 6 that controls expression of the V kappa polymorphisms and the Lyt-2 and Lyt-3 T cell alloantigens. The complete nucleotide sequence of this cloned cDNA was determined and compared with the nucleotide sequences the most closely related BALB/c myeloma L chains known. Results indicated significant differences throughout the variable region, but particularly toward the 5' portion of the sequence. A probe corresponding to 200 bp of the 5' end of the cloned V kappa Ser cDNA was used in Southern hybridizations of restriction digests of liver DNA from a number of inbred, recombinant, and recombinant inbred strains. Under stringent hybridization conditions, one strongly-hybridizing fragment was observed in Bam HI, Hind III, and Eco RI digests, and based on the size of the fragments, strains could be organized into two groups. The presence of strongly hybridizing Bam HI, Hind III, and Eco RI fragments of 3.2, 2.8, and 2.1 kb, respectively, was found to correlate completely with expression by the strain of the IB-peptide and Efla markers. All nonexpressor strains yielded hybridizing fragments of 7.8, 8.4, and 2.8 kb, respectively. Possible explanations for strain- specific expression of V kappa Ser-associated phenotypic markers are discussed. PMID:3926938

Genetic variability in biochemical characters of Brazilian field populations of the Leishmania vector, Lutzomyia longipalpis (Diptera: Psychodidae).

PubMed

Mukhopadhyay, J; Ghosh, K; Rangel, E F; Munstermann, L E

1998-12-01

The phlebotomine sand fly Lutzomyia longipalpis is the insect vector of visceral leishmaniasis, a protozoan disease of increasing incidence and distribution in Central and South America. Electrophoretic allele frequencies of 15 enzyme loci were compared among the L. longipalpis populations selected across its distribution range in Brazil. The mean heterozygosity of two colonized geographic strains (one each from Colombia and Brazil) were 6% and 13% respectively, with 1.6-1.9 alleles detected per locus. In contrast, among the seven widely separated field populations, the mean heterozygosity ranged from 11% to 16% with 2.1-2.9 alleles per locus. No locus was recovered that was diagnostic for any of the field populations. Allelic frequency differences among five field strains from the Amazon basin and eastern coastal Brazil were very low, with Nei's genetic distances of less than 0.01 separating them. The two inland and southerly samples from Minas Gerais (Lapinha) and Bahia (Jacobina) states were more distinctive with genetic distances of 0.024-0.038 and 0.038-0.059, respectively, when compared with the five other samples. These differences were the consequence of several high frequency alleles (glycerol-3-phosphate dehydrogenase [Gpd1.69] and phosphoglucomutase [Pgm1.69]) relatively uncommon in other strains. The low genetic distances, absence of diagnostic loci, and the distribution of genes in geographic space indicate L. longipalpis of Brazil to be a single, but genetically heterogeneous, polymorphic species.

Evaluation of genetic polymorphism among Lactobacillus rhamnosus non-starter Parmigiano Reggiano cheese strains.

PubMed

Bove, Claudio Giorgio; De Dea Lindner, Juliano; Lazzi, Camilla; Gatti, Monica; Neviani, Erasmo

2011-01-05

Parmigiano Reggiano (PR) is an Italian cooked, long-ripened cheese made with unheated cow's milk and natural whey starter. The microflora is involved in the manufacturing of this cheese, arising from the natural whey starter, the raw milk and the environment. Molecular studies have shown that mesophilic non-starter lactic acid bacteria (NSLAB) are the dominant microflora present during the ripening of PR. In this study, a characterisation of Lactobacillus rhamnosus isolated from a single PR manufacturing and ripening process is reported, using a combination of genotypic fingerprinting techniques (RAPD-PCR and REP-PCR). The intraspecies heterogeneity evidenced for 66 strains is correlated to their abilities to adapt to specific environmental and technological conditions. The detection of biotypes that correlate with specific moments in cheese ripening or differential development throughout this process suggests that these strains may have specific roles closely linked to their peculiar technological properties. Copyright © 2010 Elsevier B.V. All rights reserved.

Systemic Screening of Strains of the Lion's Mane Medicinal Mushroom Hericium erinaceus (Higher Basidiomycetes) and Its Protective Effects on Aβ-Triggered Neurotoxicity in PC12 Cells.

PubMed

Liu, Zongying; Wang, Qinglong; Cui, Jian; Wang, Lili; Xiong, Lili; Wang, Wei; Li, Diqiang; Liu, Na; Wu, Yiran; Mao, Canquan

2015-01-01

Hericium erinaceus possesses multiple medicinal values. To date, however, there have been few studies of the systemic screening of H. erinaceus strains, and the neuroprotective effects of H. erinaceus prepared from homogenized, fresh fruiting bodies are not fully understood. In this study, 4 random primers were selected and used in random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) to screen and evaluate the genetic diversity of 19 commercial strains of H. erinaceus from different localities in China. A total of 66 bands were obtained, and the percentage of polymorphic loci reached 80.30%. Five dendrograms were constructed based on RAPD by Jaccard cluster and within-group linkage analysis. Primer S20 as well as all 4 primers had great potential as specific primers for RAPD-PCR molecular identification and differentiation of H. erinaceus strains. Based on the results of submerged culture and fruiting body cultivation, strains HT-N, HT-J1, HT-C, and HT-M were identified as superior among the 19 H. erinaceus strains. Further study showed that the oral preparation of homogenized, fresh fruiting bodies of H. erinaceus could attenuate the Aβ25-35-triggered damage in PC12 cells by significantly increasing cell viability and by decreasing the release of lactate dehydrogenase. In conclusion, RAPD-PCR combined with liquid and solid cultures can be used well in the screening and identification of H. erinaceus strains, and products prepared from homogenized, fresh fruiting bodies of H. erinaceus had neuroprotective effects on PC12 cells.

Sequence variations of the bovine prion protein gene (PRNP) in native Korean Hanwoo cattle

PubMed Central

Choi, Sangho

2012-01-01

Bovine spongiform encephalopathy (BSE) is one of the fatal neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs) caused by infectious prion proteins. Genetic variations correlated with susceptibility or resistance to TSE in humans and sheep have not been reported for bovine strains including those from Holstein, Jersey, and Japanese Black cattle. Here, we investigated bovine prion protein gene (PRNP) variations in Hanwoo cattle [Bos (B.) taurus coreanae], a native breed in Korea. We identified mutations and polymorphisms in the coding region of PRNP, determined their frequency, and evaluated their significance. We identified four synonymous polymorphisms and two non-synonymous mutations in PRNP, but found no novel polymorphisms. The sequence and number of octapeptide repeats were completely conserved, and the haplotype frequency of the coding region was similar to that of other B. taurus strains. When we examined the 23-bp and 12-bp insertion/deletion (indel) polymorphisms in the non-coding region of PRNP, Hanwoo cattle had a lower deletion allele and 23-bp del/12-bp del haplotype frequency than healthy and BSE-affected animals of other strains. Thus, Hanwoo are seemingly less susceptible to BSE than other strains due to the 23-bp and 12-bp indel polymorphisms. PMID:22705734

DNA polymorphism in recombining and non-recombing mating-type-specific loci of the smut fungus Microbotryum

PubMed Central

Votintseva, A A; Filatov, D A

2011-01-01

The population-genetic processes leading to the genetic degeneration of non-recombining regions have mainly been studied in animal and plant sex chromosomes. Here, we report population genetic analysis of the processes in the non-recombining mating-type-specific regions of the smut fungus Microbotryum violaceum. M. violaceum has A1 and A2 mating types, determined by mating-type-specific ‘sex chromosomes' that contain 1–2 Mb long non-recombining regions. If genetic degeneration were occurring, then one would expect reduced DNA polymorphism in the non-recombining regions of this fungus. The analysis of DNA diversity among 19 M. violaceum strains, collected across Europe from Silene latifolia flowers, revealed that (i) DNA polymorphism is relatively low in all 20 studied loci (π∼0.15%), (ii) it is not significantly different between the two mating-type-specific chromosomes nor between the non-recombining and recombining regions, (iii) there is substantial population structure in M. violaceum populations, which resembles that of its host species, S. latifolia, and (iv) there is significant linkage disequilibrium, suggesting that widespread selfing in this species results in a reduction of the effective recombination rate across the genome. We hypothesise that selfing-related reduction of recombination across the M. violaceum genome negates the difference in the level of DNA polymorphism between the recombining and non-recombining regions, and may possibly lead to similar levels of genetic degeneration in the mating-type-specific regions of the non-recombining ‘sex chromosomes' and elsewhere in the genome. PMID:21081967

Multilocus enzyme electrophoresis on agarose gel as an aid to the identification of entomopathogenic Bacillus sphaericus strains.

PubMed

Zahner, V; Rabinovitch, L; Cavados, C F; Momen, H

1994-04-01

Sixty strains of Bacillus sphaericus, including 31 insect pathogens were studied by multilocus enzyme electrophoresis and were classified into 44 zymovars (electrophoretic types). Among the entomopathogenic strains, 11 belong to the same zymovar (Z59) indicating a widespread frequent genotype. Bands of enzyme activity were not detected among the strains for the loci GPI (E.C.5.3.1.9), G6P (E.C.1.1.1.49), 6PG (E.C.1.1.1.44) and ME (E.C.1.1.1.40). The enzymatic loci NP (E.C.2.4.2.1) and ACON (E.C.4.2.1.3) were monomorphic while the other enzymes, MDH (E.C.1.1.1.37), LeDH (E.C.1.4.1.9), ADH (E.C.1.4.1.1), EST (E.C.3.1.1.1), PEP-2 (E.C.3.4.11.1), PEP-3 (E.C.3.4.11) and PEP-D (E.C. 3.4.13.9) were polymorphic. The genetic variation in the non-insect pathogenic group seemed to be greater than in the entomopathogenic group. This latter group appears to be distinct from other strains of these species. All insect pathogens were recovered in the same phenetic cluster and a diagnostic allele is reported for the identification of entomopathogenic strains.

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 diagnostic real-time polymerase chain reaction assay for the detection of invasive Haemophilus influenzae in clinical samples.

PubMed

Meyler, Kenneth L; Meehan, Mary; Bennett, Desiree; Cunney, Robert; Cafferkey, Mary

2012-12-01

Since the introduction of the Haemophilus influenzae serotype b vaccine, invasive H. influenzae disease has become dominated by nontypeable (NT) strains. Several widely used molecular diagnostic methods have been shown to lack sensitivity or specificity in the detection of some of these strains. Novel real-time assays targeting the fucK, licA, and ompP2 genes were developed and evaluated. The fucK assay detected all strains of H. influenzae tested (n = 116) and had an analytical sensitivity of 10 genome copies/polymerase chain reaction (PCR). This assay detected both serotype b and NT H. influenzae in 12 previously positive specimens (culture and/or bexA PCR) and also detected H. influenzae in a further 5 of 883 culture-negative blood and cerebrospinal fluid (CSF) samples. The fucK assay has excellent potential as a diagnostic test for detection of typeable and nontypeable strains of invasive H. influenzae in clinical samples of blood and CSF. Copyright © 2012 Elsevier Inc. All rights reserved.

Multilocus microsatellite typing shows three different genetic clusters of Leishmania major in Iran.

PubMed

Mahnaz, Tashakori; Al-Jawabreh, Amer; Kuhls, Katrin; Schönian, Gabriele

2011-10-01

Ten polymorphic microsatellite markers were used to analyse 25 strains of Leishmania major collected from cutaneous leishmaniasis cases in different endemic areas in Iran. Nine of the markers were polymorphic, revealing 21 different genotypes. The data displayed significant microsatellite polymorphism with rare allelic heterozygosity. Bayesian statistic and distance based analyses identified three genetic clusters among the 25 strains analysed. Cluster I represented mainly strains isolated in the west and south-west of Iran, with the exception of four strains originating from central Iran. Cluster II comprised strains from the central part of Iran, and cluster III included only strains from north Iran. The geographical distribution of L. major in Iran was supported by comparing the microsatellite profiles of the 25 Iranian strains to those of 105 strains collected in 19 Asian and African countries. The Iranian clusters I and II were separated from three previously described populations comprising strains from Africa, the Middle East and Central Asia whereas cluster III grouped together with the Central Asian population. The considerable genetic variability of L. major might be related to the existence of different populations of Phlebotomus papatasi and/or to differences in reservoir host abundance in different parts of Iran. Copyright © 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Complete Genome Sequence of Yersinis pestis Strains Antiqua and Nepa1516: Evidence of Gene Reduction in an Emerging Pathogen

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

Chain, Patrick S; Hu, Ping; Malfatti, Stephanie

2006-01-01

Yersinia pestis, the causative agent of bubonic and pneumonic plagues, has undergone detailed study at the molecular level. To further investigate the genomic diversity among this group and to help characterize lineages of the plague organism that have no sequenced members, we present here the genomes of two isolates of the ''classical'' antiqua biovar, strains Antiqua and Nepal516. The genomes of Antiqua and Nepal516 are 4.7 Mb and 4.5 Mb and encode 4,138 and 3,956 open reading frames, respectively. Though both strains belong to one of the three classical biovars, they represent separate lineages defined by recent phylogenetic studies. Wemore » compare all five currently sequenced Y. pestis genomes and the corresponding features in Yersinia pseudotuberculosis. There are strain-specific rearrangements, insertions, deletions, single nucleotide polymorphisms, and a unique distribution of insertion sequences. We found 453 single nucleotide polymorphisms in protein-coding regions, which were used to assess the evolutionary relationships of these Y. pestis strains. Gene reduction analysis revealed that the gene deletion processes are under selective pressure, and many of the inactivations are probably related to the organism's interaction with its host environment. The results presented here clearly demonstrate the differences between the two biovar antiqua lineages and support the notion that grouping Y. pestis strains based strictly on the classical definition of biovars (predicated upon two biochemical assays) does not accurately reflect the phylogenetic relationships within this species. A comparison of four virulent Y. pestis strains with the human-avirulent strain 91001 provides further insight into the genetic basis of virulence to humans.« less

South-East Asian strains of Plasmodium falciparum display higher ratio of non-synonymous to synonymous polymorphisms compared to African strains

PubMed Central

Singh, Gajinder Pal; Sharma, Amit

2016-01-01

Resistance to frontline anti-malarial drugs, including artemisinin, has repeatedly arisen in South-East Asia, but the reasons for this are not understood. Here we test whether evolutionary constraints on Plasmodium falciparum strains from South-East Asia differ from African strains. We find a significantly higher ratio of non-synonymous to synonymous polymorphisms in P. falciparum from South-East Asia compared to Africa, suggesting differences in the selective constraints on P. falciparum genome in these geographical regions. Furthermore, South-East Asian strains showed a higher proportion of non-synonymous polymorphism at conserved positions, suggesting reduced negative selection. There was a lower rate of mixed infection by multiple genotypes in samples from South-East Asia compared to Africa. We propose that a lower mixed infection rate in South-East Asia reduces intra-host competition between the parasite clones, reducing the efficiency of natural selection. This might increase the probability of fixation of fitness-reducing mutations including drug resistant ones. PMID:27853513

Development of a Sequence-Characterized Amplified Region Marker-Targeted Quantitative PCR Assay for Strain-Specific Detection of Oenococcus oeni during Wine Malolactic Fermentation▿

PubMed Central

Solieri, Lisa; Giudici, Paolo

2010-01-01

Control over malolactic fermentation (MLF) is a difficult goal in winemaking and needs rapid methods to monitor Oenococcus oeni malolactic starters (MLS) in a stressful environment such as wine. In this study, we describe a novel quantitative PCR (QPCR) assay enabling the detection of an O. oeni strain during MLF without culturing. O. oeni strain LB221 was used as a model to develop a strain-specific sequence-characterized amplified region (SCAR) marker derived from a discriminatory OPA20-based randomly amplified polymorphic DNA (RAPD) band. The 5′ and 3′ flanking regions and the copy number of the SCAR marker were characterized using inverse PCR and Southern blotting, respectively. Primer pairs targeting the SCAR sequence enabled strain-specific detection without cross amplification of other O. oeni strains or wine species of lactic acid bacteria (LAB), acetic acid bacteria (AAB), and yeasts. The SCAR-QPCR assay was linear over a range of cell concentrations (7 log units) and detected as few as 2.2 × 102 CFU per ml of red wine with good quantification effectiveness, as shown by the correlation of QPCR and plate counting results. Therefore, the cultivation-independent monitoring of a single O. oeni strain in wine based on a SCAR marker represents a rapid and effective strain-specific approach. This strategy can be adopted to develop easy and rapid detection techniques for monitoring the implantation of inoculated O. oeni MLS on the indigenous LAB population, reducing the risk of unsuccessful MLF. PMID:20935116

Two hybridization events define the population structure of Trypanosoma cruzi.

PubMed

Westenberger, Scott J; Barnabé, Christian; Campbell, David A; Sturm, Nancy R

2005-10-01

Genetic variation in Trypanosoma cruzi is likely a key determinant in transmission and pathogenesis of Chagas disease. We have examined nine loci as markers for the extant T. cruzi strains. Four distinct alleles were found for each locus, corresponding to the sequence classes present in the homozygous discrete typing units (DTUs) I, IIa, IIb, and IIc. The alleles in DTUs IIa and IIc showed a spectrum of polymorphism ranging from DTU I-like to DTU IIb-like, in addition to DTU-specific sequence variation. DTUs IId and IIe were indistinguishable, showing DTU homozygosity at one locus and heterozygosity with DTU IIb and IIc allelic sequences at eight loci. Recombination between the DTU IIb and IIc alleles is evidenced from mosaic polymorphisms. These data imply that two discrete hybridization events resulted in the formation of the current DTUs. We propose a model in which a fusion between ancestral DTU I and IIb strains gave rise to a heterozygous hybrid that homogenized its genome to become the homozygous progenitor of DTUs IIa and IIc. The second hybridization between DTU IIb and IIc strains that generated DTUs IId and IIe resulted in extensive heterozygosity with subsequent recombination of parental genotypes.

CGDSNPdb: a database resource for error-checked and imputed mouse SNPs.

PubMed

Hutchins, Lucie N; Ding, Yueming; Szatkiewicz, Jin P; Von Smith, Randy; Yang, Hyuna; de Villena, Fernando Pardo-Manuel; Churchill, Gary A; Graber, Joel H

2010-07-06

The Center for Genome Dynamics Single Nucleotide Polymorphism Database (CGDSNPdb) is an open-source value-added database with more than nine million mouse single nucleotide polymorphisms (SNPs), drawn from multiple sources, with genotypes assigned to multiple inbred strains of laboratory mice. All SNPs are checked for accuracy and annotated for properties specific to the SNP as well as those implied by changes to overlapping protein-coding genes. CGDSNPdb serves as the primary interface to two unique data sets, the 'imputed genotype resource' in which a Hidden Markov Model was used to assess local haplotypes and the most probable base assignment at several million genomic loci in tens of strains of mice, and the Affymetrix Mouse Diversity Genotyping Array, a high density microarray with over 600,000 SNPs and over 900,000 invariant genomic probes. CGDSNPdb is accessible online through either a web-based query tool or a MySQL public login. Database URL: http://cgd.jax.org/cgdsnpdb/

Differentiation of Erwinia amylovora and Erwinia pyrifoliae strains with single nucleotide polymorphisms and by synthesis of dihydrophenylalanine.

PubMed

Gehring, I; Geider, K

2012-07-01

Fire blight has spread from North America to New Zealand, Europe, and the Mediterranean region. We were able to differentiate strains from various origins with a novel PCR method. Three Single Nucleotide Polymorphisms (SNPs) in the Erwinia amylovora genome were characteristic of isolates from North America and could distinguish them from isolates from other parts of the world. They were derived from the galE, acrB, and hrpA genes of strains Ea273 and Ea1/79. These genes were analyzed by conventional PCR (cPCR) and quantitative PCR (qPCR) with differential primer annealing temperatures. North-American E. amylovora strains were further differentiated according to their production of L: -2,5-dihydrophenylalanine (DHP) as tested by growth inhibition of the yeast Rhodotorula glutinis. E. amylovora fruit tree (Maloideae) and raspberry (rubus) strains were also differentiated by Single Strand Conformational Polymorphism analysis. Strains from the related species Erwinia pyrifoliae isolated in Korea and Japan were all DHP positive, but were differentiated from each other by SNPs in the galE gene. Differential PCR is a rapid and simple method to distinguish E. amylovora as well as E. pyrifoliae strains according to their geographical origin.

Pathological and Molecular Characterization of Xanthomonas campestris Strains Causing Diseases of Cassava (Manihot esculenta)

PubMed Central

Verdier, Valérie; Boher, Bernard; Maraite, Henri; Geiger, Jean-Paul

1994-01-01

Fifty-one strains representing Xanthomonas campestris pv. manihotis and cassavae and different pathovars occurring on plants of the family Euphorbiaceae were characterized by ribotyping with a 16S+23S rRNA probe of Escherichia coli and by restriction fragment length polymorphism analysis with a plasmid probe from X. campestris pv. manihotis. Pathogenicity tests were performed on cassava (Manihot esculenta). Histological comparative studies were conducted on strains of two pathovars of X. campestris (vascular and mesophyllic) that attack cassava. Our results indicated that X. campestris pv. manihotis and cassavae have different modes of action in the host and supplemented the taxonomic data on restriction fragment length polymorphism that clearly separate the two pathovars. The plasmid probe could detect multiple restriction fragment length polymorphisms among strains of the pathovar studied. Ribotyping provides a useful tool for rapid identification of X. campestris pathovars on cassava. Images PMID:16349463

Phylogenetic Analysis and Molecular Characterization of Xanthium sibiricum Using DNA Barcoding, PCR-RFLP, and Specific Primers.

PubMed

Tomasello, Salvatore; Heubl, Günther

2017-07-01

The fruits of Xanthium sibiricum have been widely used in traditional Chinese medicine for the treatment of nasal sinusitis and headaches. The genus Xanthium (cocklebur) is a taxonomically complex genus. Different taxonomic concepts have been proposed, some including several species, others lumping the different taxa in a few extremely polymorphic species. Due to the morphological similarities between species, the correct authentication of X. sibiricum is very difficult. Therefore, we established a polymerase chain reaction-restriction fragment length polymorphism method and diagnostic PCR based on nuclear internal transcribed spacer and chloroplast trnQ-rps16 barcodes to differentiate X. sibirium from related species.Results from the phylogenetic analyses based on sequence information from four marker regions (plastidal psbA-trnH and trnQ-rps16 and nuclear ITS and D35 ) support those taxonomic concepts accepting a reduced number of species, as four to five major clades are revealed in the phylogenetic reconstructions. X. sibiricum , together with some accessions from closely related taxa, is always supported as monophyletic, constituting a well-defined genetic entity. Allele-specific primer pairs for ITS and trnQ-rps16 were designed to amplify diagnostic products from the genomic DNA of X. sibiricum . Specific PCR in combination with digestion using the restriction enzyme Mse I allowed for the identification of X. sibiricum by producing specific restriction patterns. The results demonstrate that the applied techniques provide effective and accurate authentication of X. sibiricum . Georg Thieme Verlag KG Stuttgart · New York.

Mycobacterium leprae: genes, pseudogenes and genetic diversity

PubMed Central

Singh, Pushpendra; Cole, Stewart T

2011-01-01

Leprosy, which has afflicted human populations for millenia, results from infection with Mycobacterium leprae, an unculturable pathogen with an exceptionally long generation time. Considerable insight into the biology and drug resistance of the leprosy bacillus has been obtained from genomics. M. leprae has undergone reductive evolution and pseudogenes now occupy half of its genome. Comparative genomics of four different strains revealed remarkable conservation of the genome (99.995% identity) yet uncovered 215 polymorphic sites, mainly single nucleotide polymorphisms, and a handful of new pseudogenes. Mapping these polymorphisms in a large panel of strains defined 16 single nucleotide polymorphism-subtypes that showed strong geographical associations and helped retrace the evolution of M. leprae. PMID:21162636

Deep sequencing revealed genome-wide single-nucleotide polymorphism and plasmid content of Erwinia amylovora strains isolated in Middle Atlas, Morocco.

PubMed

Hannou, Najat; Mondy, Samuel; Planamente, Sara; Moumni, Mohieddine; Llop, Pablo; López, María; Manceau, Charles; Barny, Marie-Anne; Faure, Denis

2013-10-01

Erwinia amylovora causes economic losses that affect pear and apple production in Morocco. Here, we report comparative genomics of four Moroccan E. amylovora strains with the European strain CFBP1430 and North-American strain ATCC49946. Analysis of single nucleotide polymorphisms (SNPs) revealed genetic homogeneity of Moroccan's strains and their proximity to the European strain CFBP1430. Moreover, the collected sequences allowed the assembly of a 65 kpb plasmid, which is highly similar to the plasmid pEI70 harbored by several European E. amylovora isolates. This plasmid was found in 33% of the 40 E. amylovora strains collected from several host plants in 2009 and 2010 in Morocco. Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Detection of two fungal biocontrol agents against root-knot nematodes by RAPD markers.

PubMed

Zhu, Ming Liang; Mo, Ming He; Xia, Zhen Yuan; Li, Yun Hua; Yang, Shu Jun; Li, Tian Fei; Zhang, Ke Qin

2006-05-01

The strain ZK7 of Pochonia chlamydosporia var. chlamydosporia and IPC of Paecilomyces lilacinus are highly effective in the biological control against root-knot nematodes infecting tobacco. When applied, they require a specific monitoring method to evaluate the colonization and dispersal in soil. In this work, the randomly amplified polymorphic DNA (RAPD) technique was used to differentiate between the two individual strains and 95 other isolates, including isolates of the same species and common soil fungi. This approach allowed the selection of specific fragments of 1.2 kb (Vc1200) and 2.0 kb (Vc2000) specific for ZK7, 1.4 kb (P1400) and 0.85 kb (P850) specific for IPC, using the random Primers OPL-02, OPD-05, OPD-05 and OPC-11, respectively. These fragments were cloned, sequenced, and used to design sequence-characterized amplification region (SCAR) primers specific for the two strains. In classical polymerase chain reaction (PCR), with serial dilution of ZK7 and IPC pure culture DNAs template, the detection limits of these oligonucleotide SCAR-PCR primers were found to be 10, 1000, 500, 100 pg, respectively. In the dot blotting, digoxigenin (DIG)-labeled amplicons from these four primers specifically recognized the corresponding fragments in the DNAs template of these two strains. The detection limit of these amplicons were 0.2, 0.2, 0.5, 0.5 mug, respectively.

Using of methods of speckle optics for Chlamydia trachomatis typing

NASA Astrophysics Data System (ADS)

Ulyanov, Sergey S.; Zaytsev, Sergey S.; Ulianova, Onega V.; Saltykov, Yury V.; Feodorova, Valentina A.

2017-03-01

Specific method of transformation of nucleotide of gene into speckle pattern is suggested. Reference speckle pattern of omp1 gene of typical wild strains of Chlamydia trachomatis of genovars D, E, F, G, J and K and Chlamydia psittaci as well is generated. Perspectives of proposed technique in the gene identification and detection of natural genetic mutations as single nucleotide polymorphism (SNP) are demonstrated.

[The diagnostic value of ultrasonic elastography and ultrasonography comprehensive score in cervical lesions].

PubMed

Lu, R; Xiao, Y

2017-07-18

Objective: To evaluate the clinical value of ultrasonic elastography and ultrasonography comprehensive scoring method in the diagnosis of cervical lesions. Methods: A total of 116 patients were selected from the Department of Gynecology of the first hospital affiliated with Central South University from March 2014 to September 2015.All of the lesions were preoperatively examined by Doppler Ultrasound and elastography.The elasticity score was determined by a 5-point scoring method. Calculation of the strain ratio was based on a comparison of the average strain measured in the lesion with the adjacent tissue of the same depth, size, and shape.All these ultrasonic parameters were quantified, added, and arrived at ultrasonography comprehensive scores.To use surgical pathology as the gold standard, the sensitivity, specificity, accuracy of Doppler Ultrasound, elasticity score and strain ratio methods and ultrasonography comprehensive scoring method were comparatively analyzed. Results: (1) The sensitivity, specificity, and accuracy of Doppler Ultrasound in diagnosing cervical lesions were 82.89% (63/76), 85.0% (34/40), and 83.62% (97/116), respectively.(2) The sensitivity, specificity, and accuracy of the elasticity score method were 77.63% (59/76), 82.5% (33/40), and 79.31% (92/116), respectively; the sensitivity, specificity, and accuracy of the strain ratio measure method were 84.21% (64/76), 87.5% (35/40), and 85.34% (99/116), respectively.(3) The sensitivity, specificity, and accuracy of ultrasonography comprehensive scoring method were 90.79% (69/76), 92.5% (37/40), and 91.38% (106/116), respectively. Conclusion: (1) It was obvious that ultrasonic elastography had certain diagnostic value in cervical lesions. Strain ratio measurement can be more objective than elasticity score method.(2) The combined application of ultrasonography comprehensive scoring method, ultrasonic elastography and conventional sonography was more accurate than single parameter.

[Use of ITS and ISSR markers in the molecular characterisation of Pleurotus djamor hybrid strains].

PubMed

Aguilar Doroteo, Leticia; Zárate Segura, Paola Berenice; Villanueva Arce, Ramón; Yáñez Fernández, Jorge; Garín Aguilar, María Eugenia; Guadarrama Mendoza, Paula Cecilia; Valencia Del Toro, Gustavo

Molecular characterisation of wild type Pleurotus species is important for germplasm conservation and its further use for genetic improvement. No molecular studies have been performed with monokaryons used for producing hybrid strains, either with the reconstituted strains obtained by pairing those monokaryons. The molecular characterisation of parental dikaryons, hybrid, and reconstituted strains as well as monokaryotic strains, is therefore of utmost importance. To carry out the molecular identification of Pleurotus djamor strains, i.e. dikaryotic wild type strains, hybrid strains, and the monokaryotic strains used for the hybrid formation. Five wild type strains of P. djamor from different states in Mexico were collected and molecularly identified by sequencing the ITS1-5.8-ITS2 region using ITS1 and ITS4 universal oligonucleotides. Four hybrid strains were obtained by pairing neohaplonts of two wild type strains selected. Six ISSR markers were used for the molecular characterisation of monokaryotic and dikaryotic strains. Using the ITS markers, an amplified product of 700bp was obtained in five wild type strains, with a 99-100% similarity with P. djamor. A total of 95 fragments were obtained using the ISSR markers, with 99% of polymorphism. Wild type strains were identified as P. djamor, and were clearly grouped with Mexican strains from other states of Mexico. ISSR markers allowed the generation of polymorphic bands in monokaryotic and dikaryotic strains, splitting both types of strains. The high degree of polymorphism indicates the genetic diversity of P. djamor, an advantage in mushroom production and in the improving of the species. Copyright © 2017 Asociación Española de Micología. Publicado por Elsevier España, S.L.U. All rights reserved.

Systematic analysis of protein identity between Zika virus and other arthropod-borne viruses.

PubMed

Chang, Hsiao-Han; Huber, Roland G; Bond, Peter J; Grad, Yonatan H; Camerini, David; Maurer-Stroh, Sebastian; Lipsitch, Marc

2017-07-01

To analyse the proportions of protein identity between Zika virus and dengue, Japanese encephalitis, yellow fever, West Nile and chikungunya viruses as well as polymorphism between different Zika virus strains. We used published protein sequences for the Zika virus and obtained protein sequences for the other viruses from the National Center for Biotechnology Information (NCBI) protein database or the NCBI virus variation resource. We used BLASTP to find regions of identity between viruses. We quantified the identity between the Zika virus and each of the other viruses, as well as within-Zika virus polymorphism for all amino acid k -mers across the proteome, with k ranging from 6 to 100. We assessed accessibility of protein fragments by calculating the solvent accessible surface area for the envelope and nonstructural-1 (NS1) proteins. In total, we identified 294 Zika virus protein fragments with both low proportion of identity with other viruses and low levels of polymorphisms among Zika virus strains. The list includes protein fragments from all Zika virus proteins, except NS3. NS4A has the highest number (190 k -mers) of protein fragments on the list. We provide a candidate list of protein fragments that could be used when developing a sensitive and specific serological test to detect previous Zika virus infections.

High-Resolution Melting Analysis as a Powerful Tool to Discriminate and Genotype Pseudomonas savastanoi Pathovars and Strains

PubMed Central

Gori, Andrea; Cerboneschi, Matteo; Tegli, Stefania

2012-01-01

Pseudomonas savastanoi is a serious pathogen of Olive, Oleander, Ash, and several other Oleaceae. Its epiphytic or endophytic presence in asymptomatic plants is crucial for the spread of Olive and Oleander knot disease, as already ascertained for P. savastanoi pv. savastanoi (Psv) on Olive and for pv. nerii (Psn) on Oleander, while no information is available for pv. fraxini (Psf) on Ash. Nothing is known yet about the distribution on the different host plants and the real host range of these pathovars in nature, although cross-infections were observed following artificial inoculations. A multiplex Real-Time PCR assay was recently developed to simultaneously and quantitatively discriminate in vitro and in planta these P. savastanoi pathovars, for routine culture confirmation and for epidemiological and diagnostical studies. Here an innovative High-Resolution Melting Analysis (HRMA)-based assay was set up to unequivocally discriminate Psv, Psn and Psf, according to several single nucleotide polymorphisms found in their Type Three Secretion System clusters. The genetic distances among 56 P. savastanoi strains belonging to these pathovars were also evaluated, confirming and refining data previously obtained by fAFLP. To our knowledge, this is the first time that HRMA is applied to a bacterial plant pathogen, and one of the few multiplex HRMA-based assays developed so far. This protocol provides a rapid, sensitive, specific tool to differentiate and detect Psv, Psn and Psf strains, also in vivo and against other related bacteria, with lower costs than conventional multiplex Real-Time PCR. Its application is particularly suitable for sanitary certification programs for P. savastanoi, aimed at avoiding the spreading of this phytopathogen through asymptomatic plants. PMID:22295075

RAD sequencing yields a high success rate for westslope cutthroat and rainbow trout species-diagnostic SNP assays

USGS Publications Warehouse

Stephen J. Amish,; Paul A. Hohenlohe,; Sally Painter,; Robb F. Leary,; Muhlfeld, Clint C.; Fred W. Allendorf,; Luikart, Gordon

2012-01-01

Hybridization with introduced rainbow trout threatens most native westslope cutthroat trout populations. Understanding the genetic effects of hybridization and introgression requires a large set of high-throughput, diagnostic genetic markers to inform conservation and management. Recently, we identified several thousand candidate single-nucleotide polymorphism (SNP) markers based on RAD sequencing of 11 westslope cutthroat trout and 13 rainbow trout individuals. Here, we used flanking sequence for 56 of these candidate SNP markers to design high-throughput genotyping assays. We validated the assays on a total of 92 individuals from 22 populations and seven hatchery strains. Forty-six assays (82%) amplified consistently and allowed easy identification of westslope cutthroat and rainbow trout alleles as well as heterozygote controls. The 46 SNPs will provide high power for early detection of population admixture and improved identification of hybrid and nonhybridized individuals. This technique shows promise as a very low-cost, reliable and relatively rapid method for developing and testing SNP markers for nonmodel organisms with limited genomic resources.

Delamanid susceptibility testing of Mycobacterium tuberculosis using the resazurin microtitre assay and the BACTEC™ MGIT™ 960 system.

PubMed

Schena, Elisa; Nedialkova, Lubov; Borroni, Emanuele; Battaglia, Simone; Cabibbe, Andrea Maurizio; Niemann, Stefan; Utpatel, Christian; Merker, Matthias; Trovato, Alberto; Hofmann-Thiel, Sabine; Hoffmann, Harald; Cirillo, Daniela Maria

2016-06-01

The objective of this study was to develop standardized protocols for rapid delamanid drug susceptibility testing (DST) using the colorimetric resazurin microtitre assay (REMA) and semi-automated BACTEC™ MGIT™ 960 system (MGIT) by establishing breakpoints that accurately discriminate between susceptibility and resistance of Mycobacterium tuberculosis to delamanid. MICs of delamanid were determined by the MGIT, the REMA and the solid agar method for 19 pre-characterized strains. The MIC distribution of delamanid was then established for a panel of clinical strains never exposed to the drug and characterized by different geographical origins and susceptibility patterns. WGS was used to investigate genetic polymorphisms in five genes (ddn, fgd1, fbiA, fbiB and fbiC) involved in intracellular delamanid activation. We demonstrated that the REMA and MGIT can both be used for the rapid and accurate determination of delamanid MIC, showing excellent concordance with the solid agar reference method, as well as high reproducibility and repeatability. We propose the tentative breakpoint of 0.125 mg/L for the REMA and MGIT, allowing reliable discrimination between M. tuberculosis susceptible and resistant to delamanid. Stop codon mutations in ddn (Trp-88 → STOP) and fbiA (Lys-250 → STOP) have only been observed in strains resistant to delamanid. We established protocols for DST of delamanid in the MGIT and REMA, confirming their feasibility in routine TB diagnostics, utilizing the same discriminative concentration for both methods. Moreover, taking advantage of WGS analysis, we identified polymorphisms potentially associated with resistance in two genes involved in delamanid activation. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Microbiological characterisation of Burkholderia cepacia isolates from cystic fibrosis patients: investigation of the exopolysaccharides produced.

PubMed

Lagatolla, Cristina; Skerlavaj, Silvia; Dolzani, Lucilla; Tonin, Enrico A; Monti Bragadin, Carlo; Bosco, Marco; Rizzo, Roberto; Giglio, Luisella; Cescutti, Paola

2002-03-19

Eleven strains of Burkholderia cepacia were isolated directly from clinical specimens: 10 from sputum of cystic fibrosis patients, and one from a vaginal swab. They were biochemically identified using API20NE and confirmed by a PCR-based assay. The genomovar characterisation obtained by specific PCR amplification revealed seven strains belonging to genomovar I, three belonging to genomovar IIIA and one belonging to genomovar IV. All isolates were also typed by ribotyping and random amplification of polymorphic DNA analysis. Some of the characterised strains were examined for the ability to produce exopolysaccharides, with the aim of correlating the genomovar with the exopolysaccharide structure. The polysaccharides were analysed by means of methylation analysis and 1H-NMR spectroscopy in order to determine structural similarities. It was shown that different strains are capable of producing chemically different polysaccharides.

Discrimination of the Lactobacillus acidophilus group using sequencing, species-specific PCR and SNaPshot mini-sequencing technology based on the recA gene.

PubMed

Huang, Chien-Hsun; Chang, Mu-Tzu; Huang, Mu-Chiou; Wang, Li-Tin; Huang, Lina; Lee, Fwu-Ling

2012-10-01

To clearly identify specific species and subspecies of the Lactobacillus acidophilus group using phenotypic and genotypic (16S rDNA sequence analysis) techniques alone is difficult. The aim of this study was to use the recA gene for species discrimination in the L. acidophilus group, as well as to develop a species-specific primer and single nucleotide polymorphism primer based on the recA gene sequence for species and subspecies identification. The average sequence similarity for the recA gene among type strains was 80.0%, and most members of the L. acidophilus group could be clearly distinguished. The species-specific primer was designed according to the recA gene sequencing, which was employed for polymerase chain reaction with the template DNA of Lactobacillus strains. A single 231-bp species-specific band was found only in L. delbrueckii. A SNaPshot mini-sequencing assay using recA as a target gene was also developed. The specificity of the mini-sequencing assay was evaluated using 31 strains of L. delbrueckii species and was able to unambiguously discriminate strains belonging to the subspecies L. delbrueckii subsp. bulgaricus. The phylogenetic relationships of most strains in the L. acidophilus group can be resolved using recA gene sequencing, and a novel method to identify the species and subspecies of the L. delbrueckii and L. delbrueckii subsp. bulgaricus was developed by species-specific polymerase chain reaction combined with SNaPshot mini-sequencing. Copyright © 2012 Society of Chemical Industry.

Diagnostic multiplex PCR for toxin genotyping of Clostridium perfringens isolates.

PubMed

Baums, Christoph G; Schotte, Ulrich; Amtsberg, Gunter; Goethe, Ralph

2004-05-20

In this study we provide a protocol for genotyping Clostridium perfringens with a new multiplex PCR. This PCR enables reliable and specific detection of the toxin genes cpa, cpb, etx, iap, cpe and cpb2 from heat lysed bacterial suspensions. The efficiency of the protocol was demonstrated by typing C. perfringens reference strains and isolates from veterinary bacteriological routine diagnostic specimens.

A diagnostic PCR assay for the detection of an Australian epidemic strain of Pseudomonas aeruginosa

PubMed Central

2010-01-01

Background Chronic lung infection with the bacterium Pseudomonas aeruginosa is one of the hallmarks of cystic fibrosis (CF) and is associated with worsening lung function, increased hospitalisation and reduced life expectancy. A virulent clonal strain of P. aeruginosa (Australian epidemic strain I; AES-I) has been found to be widespread in CF patients in eastern Australia. Methods Suppression subtractive hybridization (SSH) was employed to identify genetic sequences that are present in the AES-I strain but absent from the sequenced reference strain PAO1. We used PCR to evaluate the distribution of several of the AES-I loci amongst a collection of 188 P. aeruginosa isolates which was comprised of 35 AES-I isolates (as determined by PFGE), 78 non-AES-I CF isolates including other epidemic CF strains as well as 69 P. aeruginosa isolates from other clinical and environmental sources. Results We have identified a unique AES-I genetic locus that is present in all 35 AES-I isolates tested and not present in any of the other 153 P. aeruginosa strains examined. We have used this unique AES-I locus to develop a diagnostic PCR and a real-time PCR assay to detect the presence of P. aeruginosa and AES-I in patient sputum samples. Conclusions We have developed diagnostic PCR assays that are 100% sensitive and 100% specific for the P. aeruginosa strain AES-I. We have also shown that Whatman FTA® Elute cards may be used with PCR-based assays to rapidly detect the presence of P. aeruginosa strains in CF sputum. PMID:20637114

Parasite genetics and the immune host: recombination between antigenic types of Eimeria maxima as an entrée to the identification of protective antigens.

PubMed

Blake, Damer P; Hesketh, Patricia; Archer, Andrew; Carroll, Fionnadh; Smith, Adrian L; Shirley, Martin W

2004-11-01

The genomes of protozoan parasites encode thousands of gene products and identification of the subset that stimulates a protective immune response is a daunting task. Most screens for vaccine candidates identify molecules by capacity to induce immune responses rather than protection. This paper describes the core findings of a strategy developed with the coccidial parasite Eimeria maxima to rationally identify loci within its genome that encode immunoprotective antigens. Our strategy uses a novel combination of parasite genetics, DNA fingerprinting, drug-resistance and strain-specific immunity and centres on two strains of E. maxima that each induce a lethal strain-specific protective immune response in the host and show a differential response to anti-Eimeria chemotherapy. Through classical mating studies with these strains we have demonstrated that loci encoding molecules stimulating strain-specific protective immunity or resistance to the anti-coccidial drug robenidine segregate independently. Furthermore, passage of populations of recombinant parasites in the face of killing in the immune host was accompanied by the elimination of some polymorphic DNA markers defining the parent strain used to immunise the host. Consideration of the numbers of parasites recombinant for the two traits implicates very few antigen-encoding loci. Our data provide a potential strategy to identify putative antigen-encoding loci in other parasites.

Identification of Plasmodium falciparum reticulocyte binding protein homologue 5-interacting protein, PfRipr, as a highly conserved blood-stage malaria vaccine candidate.

PubMed

Ntege, Edward H; Arisue, Nobuko; Ito, Daisuke; Hasegawa, Tomoyuki; Palacpac, Nirianne M Q; Egwang, Thomas G; Horii, Toshihiro; Takashima, Eizo; Tsuboi, Takafumi

2016-11-04

Genetic variability in Plasmodium falciparum malaria parasites hampers current malaria vaccine development efforts. Here, we hypothesize that to address the impact of genetic variability on vaccine efficacy in clinical trials, conserved antigen targets should be selected to achieve robust host immunity across multiple falciparum strains. Therefore, suitable vaccine antigens should be assessed for levels of polymorphism and genetic diversity. Using a total of one hundred and two clinical isolates from a region of high malaria transmission in Uganda, we analyzed extent of polymorphism and genetic diversity in four recently reported novel blood-stage malaria vaccine candidate proteins: Rh5 interacting protein (PfRipr), GPI anchored micronemal antigen (PfGAMA), rhoptry-associated leucine zipper-like protein 1 (PfRALP1) and Duffy binding-like merozoite surface protein 1 (PfMSPDBL1). In addition, utilizing the wheat germ cell-free system, we expressed recombinant proteins for the four candidates based on P. falciparum laboratory strain 3D7 sequences, immunized rabbits to obtain specific antibodies (Abs) and performed functional growth inhibition assay (GIA). The GIA activity of the raised Abs was demonstrated using both homologous 3D7 and heterologous FVO strains in vitro. Both pfripr and pfralp1 are less polymorphic but the latter is comparatively more diverse, with varied number of regions having insertions and deletions, asparagine and 6-mer repeats in the coding sequences. Pfgama and pfmspdbl1 are polymorphic and genetically diverse among the isolates with antibodies against the 3D7-based recombinant PfGAMA and PfMSPDBL1 inhibiting merozoite invasion only in the 3D7 but not FVO strain. Moreover, although Abs against the 3D7-based recombinant PfRipr and PfRALP1 proteins potently inhibited merozoite invasion of both 3D7 and FVO, the GIA activity of anti-PfRipr was much higher than that of anti-PfRALP1. Thus, PfRipr is regarded as a promising blood-stage vaccine candidate for next-generation vaccines against P. falciparum. Copyright © 2016 Elsevier Ltd. All rights reserved.

Use of restriction fragment length polymorphisms to investigate strain variation within Neisseria meningitidis

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

Williams, S.D.

1989-01-01

Similarity within bacterial populations is difficult to assess due to the limited number of characters available for evaluation and the heterogeneity of bacterial species. Currently, the preferred method used to evaluate the structure of bacterial populations is multilocus enzyme electrophoresis. However, this method is extremely cumbersome and only offers an indirect measure of genetic similarities. The development of a more direct and less cumbersome method for this purpose is warranted. Restriction fragment length polymorphism analysis was evaluated as a tool for use in the study of bacterial population structures and in the epidemiology and surveillance of infectious disease. A collectionmore » of Neisseria meningitidis was available for use in the investigation of this technique. Neisseria meningitidis is the causative agent of epidemic cerebrospinal meningitis and septicemia as well as a variety of other clinical manifestations. Each isolate in the collection was defined in terms of serogroup specificity, clinical history, geographic source, and date of isolation. Forty-six strains were chosen for this study. The DNA from each strain was restricted with Pst1 and EcoR1 and electrophoresed on agarose gels. The DNA was transferred to nylon filters and hybridized with P{sup 32} labeled DNA probes. Two randomly generated probes and a gene-specific probe were used to estimate the genetic similarities between and among the strains in the study population. A total of 28 different restriction fragment migration types were detected by the probes used. Data obtained from the RFLP analysis was analyzed by cluster analysis and multivariate statistical methods. A total of 7 clones groups were detected. Two of these appear to be major clones that comprise 35% of the population.« less

Genomic Variability of Mycobacterium tuberculosis Strains of the Euro-American Lineage Based on Large Sequence Deletions and 15-Locus MIRU-VNTR Polymorphism

PubMed Central

Rindi, Laura; Medici, Chiara; Bimbi, Nicola; Buzzigoli, Andrea; Lari, Nicoletta; Garzelli, Carlo

2014-01-01

A sample of 260 Mycobacterium tuberculosis strains assigned to the Euro-American family was studied to identify phylogenetically informative genomic regions of difference (RD). Mutually exclusive deletions of regions RD115, RD122, RD174, RD182, RD183, RD193, RD219, RD726 and RD761 were found in 202 strains; the RDRio deletion was detected exclusively among the RD174-deleted strains. Although certain deletions were found more frequently in certain spoligotype families (i.e., deletion RD115 in T and LAM, RD174 in LAM, RD182 in Haarlem, RD219 in T and RD726 in the “Cameroon” family), the RD-defined sublineages did not specifically match with spoligotype-defined families, thus arguing against the use of spoligotyping for establishing exact phylogenetic relationships between strains. Notably, when tested for katG463/gyrA95 polymorphism, all the RD-defined sublineages belonged to Principal Genotypic Group (PGG) 2, except sublineage RD219 exclusively belonging to PGG3; the 58 Euro-American strains with no deletion were of either PGG2 or 3. A representative sample of 197 isolates was then analyzed by standard 15-locus MIRU-VNTR typing, a suitable approach to independently assess genetic relationships among the strains. Analysis of the MIRU-VNTR typing results by using a minimum spanning tree (MST) and a classical dendrogram showed groupings that were largely concordant with those obtained by RD-based analysis. Isolates of a given RD profile show, in addition to closely related MIRU-VNTR profiles, related spoligotype profiles that can serve as a basis for better spoligotype-based classification. PMID:25197794

Novel primer specific false terminations during DNA sequencing reactions: danger of inaccuracy of mutation analysis in molecular diagnostics

PubMed Central

Anwar, R; Booth, A; Churchill, A J; Markham, A F

1996-01-01

The determination of nucleotide sequence is fundamental to the identification and molecular analysis of genes. Direct sequencing of PCR products is now becoming a commonplace procedure for haplotype analysis, and for defining mutations and polymorphism within genes, particularly for diagnostic purposes. A previously unrecognised phenomenon, primer related variability, observed in sequence data generated using Taq cycle sequencing and T7 Sequenase sequencing, is reported. This suggests that caution is necessary when interpreting DNA sequence data. This is particularly important in situations where treatment may be dependent on the accuracy of the molecular diagnosis. Images PMID:16696096

A Brillouin smart FRP material and a strain data post processing software for structural health monitoring through laboratory testing and field application on a highway bridge

NASA Astrophysics Data System (ADS)

Bastianini, Filippo; Matta, Fabio; Galati, Nestore; Nanni, Antonio

2005-05-01

Strain and temperature sensing obtained through frequency shift evaluation of Brillouin scattered light is a technology that seems extremely promising for Structural Health Monitoring (SHM). Due to the intrinsic distributed sensing capability, Brillouin can measure the deformation of any individual segment of huge lengths of inexpensive single-mode fiber. In addition, Brillouin retains other typical advantages of Fiber Optic Sensors (FOS), such as harsh environment durability and interference rejection. Despite these advantages, the diffusion of Brillouin for SHM is constrained by different factors, such as the high equipment cost, the commercial unavailability of specific SHM oriented fiber products and even some prejudices on the required sensitivity performances. In the present work, a complete SHM pilot application was developed, installed and successfully operated during a diagnostic load test on the High Performance Steel (HPS) bridge A6358 located at the Lake of the Ozarks (Miller County, MO, USA). Four out of five girders were extensively instrumented with a "smart" Glass Fiber Reinforced Polymer (GFRP) tape having embedded fibers for strain sensing and thermal compensation. Data collected during a diagnostic load test were elaborated through a specific post-processing software, and the strain profiles retrieved were compared to traditional strain gauges and theoretical results based on the AASHTO LRFD Bridge Design Specifications for structural assessment purposes. The excellent results obtained confirm the effectiveness of Brillouin SHM systems for the monitoring of real applications.

Characterization of Satellite DNA Sequences from the Commercially Important Marine Rotifers Brachionus rotundiformis and Brachionus plicatilis.

PubMed

Boehm; Gibson; Lubzens

2000-01-01

This study was initiated to search for species-specific and strain-specific satellite DNA sequences for which oligonucleotide primers could be designed to differentiate between various commercially important strains of the marine monogonont rotifers Brachionus rotundiformis and Brachionus plicatilis. Two unrelated, highly reiterated satellite sequences were cloned and characterized. The eight sequenced monomers from B. rotundiformis and six from B. plicatilis had low intrarepeat variability and were similar in their overall lengths, A + T compositions, and high degrees of repeated motif substructure. However, hybridizations to 19 representative strains, sequence characterizations, and GenBank searches indicated that these two satellites are morphotype-specific and population-specific, respectively, and share little homology to each other or to other characterized sequences in the database. Primer pairs designed for the B. rotundiformis satellite confirmed hybridization specificities on polymerase chain reaction and could serve as a useful molecular diagnostic tool to identify strains belonging to the SS morphotype, which are gaining widespread usage as first feeds for marine fish in commercial production.

Analysis of the QTL for sleep homeostasis in mice: Homer1a is a likely candidate.

PubMed

Mackiewicz, M; Paigen, B; Naidoo, N; Pack, A I

2008-03-14

Electroencephalographic oscillations in the frequency range of 0.5-4 Hz, characteristic of slow-wave sleep (SWS), are often referred to as the delta oscillation or delta power. Delta power reflects sleep intensity and correlates with the homeostatic response to sleep loss. A published survey of inbred strains of mice demonstrated that the time course of accumulation of delta power varied among inbred strains, and the segregation of the rebound of delta power in BxD recombinant inbred strains identified a genomic region on chromosome 13 referred to as the delta power in SWS (or Dps1). The quantitative trait locus (QTL) contains genes that modify the accumulation of delta power after sleep deprivation. Here, we narrow the QTL using interval-specific haplotype analysis and present a comprehensive annotation of the remaining genes in the Dps1 region with sequence comparisons to identify polymorphisms within the coding and regulatory regions. We established the expression pattern of selected genes located in the Dps1 interval in sleep and wakefulness in B6 and D2 parental strains. Taken together, these steps reduced the number of potential candidate genes that may underlie the accumulation of delta power after sleep deprivation and explain the Dps1 QTL. The strongest candidate gene is Homer1a, which is supported by expression differences between sleep and wakefulness and the SNP polymorphism in the upstream regulatory regions.

[PSI+] Prion transmission barriers protect Saccharomyces cerevisiae from infection: intraspecies 'species barriers'.

PubMed

Bateman, David A; Wickner, Reed B

2012-02-01

[PSI+] is a prion of Sup35p, an essential translation termination and mRNA turnover factor. The existence of lethal [PSI+] variants, the absence of [PSI+] in wild strains, the mRNA turnover function of the Sup35p prion domain, and the stress reaction to prion infection suggest that [PSI+] is a disease. Nonetheless, others have proposed that [PSI+] and other yeast prions benefit their hosts. We find that wild Saccharomyces cerevisiae strains are polymorphic for the sequence of the prion domain and particularly in the adjacent M domain. Here we establish that these variations within the species produce barriers to prion transmission. The barriers are partially asymmetric in some cases, and evidence for variant specificity in barriers is presented. We propose that, as the PrP 129M/V polymorphism protects people from Creutzfeldt-Jakob disease, the Sup35p polymorphisms were selected to protect yeast cells from prion infection. In one prion incompatibility group, the barrier is due to N109S in the Sup35 prion domain and several changes in the middle (M) domain, with either the single N109S mutation or the group of M changes (without the N109S) producing a barrier. In another, the barrier is due to a large deletion in the repeat domain. All are outside the region previously believed to determine transmission compatibility. [SWI+], a prion of the chromatin remodeling factor Swi1p, was also proposed to benefit its host. We find that none of 70 wild strains carry this prion, suggesting that it is not beneficial.

Interferometric Reflectance Imaging Sensor (IRIS)—A Platform Technology for Multiplexed Diagnostics and Digital Detection

PubMed Central

Avci, Oguzhan; Lortlar Ünlü, Nese; Yalçın Özkumur, Ayça; Ünlü, M. Selim

2015-01-01

Over the last decade, the growing need in disease diagnostics has stimulated rapid development of new technologies with unprecedented capabilities. Recent emerging infectious diseases and epidemics have revealed the shortcomings of existing diagnostics tools, and the necessity for further improvements. Optical biosensors can lay the foundations for future generation diagnostics by providing means to detect biomarkers in a highly sensitive, specific, quantitative and multiplexed fashion. Here, we review an optical sensing technology, Interferometric Reflectance Imaging Sensor (IRIS), and the relevant features of this multifunctional platform for quantitative, label-free and dynamic detection. We discuss two distinct modalities for IRIS: (i) low-magnification (ensemble biomolecular mass measurements) and (ii) high-magnification (digital detection of individual nanoparticles) along with their applications, including label-free detection of multiplexed protein chips, measurement of single nucleotide polymorphism, quantification of transcription factor DNA binding, and high sensitivity digital sensing and characterization of nanoparticles and viruses. PMID:26205273

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.

Analysis on the DNA Fingerprinting of Aspergillus Oryzae Mutant Induced by High Hydrostatic Pressure

NASA Astrophysics Data System (ADS)

Wang, Hua; Zhang, Jian; Yang, Fan; Wang, Kai; Shen, Si-Le; Liu, Bing-Bing; Zou, Bo; Zou, Guang-Tian

2011-01-01

The mutant strains of aspergillus oryzae (HP300a) are screened under 300 MPa for 20 min. Compared with the control strains, the screened mutant strains have unique properties such as genetic stability, rapid growth, lots of spores, and high protease activity. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) are used to analyze the DNA fingerprinting of HP300a and the control strains. There are 67.9% and 51.3% polymorphic bands obtained by these two markers, respectively, indicating significant genetic variations between HP300a and the control strains. In addition, comparison of HP300a and the control strains, the genetic distances of random sequence and simple sequence repeat of DNA are 0.51 and 0.34, respectively.

DNA detection and single nucleotide mutation identification using SERS for molecular diagnostics and global health

NASA Astrophysics Data System (ADS)

Ngo, Hoan T.; Gandra, Naveen; Fales, Andrew M.; Taylor, Steve M.; Vo-Dinh, Tuan

2017-02-01

Nucleic acid-based molecular diagnostics at the point-of-care (POC) and in resource-limited settings is still a challenge. We present a sensitive yet simple DNA detection method with single nucleotide polymorphism (SNP) identification capability. The detection scheme involves sandwich hybridization of magnetic beads conjugated with capture probes, target sequences, and ultrabright surface-enhanced Raman Scattering (SERS) nanorattles conjugated with reporter probes. Upon hybridization, the sandwich probes are concentrated at the detection focus controlled by a magnetic system for SERS measurements. The ultrabright SERS nanorattles, consisting of a core and a shell with resonance Raman reporters loaded in the gap space between the core and the shell, serve as SERS tags for ultrasensitive signal detection. Specific DNA sequences of the malaria parasite Plasmodium falciparum and dengue virus 1 (DENV1) were used as the model marker system. Detection limit of approximately 100 attomoles was achieved. Single nucleotide polymorphism (SNP) discrimination of wild type malaria DNA and mutant malaria DNA, which confers resistance to artemisinin drugs, was also demonstrated. The results demonstrate the molecular diagnostic potential of the nanorattle-based method to both detect and genotype infectious pathogens. The method's simplicity makes it a suitable candidate for molecular diagnosis at the POC and in resource-limited settings.

Computational Approach for Epitaxial Polymorph Stabilization through Substrate Selection.

PubMed

Ding, Hong; Dwaraknath, Shyam S; Garten, Lauren; Ndione, Paul; Ginley, David; Persson, Kristin A

2016-05-25

With the ultimate goal of finding new polymorphs through targeted synthesis conditions and techniques, we outline a computational framework to select optimal substrates for epitaxial growth using first principle calculations of formation energies, elastic strain energy, and topological information. To demonstrate the approach, we study the stabilization of metastable VO2 compounds which provides a rich chemical and structural polymorph space. We find that common polymorph statistics, lattice matching, and energy above hull considerations recommends homostructural growth on TiO2 substrates, where the VO2 brookite phase would be preferentially grown on the a-c TiO2 brookite plane while the columbite and anatase structures favor the a-b plane on the respective TiO2 phases. Overall, we find that a model which incorporates a geometric unit cell area matching between the substrate and the target film as well as the resulting strain energy density of the film provide qualitative agreement with experimental observations for the heterostructural growth of known VO2 polymorphs: rutile, A and B phases. The minimal interfacial geometry matching and estimated strain energy criteria provide several suggestions for substrates and substrate-film orientations for the heterostructural growth of the hitherto hypothetical anatase, brookite, and columbite polymorphs. These criteria serve as a preliminary guidance for the experimental efforts stabilizing new materials and/or polymorphs through epitaxy. The current screening algorithm is being integrated within the Materials Project online framework and data and hence publicly available.

Computational Approach for Epitaxial Polymorph Stabilization through Substrate Selection

DOE PAGES

Ding, Hong; Dwaraknath, Shyam S.; Garten, Lauren; ...

2016-05-04

With the ultimate goal of finding new polymorphs through targeted synthesis conditions and techniques, we outline a computational framework to select optimal substrates for epitaxial growth using first principle calculations of formation energies, elastic strain energy, and topological information. To demonstrate the approach, we study the stabilization of metastable VO 2 compounds which provides a rich chemical and structural polymorph space. Here, we find that common polymorph statistics, lattice matching, and energy above hull considerations recommends homostructural growth on TiO 2 substrates, where the VO 2 brookite phase would be preferentially grown on the a-c TiO 2 brookite plane whilemore » the columbite and anatase structures favor the a-b plane on the respective TiO 2 phases. Overall, we find that a model which incorporates a geometric unit cell area matching between the substrate and the target film as well as the resulting strain energy density of the film provide qualitative agreement with experimental observations for the heterostructural growth of known VO 2 polymorphs: rutile, A and B phases. The minimal interfacial geometry matching and estimated strain energy criteria provide several suggestions for substrates and substrate-film orientations for the heterostructural growth of the hitherto hypothetical anatase, brookite, and columbite polymorphs. Our criteria serve as a preliminary guidance for the experimental efforts stabilizing new materials and/or polymorphs through epitaxy. The current screening algorithm is being integrated within the Materials Project online framework and data and hence publicly available.« less

Computational Approach for Epitaxial Polymorph Stabilization through Substrate Selection

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

Ding, Hong; Dwaraknath, Shyam S.; Garten, Lauren

With the ultimate goal of finding new polymorphs through targeted synthesis conditions and techniques, we outline a computational framework to select optimal substrates for epitaxial growth using first principle calculations of formation energies, elastic strain energy, and topological information. To demonstrate the approach, we study the stabilization of metastable VO2 compounds which provides a rich chemical and structural polymorph space. We find that common polymorph statistics, lattice matching, and energy above hull considerations recommends homostructural growth on TiO2 substrates, where the VO2 brookite phase would be preferentially grown on the a-c TiO2 brookite plane while the columbite and anatase structuresmore » favor the a-b plane on the respective TiO2 phases. Overall, we find that a model which incorporates a geometric unit cell area matching between the substrate and the target film as well as the resulting strain energy density of the film provide qualitative agreement with experimental observations for the heterostructural growth of known VO2 polymorphs: rutile, A and B phases. The minimal interfacial geometry matching and estimated strain energy criteria provide several suggestions for substrates and substrate-film orientations for the heterostructural growth of the hitherto hypothetical anatase, brookite, and columbite polymorphs. These criteria serve as a preliminary guidance for the experimental efforts stabilizing new materials and/or polymorphs through epitaxy. The current screening algorithm is being integrated within the Materials Project online framework and data and hence publicly available.« less

Computational Approach for Epitaxial Polymorph Stabilization through Substrate Selection

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

Ding, Hong; Dwaraknath, Shyam S.; Garten, Lauren

With the ultimate goal of finding new polymorphs through targeted synthesis conditions and techniques, we outline a computational framework to select optimal substrates for epitaxial growth using first principle calculations of formation energies, elastic strain energy, and topological information. To demonstrate the approach, we study the stabilization of metastable VO 2 compounds which provides a rich chemical and structural polymorph space. Here, we find that common polymorph statistics, lattice matching, and energy above hull considerations recommends homostructural growth on TiO 2 substrates, where the VO 2 brookite phase would be preferentially grown on the a-c TiO 2 brookite plane whilemore » the columbite and anatase structures favor the a-b plane on the respective TiO 2 phases. Overall, we find that a model which incorporates a geometric unit cell area matching between the substrate and the target film as well as the resulting strain energy density of the film provide qualitative agreement with experimental observations for the heterostructural growth of known VO 2 polymorphs: rutile, A and B phases. The minimal interfacial geometry matching and estimated strain energy criteria provide several suggestions for substrates and substrate-film orientations for the heterostructural growth of the hitherto hypothetical anatase, brookite, and columbite polymorphs. Our criteria serve as a preliminary guidance for the experimental efforts stabilizing new materials and/or polymorphs through epitaxy. The current screening algorithm is being integrated within the Materials Project online framework and data and hence publicly available.« less

Application of loop-mediated isothermal amplification (LAMP) assays for the detection of bovine herpesvirus 1.

PubMed

Socha, W; Rola, J; Urban-Chmiel, R; Żmudziński, J F

2017-09-26

Bovine herpesvirus-1 (BoHV-1), a causative agent of Infectious Bovine Rhinotracheitis (IBR), is responsible for high economic losses in cattle farming industry. The use of testing methods that allow early detection of BoHV-1-infected animals is a key element of each program of IBR eradication. The aim of the study was to design and evaluate two variants of LAMP isothermal tests with SYBR Green fluorescence probes, specific to the genes encoding gD and gE glycoproteins of BoHV-1. LAMP gE BoHV-1 assay was able to distinguish between gE- and gE+ strains of the virus. Both LAMP gD and gE assays were specific to BoHV-1 and did not react with other related to BoHV-1 alphaherpesviruses. Sensitivity of LAMP gD was 2x104 copies of the viral genome whereas for LAMP gE it was 2x105. Diagnostic sensitivity calculated for LAMP gD was 64.7% whereas for LAMP gE it was 80%. Diagnostic specificity for LAMP gD and LAMP gE was 78.9% and 89.3%, respectively. LAMP assay can be a rapid and simple method of diagnosis of acute BoHV-1 infections and discrimination of gE- strains. However, relatively low diagnostic sensitivity of the method can limit its use in routine diagnostics.

Mitochondrial pathogenic mutations are population-specific.

PubMed

Breen, Michael S; Kondrashov, Fyodor A

2010-12-31

Surveying deleterious variation in human populations is crucial for our understanding, diagnosis and potential treatment of human genetic pathologies. A number of recent genome-wide analyses focused on the prevalence of segregating deleterious alleles in the nuclear genome. However, such studies have not been conducted for the mitochondrial genome. We present a systematic survey of polymorphisms in the human mitochondrial genome, including those predicted to be deleterious and those that correspond to known pathogenic mutations. Analyzing 4458 completely sequenced mitochondrial genomes we characterize the genetic diversity of different types of single nucleotide polymorphisms (SNPs) in African (L haplotypes) and non-African (M and N haplotypes) populations. We find that the overall level of polymorphism is higher in the mitochondrial compared to the nuclear genome, although the mitochondrial genome appears to be under stronger selection as indicated by proportionally fewer nonsynonymous than synonymous substitutions. The African mitochondrial genomes show higher heterozygosity, a greater number of polymorphic sites and higher frequencies of polymorphisms for synonymous, benign and damaging polymorphism than non-African genomes. However, African genomes carry significantly fewer SNPs that have been previously characterized as pathogenic compared to non-African genomes. Finding SNPs classified as pathogenic to be the only category of polymorphisms that are more abundant in non-African genomes is best explained by a systematic ascertainment bias that favours the discovery of pathogenic polymorphisms segregating in non-African populations. This further suggests that, contrary to the common disease-common variant hypothesis, pathogenic mutations are largely population-specific and different SNPs may be associated with the same disease in different populations. Therefore, to obtain a comprehensive picture of the deleterious variability in the human population, as well as to improve the diagnostics of individuals carrying African mitochondrial haplotypes, it is necessary to survey different populations independently. This article was reviewed by Dr Mikhail Gelfand, Dr Vasily Ramensky (nominated by Dr Eugene Koonin) and Dr David Rand (nominated by Dr Laurence Hurst).

Flor yeasts of Saccharomyces cerevisiae--their ecology, genetics and metabolism.

PubMed

Alexandre, Hervé

2013-10-15

The aging of certain white wines is dependent on the presence of yeast strains that develop a biofilm on the wine surface after the alcoholic fermentation. These strains belong to the genus Saccharomyces and are called flor yeasts. These strains possess distinctive characteristics compared with Saccharomyces cerevisiae fermenting strain. The most important one is their capacity to form a biofilm on the air-liquid interface of the wine. The major gene involved in this phenotype is FLO11, however other genes are also involved in velum formation by these yeast and will be detailed. Other striking features presented in this review are their aneuploidy, and their mitochondrial DNA polymorphism which seems to reflect adaptive evolution of the yeast to a stressful environment where acetaldehyde and ethanol are present at elevated concentration. The biofilm assures access to oxygen and therefore permits continued growth on non-fermentable ethanol. This specific metabolism explains the peculiar organoleptic profile of these wines, especially their content in acetaldehyde and sotolon. This review deals with these different specificities of flor yeasts and will also underline the existing gaps regarding these astonishing yeasts. © 2013.

Performance of an ultra-sensitive Plasmodium falciparum HRP2-based rapid diagnostic test with recombinant HRP2, culture parasites, and archived whole blood samples.

PubMed

Das, Smita; Peck, Roger B; Barney, Rebecca; Jang, Ihn Kyung; Kahn, Maria; Zhu, Meilin; Domingo, Gonzalo J

2018-03-17

As malaria endemic countries shift from control to elimination, the proportion of low density Plasmodium falciparum infections increases. Current field diagnostic tools, such as microscopy and rapid diagnostic tests (RDT), with detection limits of approximately 100-200 parasites/µL (p/µL) and 800-1000 pg/mL histidine-rich protein 2 (HRP2), respectively, are unable to detect these infections. A novel ultra-sensitive HRP2-based Alere™ Malaria Ag P.f RDT (uRDT) was evaluated in laboratory conditions to define the test's performance against recombinant HRP2 and native cultured parasites. The uRDT detected dilutions of P. falciparum recombinant GST-W2 and FliS-W2, as well as cultured W2 and ITG, diluted in whole blood down to 10-40 pg/mL HRP2, depending on the protein tested. uRDT specificity was 100% against 123 archived frozen whole blood samples. Rapid test cross-reactivity with HRP3 was investigated using pfhrp2 gene deletion strains D10 and Dd2, pfhrp3 gene deletion strain HB3, and controls pfhrp2 and pfhrp3 double deletion strain 3BD5 and pfhrp2 and pfhrp3 competent strain ITG. The commercial Standard Diagnostics, Inc. BIOLINE Malaria Ag P.f RDT (SD-RDT) and uRDT detected pfhrp2 positive strains down to 49 and 3.13 p/µL, respectively. The pfhrp2 deletion strains were detected down to 98 p/µL by both tests. The performance of the uRDT was variable depending on the protein, but overall showed a greater than 10-fold improvement over the SD-RDT. The uRDT also exhibited excellent specificity and showed the same cross-reactivity with HRP3 as the SD-RDT. Together, the results support the uRDT as a more sensitive HRP2 test that could be a potentially effective tool in elimination campaigns. Further clinical evaluations for this purpose are merited.

Genetic variability of citrinin-producing Penicillium citrinum strains as occupational health hazards in northern Iran.

PubMed

Khosravi, Ali Reza; Sheikhkarami, Mojgan; Shokri, Hojjatollah; Sabokbar, Azar

2012-12-01

We evaluated the ability of randomly amplified polymorphic DNA (RAPD) to type citrinin-producing Penicillium citrinum (P. citrinum) strains recovered from the forest's air in northern Iran. A total of 12 P. citrinum strains (P1-P12) were characterised by citrinin production and random amplification of polymorphic DNA (RAPD) technique. All the strains produced citrinin with levels ranging from 1.5 μg mL(-1) to 39.6 μg mL(-1) (average value: 12.68 μg mL(-1)). Of 11 primers tested, eight primers produced polymorphic amplification patterns. These primers generated a total of 105 reproducible RAPD bands, averaging to 13.1 bands per primer. Dendrogram for each primer indicating the distance of the strains to each other was constructed. RAPD results showed that the collected strains constituted four different clusters. The first cluster included two isolates (P1 and P3). The second cluster included seven isolates (P2, P4, P5, P6, P7, P8, and P10). The third and fourth clusters included one isolate (P9) and two isolates (P11 and P12), respectively. We concluded that RAPD analysis might be used in providing genotypic characters for toxigenic P. citrinum strains typing in epidemiological investigations and public health related risk assessment.

Investigating pulmonary embolism in the emergency department with lower limb plethysmography: the Manchester Investigation of Pulmonary Embolism Diagnosis (MIOPED) study

PubMed Central

Hogg, K; Dawson, D; Mackway‐Jones, K

2006-01-01

Objectives To measure the diagnostic accuracy of computerised strain gauge plethysmography in the diagnosis of pulmonary embolism (PE). Methods Two researchers prospectively recruited 425 patients with pleuritic chest pain presenting to the emergency department (ED). Lower limb computerised strain gauge plethysmography was performed in the ED. All patients underwent an independent reference standard diagnostic algorithm to establish the presence or absence of PE. A low modified Wells' clinical probability combined with a normal D‐dimer excluded PE. All others required diagnostic imaging with PIOPED interpreted ventilation perfusion scanning and/or computerised tomography (CT) pulmonary angiography. Patients with a nondiagnostic CT had digital subtraction pulmonary angiography. All patients were followed up clinically for 3 months. Results The sensitivity of computerised strain gauge plethysmography was 33.3% (95% confidence interval (CI) 16.3 to 56.2%) and specificity 64.1% (95% CI 59.0 to 68.8%). The negative likelihood ratio was 1.04 (95% CI 0.68 to 1.33) and positive likelihood ratio 0.93 (95% CI 0.45 to 1.60). Conclusions Lower limb computerised strain gauge plethysmography does not aid in the diagnosis of PE. PMID:16439734

The murine Cd48 gene: allelic polymorphism in the IgV-like region.

PubMed

Cabrero, J G; Freeman, G J; Reiser, H

1998-12-01

The murine CD48 molecule is a member of the immunoglobulin superfamily which regulates the activation of T lymphocytes. prior cloning experiments using mRNA from two different mouse strains had yielded discrepant sequences within the IgV-like domain of murine CD48. To resolve this issue, we have directly sequenced genomic DNA of 10 laboratory strains and two inbred strains of wild origin. The results of our analysis reveal an allelic polymorphism within the IgV-like domain of murine CD48.

[The use of multilocus sequence typing (MLST) and randomly amplified polymorphic DNA (RAPD) for the differentiation between strains of Burkholderia mallei].

PubMed

Antonov, V A; Altukhova, V V; Savchenko, S S; Zamaraev, V S; Iliukhin, V I; Alekseev, V V

2007-01-01

Burkholderia mallei is highly pathogenic microorganism for both humans and animals. In this work, the possibility of the use of the genotyping method for differentiation between strains of B. mallei was studied. A collection of 14 isolates of B. mallei was characterized using randomly amplified polymorphic DNA (RAPD) and multilocus sequence typing (MLST). RAPD was the best method used for detecting strain differences of B. mallei. It was suggested that this method would be an increasingly useful molecular epidemiological tool.

[Clustered regularly interspaced short palindromic repeats (CRISPR) site in Bacillus anthracis].

PubMed

Gao, Zhiqi; Wang, Dongshu; Feng, Erling; Wang, Bingxiang; Hui, Yiming; Han, Shaobo; Jiao, Lei; Liu, Xiankai; Wang, Hengliang

2014-11-04

To investigate the polymorphism of clustered regularly interspaced short palindromic repeats (CRISPR) in Bacillu santhracis and the application to molecular typing based on the polymorphism of CRISPR in B. anthracis. We downloaded the whole genome sequence of 6 B. anthracis strains and extracted the CRISPR sites. We designed the primers of CRISPR sites and amplified the CRISPR fragments in 193 B. anthracis strains by PCR and sequenced these fragments. In order to reveal the polymorphism of CRISPR in B. anthracis, wealigned all the extracted sequences and sequenced results by local blasting. At the same time, we also analyzed the CRISPR sites in B. cereus and B. thuringiensis. We did not find any polymorphism of CRISPR in B. anthracis. The molecular typing approach based on CRISPR polymorphism is not suitable for B. anthracis, but it is possible for us to distinguish B. anthracis from B. cereus and B. thuringiensis.

Loop-mediated isothermal amplification assay targeting the mpb70 gene for rapid differential detection of Mycobacterium bovis.

PubMed

Zhang, Hui; Wang, Zhen; Cao, Xudong; Wang, Zhengrong; Sheng, Jinliang; Wang, Yong; Zhang, Jing; Li, Zhiqiang; Gu, Xinli; Chen, Chuangfu

2016-11-01

Loop-mediated isothermal amplification (LAMP) is a highly sensitive, rapid, cost-effective nucleic acid amplification method. Tuberculosis (TB) is widely popular in the world and it is difficult to cure. The fundamental treatment is to clear the types of TB pathogens such as Mycobacterium bovis (M. bovis), Mycobacterium tuberculosis (M. tuberculosis). In order to detect and diagnose TB early, we constructed the differential diagnostic method of TB. In this study, we used LAMP for detection of M. bovis, based on amplification of the mpb70 gene which is a unique gene in M. bovis strain. The LAMP assay was able to detect only seven copies of the gene per reaction, whereas for the conventional PCR, it was 70 copies. The LAMP was evaluated for its specificity using six strains of five Mycobacterium species and 18 related non-Mycobacterium microorganism strains as controls. The target three Mycobacterium strains were all amplified, and no cross-reaction was found with 18 non-Mycobacterium microorganism strains. TB was detected by two methods, LAMP and conventional PCR (based on mpb70 gene); the positive rates of the two methods were 9.55 and 7.01 %, respectively. Our results indicate that the LAMP method should be a potential tool with high convenience, rapidity, sensitivity and specificity for the diagnosis of TB caused by M. bovis. Most importance is that the use of LAMP as diagnostic method in association with diagnostic tests based on mpb70 gene would allow the differentiation between M. bovis and other Mycobacterium in humans or animals. The LAMP method is actually in order to detect human TB, and it can be used for differential diagnosis in this paper.

Characterization of natural polymorphic sites of the HIV-1 integrase before the introduction of HIV-1 integrase inhibitors in Germany

PubMed Central

Meixenberger, Karolin; Pouran Yousef, Kaveh; Somogyi, Sybille; Fiedler, Stefan; Bartmeyer, Barbara; von Kleist, Max; Kücherer, Claudia

2014-01-01

Introduction The aim of our study was to analyze the occurrence and evolution of HIV-1 integrase polymorphisms during the HIV-1 epidemic in Germany prior to the introduction of the first integrase inhibitor raltegravir in 2007. Materials and Methods Plasma samples from drug-naïve HIV-1 infected individuals newly diagnosed between 1986 and 2006 were used to determine PCR-based population sequences of the HIV-1 integrase (amino acids 1–278). The HIV-1 subtype was determined using the REGA HIV-1 subtyping tool. We calculated the frequency of amino acids at each position of the HIV-1 integrase in 337 subtype B strains for the time periods 1986–1989, 1991–1994, 1995–1998, 1999–2002, and 2003–2006. Positions were defined as polymorphic if amino acid variation was >1% in any period. Logistic regression was used to identify trends in amino acid variation over time. Resistance-associated mutations were identified according to the IAS 2013 list and the HIVdb, ANRS and GRADE algorithms. Results Overall, 56.8% (158/278) amino acid positions were polymorphic and 15.8% (25/158) of these positions exhibited a significant trend in amino acid variation over time. Proportionately, most polymorphic positions (63.3%, 31/49) were detected in the N-terminal zinc finger domain of the HIV-1 integrase. Motifs and residues essential for HIV-1 integrase activity were little polymorphic, but within the minimal non-specific DNA binding region I220-D270 up to 18.1% amino acid variation was noticed, including four positions with significant amino acid variation over time (S230, D232, D256, A265). No major resistance mutations were identified, and minor resistance mutations were rarely observed without trend over time. E157Q considered by HIVdb, ANRS, and GRADE algorithms was the most frequent resistance-associated polymorphism with an overall prevalence of 2.4%. Conclusions Detailed knowledge of the evolutionary variation of HIV-1 integrase polymorphisms is important to understand the development of resistance in the presence of the drug. Our results will contribute to define the relevance of integrase polymorphisms in HIV-strains resistant to integrase inhibitors and to improve resistance interpretation algorithms. PMID:25397491

How direct competition shapes coexistence and vaccine effects in multi-strain pathogen systems.

PubMed

Gjini, Erida; Valente, Carina; Sá-Leão, Raquel; Gomes, M Gabriela M

2016-01-07

We describe an integrated modeling framework for understanding strain coexistence in polymorphic pathogen systems. Previous studies have debated the utility of neutral formulations and focused on cross-immunity between strains as a major stabilizing mechanism. Here we convey that direct competition for colonization mediates stable coexistence only when competitive abilities amongst pathogen clones satisfy certain pairwise asymmetries. We illustrate our ideas with nested SIS models of single and dual colonization, applied to polymorphic pneumococcal bacteria. By fitting the models to cross-sectional prevalence data from Portugal (before and after the introduction of a seven-valent pneumococcal conjugate vaccine), we are able to not only statistically compare neutral and non-neutral epidemiological formulations, but also estimate vaccine efficacy, transmission and competition parameters simultaneously. Our study highlights that the response of polymorphic pathogen populations to interventions holds crucial information about strain interactions, which can be extracted by suitable nested modeling. Copyright © 2015 Elsevier Ltd. All rights reserved.

Efficiency of PCR-based methods in discriminating Bifidobacterium longum ssp. longum and Bifidobacterium longum ssp. infantis strains of human origin.

PubMed

Srůtková, Dagmar; Spanova, Alena; Spano, Miroslav; Dráb, Vladimír; Schwarzer, Martin; Kozaková, Hana; Rittich, Bohuslav

2011-10-01

Bifidobacterium longum is considered to play an important role in health maintenance of the human gastrointestinal tract. Probiotic properties of bifidobacterial isolates are strictly strain-dependent and reliable methods for the identification and discrimination of this species at both subspecies and strain levels are thus required. Differentiation between B. longum ssp. longum and B. longum ssp. infantis is difficult due to high genomic similarities. In this study, four molecular-biological methods (species- and subspecies-specific PCRs, random amplified polymorphic DNA (RAPD) method using 5 primers, repetitive sequence-based (rep)-PCR with BOXA1R and (GTG)(5) primers and amplified ribosomal DNA restriction analysis (ARDRA)) and biochemical analysis, were compared for the classification of 30 B. longum strains (28 isolates and 2 collection strains) on subspecies level. Strains originally isolated from the faeces of breast-fed healthy infants (25) and healthy adults (3) showed a high degree of genetic homogeneity by PCR with subspecies-specific primers and rep-PCR. When analysed by RAPD, the strains formed many separate clusters without any potential for subspecies discrimination. These methods together with arabionose/melezitose fermentation analysis clearly differentiated only the collection strains into B. longum ssp. longum and B. longum ssp. infantis at the subspecies level. On the other hand, ARDRA analysis differentiated the strains into the B. longum/infantis subspecies using the cleavage analysis of genus-specific amplicon with just one enzyme, Sau3AI. According to our results the majority of the strains belong to the B. longum ssp. infantis (75%). Therefore we suggest ARDRA using Sau3AI restriction enzyme as the first method of choice for distinguishing between B. longum ssp. longum and B. longum ssp. infantis. Copyright © 2011 Elsevier B.V. All rights reserved.

Neuropsychopharmacology and Neurogenetic Aspects of Executive Functioning: Should Reward Gene Polymorphisms Constitute a Diagnostic Tool to Identify Individuals at Risk for Impaired Judgment?

PubMed Central

Bowirrat, Abdalla; Chen, Thomas JH; Oscar-Berman, Marlene; Madigan, Margaret; Chen, Amanda LH; Bailey, John A.; Braverman, Eric R.; Kerner, Mallory; Giordano, John; Morse, Siohban; Downs, B. William; Waite, Roger L.; Fornari, Frank; Armaly, Zaher; Blum, Kenneth

2013-01-01

Executive functions are processes that act in harmony to control behaviors necessary for maintaining focus and achieving outcomes. Executive dysfunction in neuropsychiatric disorders is attributed to structural or functional pathology of brain networks involving prefrontal cortex (PFC) and its connections with other brain regions. The PFC receives innervations from different neurons associated with a number of neurotransmitters, especially dopamine (DA). Here we review findings on the contribution of PFC DA to higher-order cognitive and emotional behaviors. We suggest examination of multifactorial interactions of an individual’s genetic history, along with environmental risk factors, can assist in the characterization of executive functioning for that individual. Based upon the results of genetic studies we also propose genetic mapping as a probable diagnostic tool serving as a therapeutic adjunct for augmenting executive functioning capabilities. We conclude that preservation of the neurological underpinnings of executive functions requires the integrity of complex neural systems including the influence of specific genes and associated polymorphisms to provide adequate neurotransmission. PMID:22371275

Neuropsychopharmacology and neurogenetic aspects of executive functioning: should reward gene polymorphisms constitute a diagnostic tool to identify individuals at risk for impaired judgment?

PubMed

Bowirrat, Abdalla; Chen, Thomas J H; Oscar-Berman, Marlene; Madigan, Margaret; Chen, Amanda Lh; Bailey, John A; Braverman, Eric R; Kerner, Mallory; Giordano, John; Morse, Siobhan; Downs, B William; Waite, Roger L; Fornari, Frank; Armaly, Zaher; Blum, Kenneth

2012-04-01

Executive functions are processes that act in harmony to control behaviors necessary for maintaining focus and achieving outcomes. Executive dysfunction in neuropsychiatric disorders is attributed to structural or functional pathology of brain networks involving prefrontal cortex (PFC) and its connections with other brain regions. The PFC receives innervations from different neurons associated with a number of neurotransmitters, especially dopamine (DA). Here we review findings on the contribution of PFC DA to higher-order cognitive and emotional behaviors. We suggest that examination of multifactorial interactions of an individual's genetic history, along with environmental risk factors, can assist in the characterization of executive functioning for that individual. Based upon the results of genetic studies, we also propose genetic mapping as a probable diagnostic tool serving as a therapeutic adjunct for augmenting executive functioning capabilities. We conclude that preservation of the neurological underpinnings of executive functions requires the integrity of complex neural systems including the influence of specific genes and associated polymorphisms to provide adequate neurotransmission.

Porphyromonas gingivalis Uses Specific Domain Rearrangements and Allelic Exchange to Generate Diversity in Surface Virulence Factors.

PubMed

Dashper, Stuart G; Mitchell, Helen L; Seers, Christine A; Gladman, Simon L; Seemann, Torsten; Bulach, Dieter M; Chandry, P Scott; Cross, Keith J; Cleal, Steven M; Reynolds, Eric C

2017-01-01

Porphyromonas gingivalis is a keystone pathogen of chronic periodontitis. The virulence of P. gingivalis is reported to be strain related and there are currently a number of strain typing schemes based on variation in capsular polysaccharide, the major and minor fimbriae and adhesin domains of Lys-gingipain (Kgp), amongst other surface proteins. P. gingivalis can exchange chromosomal DNA between strains by natural competence and conjugation. The aim of this study was to determine the genetic variability of P. gingivalis strains sourced from international locations over a 25-year period and to determine if variability in surface virulence factors has a phylogenetic basis. Whole genome sequencing was performed on 13 strains and comparison made to 10 previously sequenced strains. A single nucleotide polymorphism-based phylogenetic analysis demonstrated a shallow tri-lobed phylogeny. There was a high level of reticulation in the phylogenetic network, demonstrating extensive horizontal gene transfer between the strains. Two highly conserved variants of the catalytic domain of the major virulence factor the Kgp proteinase (Kgp cat I and Kgp cat II) were found. There were three variants of the fourth Kgp C-terminal cleaved adhesin domain. Specific variants of the cell surface proteins FimA, FimCDE, MfaI, RagAB, Tpr, and PrtT were also identified. The occurrence of all these variants in the P. gingivalis strains formed a mosaic that was not related to the SNP-based phylogeny. In conclusion P. gingivalis uses domain rearrangements and genetic exchange to generate diversity in specific surface virulence factors.

Diagnosis of sub-centimetre breast lesions: combining BI-RADS-US with strain elastography and contrast-enhanced ultrasound-a preliminary study in China.

PubMed

Xiao, Xiaoyun; Jiang, Qiongchao; Wu, Huan; Guan, Xiaofeng; Qin, Wei; Luo, Baoming

2017-06-01

To compare the diagnostic efficacies of B-mode ultrasound (US), strain elastography (SE), contrast-enhanced ultrasound (CEUS) and the combination of these modalities for breast lesions <1 cm in size. Between January 2013 and October 2015, 203 inpatients with 209 sub-centimetre breast lesions categorised as BI-RADS-US (Breast Imaging Reporting and Data System for Ultrasound) 3-5 were included. US, SE and CEUS were performed to evaluate each lesion. The diagnostic performances of different ultrasonic modalities were compared. The diagnostic efficacies of BI-RADS-US and our re-rating systems were also compared. The pathology findings were used as the reference standard. The specificities of US, SE and CEUS for tumour differentiation were 17.4 %, 56.2 % and 86.0 %, respectively (P < 0.05); and the sensitivities were 100 %, 93.2 % and 93.2 % for US, SE and CEUS, respectively (P < 0.05). The area under the curve (AUC) of the receiver operating characteristic (ROC) curve was 0.867 for original BI-RADS-US, 0.882 for BI-RADS-US combined with only SE, 0.953 for BI-RADS-US combined with only CEUS and 0.924 for BI-RADS-US combined with both SE and CEUS. The best combination was BI-RADS-US combined with only CEUS. Evaluating sub-centimetre breast lesions with SE and CEUS could increase the diagnostic specificity while retaining high sensitivity compared with B-mode ultrasound. • Evaluating breast lesions with SE and CEUS could increase the diagnostic specificity • SE and CEUS offer alternatives to biopsy and possibly allow shorter-interval follow-ups • BI-RADS-US combined with CEUS exhibited the best diagnostic performance.

Effect of 5-HT2A Receptor Polymorphisms, Work Stressors, and Social Support on Job Strain among Petroleum Workers in Xinjiang, China.

PubMed

Jiang, Yu; Tang, Jinhua; Li, Rong; Zhao, Junling; Song, Zhixin; Ge, Hua; Lian, Yulong; Liu, Jiwen

2016-12-19

Previous studies have shown that work stressors and social support influence job strain. However, few studies have examined the impact of individual differences on job strain. In Xinjiang, there are a large number of petroleum workers in arid deserts. The present study investigated the effects of work stressors, social support, and 5-hydroxytryptamine receptor (5-HTR2A) genotype on the etiology of job strain among petroleum workers in Xinjiang. A cross-sectional study was carried out between January and August 2013. A total of 700 workers were selected by a three-stage stratified sampling method. 5-HTR2A genotypes were determined with the SNaPshot single nucleotide polymorphism assay. Work stressors and job strain were evaluated with the Occupational Stress Inventory-Revised questionnaire. Social support was assessed with the Chinese Social Support Rating Scale. Work overload and responsibility were significantly associated with job strain. Low social support was associated with severe vocational and interpersonal strain. High social support was a protective factor against job strain (odds ratio (OR) = 0.32, 95% confidence interval (CI): 0.14-0.76). The CC genotype of rs6313 and the AA genotype of rs2070040 were linked to severe vocational strain. Ordinal logistic regression analysis revealed that the CC genotype of rs6313 was linked to higher risk of job strain than the TT genotype (OR = 1.88, 95% CI: 1.10-3.23). These data provide evidence that work stressors, low social support, and 5-HTR2A gene polymorphism contributes to the risk of job strain.

2D-speckle tracking right ventricular strain to assess right ventricular systolic function in systolic heart failure. Analysis of the right ventricular free and posterolateral walls.

PubMed

Mouton, Stéphanie; Ridon, Héléne; Fertin, Marie; Pentiah, Anju Duva; Goémine, Céline; Petyt, Grégory; Lamblin, Nicolas; Coisne, Augustin; Foucher-Hossein, Claude; Montaigne, David; de Groote, Pascal

2017-10-15

Right ventricular (RV) systolic function is a powerful prognostic factor in patients with systolic heart failure. The accurate estimation of RV function remains difficult. The aim of the study was to determine the diagnostic accuracy of 2D-speckle tracking RV strain in patients with systolic heart failure, analyzing both free and posterolateral walls. Seventy-six patients with dilated cardiopathy (left ventricular end-diastolic volume≥75ml/m 2 ) and left ventricular ejection fraction≤45% had an analysis of the RV strain. Feasibility, reproducibility and diagnostic accuracy of RV strain were analyzed and compared to other echocardiographic parameters of RV function. RV dysfunction was defined as a RV ejection fraction≤40% measured by radionuclide angiography. RV strain feasibility was 93.9% for the free-wall and 79.8% for the posterolateral wall. RV strain reproducibility was good (intra-observer and inter-observer bias and limits of agreement of 0.16±1.2% [-2.2-2.5] and 0.84±2.4 [-5.5-3.8], respectively). Patients with left heart failure have a RV systolic dysfunction that can be unmasked by advanced echocardiographic imaging: mean RV strain was -21±5.7% in patients without RV dysfunction and -15.8±5.1% in patients with RV dysfunction (p=0.0001). Mean RV strain showed the highest diagnostic accuracy to predict depressed RVEF (area under the curve (AUC) 0.75) with moderate sensitivity (60.5%) but high specificity (87.5%) using a cutoff value of -16%. RV strain seems to be a promising and more efficient measure than previous RV echocardiographic parameters for the diagnosis of RV systolic dysfunction. Copyright © 2017 Elsevier B.V. All rights reserved.

Use of Conserved Randomly Amplified Polymorphic DNA (RAPD) Fragments and RAPD Pattern for Characterization of Lactobacillus fermentum in Ghanaian Fermented Maize Dough

PubMed Central

Hayford, Alice E.; Petersen, Anne; Vogensen, Finn K.; Jakobsen, Mogens

1999-01-01

The present work describes the use of randomly amplified polymorphic DNA (RAPD) for the characterization of 172 dominant Lactobacillus isolates from present and previous studies of Ghanaian maize fermentation. Heterofermentative lactobacilli dominate the fermentation flora, since approximately 85% of the isolates belong to this group. Cluster analysis of the RAPD profiles obtained showed the presence of two main clusters. Cluster 1 included Lactobacillus fermentum, whereas cluster 2 comprised the remaining Lactobacillus spp. The two distinct clusters emerged at the similarity level of <50%. All isolates in cluster 1 showed similarity in their RAPD profile to the reference strains of L. fermentum included in the study. These isolates, yielding two distinct bands of approximately 695 and 773 bp with the primers used, were divided into four subclusters, indicating that several strains are involved in the fermentation and remain dominant throughout the process. The two distinct RAPD fragments were cloned, sequenced, and used as probes in Southern hybridization experiments. With one exception, Lactobacillus reuteri LMG 13045, the probes hybridized only to fragments of different sizes in EcoRI-digested chromosomal DNA of L. fermentum strains, thus indicating the specificity of the probes and variation within the L. fermentum isolates. PMID:10388723

Genetic relationships among strains of Xanthomonas fragariae based on random amplified polymorphic DNA PCR, repetitive extragenic palindromic PCR, and enterobacterial repetitive intergenic consensus PCR data and generation of multiplexed PCR primers useful for the identification of this phytopathogen.

PubMed Central

Pooler, M R; Ritchie, D F; Hartung, J S

1996-01-01

Genetic relationships among 25 isolates of Xanthomonas fragariae from diverse geographic regions were determined by three PCR methods that rely on different amplification priming strategies: random amplified polymorphic DNA (RAPD) PCR, repetitive extragenic palindromic (REP) PCR, and enterobacterial repetitive intergenic consensus (ERIC) PCR. The results of these assays are mutually consistent and indicate that pathogenic strains are very closely related to each other. RAPD, ERIC, and REP PCR assays identified nine, four, and two genotypes, respectively, within X. fragariae isolates. A single nonpathogenic isolate of X. fragariae was not distinguishable by these methods. The results of the PCR assays were also fully confirmed by physiological tests. There was no correlation between DNA amplification product patterns and geographic sites of isolation, suggesting that this bacterium has spread largely through exchange of infected plant germ plasm. Sequences identified through the RAPD assays were used to develop three primer pairs for standard PCR assays to identify X. fragariae. In addition, we developed a stringent multiplexed PCR assay to identify X. fragariae by simultaneously using the three independently derived sets of primers specific for pathogenic strains of the bacteria. PMID:8795198

Rheumatoid arthritis: identifying and characterising polymorphisms using rat models

PubMed Central

2016-01-01

ABSTRACT Rheumatoid arthritis is a chronic inflammatory joint disorder characterised by erosive inflammation of the articular cartilage and by destruction of the synovial joints. It is regulated by both genetic and environmental factors, and, currently, there is no preventative treatment or cure for this disease. Genome-wide association studies have identified ∼100 new loci associated with rheumatoid arthritis, in addition to the already known locus within the major histocompatibility complex II region. However, together, these loci account for only a modest fraction of the genetic variance associated with this disease and very little is known about the pathogenic roles of most of the risk loci identified. Here, we discuss how rat models of rheumatoid arthritis are being used to detect quantitative trait loci that regulate different arthritic traits by genetic linkage analysis and to positionally clone the underlying causative genes using congenic strains. By isolating specific loci on a fixed genetic background, congenic strains overcome the challenges of genetic heterogeneity and environmental interactions associated with human studies. Most importantly, congenic strains allow functional experimental studies be performed to investigate the pathological consequences of natural genetic polymorphisms, as illustrated by the discovery of several major disease genes that contribute to arthritis in rats. We discuss how these advances have provided new biological insights into arthritis in humans. PMID:27736747

Phylogeny and strain typing of Escherichia coli, inferred from variation at mononucleotide repeat loci.

PubMed

Diamant, Eran; Palti, Yniv; Gur-Arie, Riva; Cohen, Helit; Hallerman, Eric M; Kashi, Yechezkel

2004-04-01

Multilocus sequencing of housekeeping genes has been used previously for bacterial strain typing and for inferring evolutionary relationships among strains of Escherichia coli. In this study, we used shorter intergenic sequences that contained simple sequence repeats (SSRs) of repeating mononucleotide motifs (mononucleotide repeats [MNRs]) to infer the phylogeny of pathogenic and commensal E. coli strains. Seven noncoding loci (four MNRs and three non-SSRs) were sequenced in 27 strains, including enterohemorrhagic (six isolates of O157:H7), enteropathogenic, enterotoxigenic, B, and K-12 strains. The four MNRs were also sequenced in 20 representative strains of the E. coli reference (ECOR) collection. Sequence polymorphism was significantly higher at the MNR loci, including the flanking sequences, indicating a higher mutation rate in the sequences flanking the MNR tracts. The four MNR loci were amplifiable by PCR in the standard ECOR A, B1, and D groups, but only one (yaiN) in the B2 group was amplified, which is consistent with previous studies that suggested that B2 is the most ancient group. High sequence compatibility was found between the four MNR loci, indicating that they are in the same clonal frame. The phylogenetic trees that were constructed from the sequence data were in good agreement with those of previous studies that used multilocus enzyme electrophoresis. The results demonstrate that MNR loci are useful for inferring phylogenetic relationships and provide much higher sequence variation than housekeeping genes. Therefore, the use of MNR loci for multilocus sequence typing should prove efficient for clinical diagnostics, epidemiology, and evolutionary study of bacteria.

Phylogeny and Strain Typing of Escherichia coli, Inferred from Variation at Mononucleotide Repeat Loci

PubMed Central

Diamant, Eran; Palti, Yniv; Gur-Arie, Riva; Cohen, Helit; Hallerman, Eric M.; Kashi, Yechezkel

2004-01-01

Multilocus sequencing of housekeeping genes has been used previously for bacterial strain typing and for inferring evolutionary relationships among strains of Escherichia coli. In this study, we used shorter intergenic sequences that contained simple sequence repeats (SSRs) of repeating mononucleotide motifs (mononucleotide repeats [MNRs]) to infer the phylogeny of pathogenic and commensal E. coli strains. Seven noncoding loci (four MNRs and three non-SSRs) were sequenced in 27 strains, including enterohemorrhagic (six isolates of O157:H7), enteropathogenic, enterotoxigenic, B, and K-12 strains. The four MNRs were also sequenced in 20 representative strains of the E. coli reference (ECOR) collection. Sequence polymorphism was significantly higher at the MNR loci, including the flanking sequences, indicating a higher mutation rate in the sequences flanking the MNR tracts. The four MNR loci were amplifiable by PCR in the standard ECOR A, B1, and D groups, but only one (yaiN) in the B2 group was amplified, which is consistent with previous studies that suggested that B2 is the most ancient group. High sequence compatibility was found between the four MNR loci, indicating that they are in the same clonal frame. The phylogenetic trees that were constructed from the sequence data were in good agreement with those of previous studies that used multilocus enzyme electrophoresis. The results demonstrate that MNR loci are useful for inferring phylogenetic relationships and provide much higher sequence variation than housekeeping genes. Therefore, the use of MNR loci for multilocus sequence typing should prove efficient for clinical diagnostics, epidemiology, and evolutionary study of bacteria. PMID:15066845

Molecular insight into the association between cartilage regeneration and ear wound healing in genetic mouse models: targeting new genes in regeneration.

PubMed

Rai, Muhammad Farooq; Schmidt, Eric J; McAlinden, Audrey; Cheverud, James M; Sandell, Linda J

2013-11-06

Tissue regeneration is a complex trait with few genetic models available. Mouse strains LG/J and MRL are exceptional healers. Using recombinant inbred strains from a large (LG/J, healer) and small (SM/J, nonhealer) intercross, we have previously shown a positive genetic correlation between ear wound healing, knee cartilage regeneration, and protection from osteoarthritis. We hypothesize that a common set of genes operates in tissue healing and articular cartilage regeneration. Taking advantage of archived histological sections from recombinant inbred strains, we analyzed expression of candidate genes through branched-chain DNA technology directly from tissue lysates. We determined broad-sense heritability of candidates, Pearson correlation of candidates with healing phenotypes, and Ward minimum variance cluster analysis for strains. A bioinformatic assessment of allelic polymorphisms within and near candidate genes was also performed. The expression of several candidates was significantly heritable among strains. Although several genes correlated with both ear wound healing and cartilage healing at a marginal level, the expression of four genes representing DNA repair (Xrcc2, Pcna) and Wnt signaling (Axin2, Wnt16) pathways was significantly positively correlated with both phenotypes. Cluster analysis accurately classified healers and nonhealers for seven out of eight strains based on gene expression. Specific sequence differences between LG/J and SM/J were identified as potential causal polymorphisms. Our study suggests a common genetic basis between tissue healing and osteoarthritis susceptibility. Mapping genetic variations causing differences in diverse healing responses in multiple tissues may reveal generic healing processes in pursuit of new therapeutic targets designed to induce or enhance regeneration and, potentially, protection from osteoarthritis.

The Mouse Genomes Project: a repository of inbred laboratory mouse strain genomes.

PubMed

Adams, David J; Doran, Anthony G; Lilue, Jingtao; Keane, Thomas M

2015-10-01

The Mouse Genomes Project was initiated in 2009 with the goal of using next-generation sequencing technologies to catalogue molecular variation in the common laboratory mouse strains, and a selected set of wild-derived inbred strains. The initial sequencing and survey of sequence variation in 17 inbred strains was completed in 2011 and included comprehensive catalogue of single nucleotide polymorphisms, short insertion/deletions, larger structural variants including their fine scale architecture and landscape of transposable element variation, and genomic sites subject to post-transcriptional alteration of RNA. From this beginning, the resource has expanded significantly to include 36 fully sequenced inbred laboratory mouse strains, a refined and updated data processing pipeline, and new variation querying and data visualisation tools which are available on the project's website ( http://www.sanger.ac.uk/resources/mouse/genomes/ ). The focus of the project is now the completion of de novo assembled chromosome sequences and strain-specific gene structures for the core strains. We discuss how the assembled chromosomes will power comparative analysis, data access tools and future directions of mouse genetics.

Indication for Co-evolution of Lactobacillus johnsonii with its hosts

PubMed Central

2012-01-01

Background The intestinal microbiota, composed of complex bacterial populations, is host-specific and affected by environmental factors as well as host genetics. One important bacterial group is the lactic acid bacteria (LAB), which include many health-promoting strains. Here, we studied the genetic variation within a potentially probiotic LAB species, Lactobacillus johnsonii, isolated from various hosts. Results A wide survey of 104 fecal samples was carried out for the isolation of L. johnsonii. As part of the isolation procedure, terminal restriction fragment length polymorphism (tRFLP) was performed to identify L. johnsonii within a selected narrow spectrum of fecal LAB. The tRFLP results showed host specificity of two bacterial species, the Enterococcus faecium species cluster and Lactobacillus intestinalis, to different host taxonomic groups while the appearance of L. johnsonii and E. faecalis was not correlated with any taxonomic group. The survey ultimately resulted in the isolation of L. johnsonii from few host species. The genetic variation among the 47 L. johnsonii strains isolated from the various hosts was analyzed based on variation at simple sequence repeats (SSR) loci and multi-locus sequence typing (MLST) of conserved hypothetical genes. The genetic relationships among the strains inferred by each of the methods were similar, revealing three different clusters of L. johnsonii strains, each cluster consisting of strains from a different host, i.e. chickens, humans or mice. Conclusions Our typing results support phylogenetic separation of L. johnsonii strains isolated from different animal hosts, suggesting specificity of L. johnsonii strains to their hosts. Taken together with the tRFLP results, that indicated the association of specific LAB species with the host taxonomy, our study supports co-evolution of the host and its intestinal lactic acid bacteria. PMID:22827843

Indication for Co-evolution of Lactobacillus johnsonii with its hosts.

PubMed

Buhnik-Rosenblau, Keren; Matsko-Efimov, Vera; Jung, Minju; Shin, Heuynkil; Danin-Poleg, Yael; Kashi, Yechezkel

2012-07-25

The intestinal microbiota, composed of complex bacterial populations, is host-specific and affected by environmental factors as well as host genetics. One important bacterial group is the lactic acid bacteria (LAB), which include many health-promoting strains. Here, we studied the genetic variation within a potentially probiotic LAB species, Lactobacillus johnsonii, isolated from various hosts. A wide survey of 104 fecal samples was carried out for the isolation of L. johnsonii. As part of the isolation procedure, terminal restriction fragment length polymorphism (tRFLP) was performed to identify L. johnsonii within a selected narrow spectrum of fecal LAB. The tRFLP results showed host specificity of two bacterial species, the Enterococcus faecium species cluster and Lactobacillus intestinalis, to different host taxonomic groups while the appearance of L. johnsonii and E. faecalis was not correlated with any taxonomic group. The survey ultimately resulted in the isolation of L. johnsonii from few host species. The genetic variation among the 47 L. johnsonii strains isolated from the various hosts was analyzed based on variation at simple sequence repeats (SSR) loci and multi-locus sequence typing (MLST) of conserved hypothetical genes. The genetic relationships among the strains inferred by each of the methods were similar, revealing three different clusters of L. johnsonii strains, each cluster consisting of strains from a different host, i.e. chickens, humans or mice. Our typing results support phylogenetic separation of L. johnsonii strains isolated from different animal hosts, suggesting specificity of L. johnsonii strains to their hosts. Taken together with the tRFLP results, that indicated the association of specific LAB species with the host taxonomy, our study supports co-evolution of the host and its intestinal lactic acid bacteria.

Identification of salivary Lactobacillus rhamnosus species by DNA profiling and a specific probe.

PubMed

Richard, B; Groisillier, A; Badet, C; Dorignac, G; Lonvaud-Funel, A

2001-03-01

The Lactobacillus genus has been shown to be associated with the dental carious process, but little is known about the species related to the decay, although Lactobacillus rhamnosus is suspected to be the most implicated species. Conventional identification methods based on biochemical criteria lead to ambiguous results, since the Lactobacillus species found in saliva are phenotypically close. To clarify the role of this genus in the evolution of carious disease, this work aimed to find a rapid and reliable method for identifying the L. rhamnosus species. Methods based on hybridization with DNA probes and DNA amplification by PCR were used. The dominant salivary Lactobacillus species (reference strains from the ATCC) were selected for this purpose as well as some wild strains isolated from children's saliva. DNA profiling using semirandom polymorphic DNA amplification (semi-RAPD) generated specific patterns for L. rhamnosus ATCC 7469. The profiles of all L. rhamnosus strains tested were similar and could be grouped; these strains shared four common fragments. Wild strains first identified with classic methods shared common patterns with the L. rhamnosus species and could be reclassified. One fragment of the profile was purified, cloned, used as a probe and found to be specific to the L. rhamnosus species. These results may help to localize this species within its ecological niche and to elucidate the progression of the carious process.

SNP-associations and phenotype predictions from hundreds of microbial genomes without genome alignments.

PubMed

Hall, Barry G

2014-01-01

SNP-association studies are a starting point for identifying genes that may be responsible for specific phenotypes, such as disease traits. The vast bulk of tools for SNP-association studies are directed toward SNPs in the human genome, and I am unaware of any tools designed specifically for such studies in bacterial or viral genomes. The PPFS (Predict Phenotypes From SNPs) package described here is an add-on to kSNP , a program that can identify SNPs in a data set of hundreds of microbial genomes. PPFS identifies those SNPs that are non-randomly associated with a phenotype based on the χ² probability, then uses those diagnostic SNPs for two distinct, but related, purposes: (1) to predict the phenotypes of strains whose phenotypes are unknown, and (2) to identify those diagnostic SNPs that are most likely to be causally related to the phenotype. In the example illustrated here, from a set of 68 E. coli genomes, for 67 of which the pathogenicity phenotype was known, there were 418,500 SNPs. Using the phenotypes of 36 of those strains, PPFS identified 207 diagnostic SNPs. The diagnostic SNPs predicted the phenotypes of all of the genomes with 97% accuracy. It then identified 97 SNPs whose probability of being causally related to the pathogenic phenotype was >0.999. In a second example, from a set of 116 E. coli genome sequences, using the phenotypes of 65 strains PPFS identified 101 SNPs that predicted the source host (human or non-human) with 90% accuracy.

Hospital strain colonization by Staphylococcus epidermidis.

PubMed

Blum-Menezes, D; Bratfich, O J; Padoveze, M C; Moretti, M L

2009-03-01

The skin and mucous membranes of healthy subjects are colonized by strains of Staphylococcus epidermidis showing a high diversity of genomic DNA polymorphisms. Prolonged hospitalization and the use of invasive procedures promote changes in the microbiota with subsequent colonization by hospital strains. We report here a patient with prolonged hospitalization due to chronic pancreatitis who was treated with multiple antibiotics, invasive procedures and abdominal surgery. We studied the dynamics of skin colonization by S. epidermidis leading to the development of catheter-related infections and compared the genotypic profile of clinical and microbiota strains by pulsed field gel electrophoresis. During hospitalization, the normal S. epidermidis skin microbiota exhibiting a polymorphic genomic DNA profile was replaced with a hospital-acquired biofilm-producer S. epidermidis strain that subsequently caused repetitive catheter-related infections.

Strain screen and haplotype association mapping of wheel running in inbred mouse strains.

PubMed

Lightfoot, J Timothy; Leamy, Larry; Pomp, Daniel; Turner, Michael J; Fodor, Anthony A; Knab, Amy; Bowen, Robert S; Ferguson, David; Moore-Harrison, Trudy; Hamilton, Alicia

2010-09-01

Previous genetic association studies of physical activity, in both animal and human models, have been limited in number of subjects and genetically homozygous strains used as well as number of genomic markers available for analysis. Expansion of the available mouse physical activity strain screens and the recently published dense single-nucleotide polymorphism (SNP) map of the mouse genome (approximately 8.3 million SNPs) and associated statistical methods allowed us to construct a more generalizable map of the quantitative trait loci (QTL) associated with physical activity. Specifically, we measured wheel running activity in male and female mice (average age 9 wk) in 41 inbred strains and used activity data from 38 of these strains in a haplotype association mapping analysis to determine QTL associated with activity. As seen previously, there was a large range of activity patterns among the strains, with the highest and lowest strains differing significantly in daily distance run (27.4-fold), duration of activity (23.6-fold), and speed (2.9-fold). On a daily basis, female mice ran further (24%), longer (13%), and faster (11%). Twelve QTL were identified, with three (on Chr. 12, 18, and 19) in both male and female mice, five specific to males, and four specific to females. Eight of the 12 QTL, including the 3 general QTL found for both sexes, fell into intergenic areas. The results of this study further support the findings of a moderate to high heritability of physical activity and add general genomic areas applicable to a large number of mouse strains that can be further mined for candidate genes associated with regulation of physical activity. Additionally, results suggest that potential genetic mechanisms arising from traditional noncoding regions of the genome may be involved in regulation of physical activity.

Applied Genomics: Data Mining Reveals Species-Specific Malaria Diagnostic Targets More Sensitive than 18S rRNA▿†‡

PubMed Central

Demas, Allison; Oberstaller, Jenna; DeBarry, Jeremy; Lucchi, Naomi W.; Srinivasamoorthy, Ganesh; Sumari, Deborah; Kabanywanyi, Abdunoor M.; Villegas, Leopoldo; Escalante, Ananias A.; Kachur, S. Patrick; Barnwell, John W.; Peterson, David S.; Udhayakumar, Venkatachalam; Kissinger, Jessica C.

2011-01-01

Accurate and rapid diagnosis of malaria infections is crucial for implementing species-appropriate treatment and saving lives. Molecular diagnostic tools are the most accurate and sensitive method of detecting Plasmodium, differentiating between Plasmodium species, and detecting subclinical infections. Despite available whole-genome sequence data for Plasmodium falciparum and P. vivax, the majority of PCR-based methods still rely on the 18S rRNA gene targets. Historically, this gene has served as the best target for diagnostic assays. However, it is limited in its ability to detect mixed infections in multiplex assay platforms without the use of nested PCR. New diagnostic targets are needed. Ideal targets will be species specific, highly sensitive, and amenable to both single-step and multiplex PCRs. We have mined the genomes of P. falciparum and P. vivax to identify species-specific, repetitive sequences that serve as new PCR targets for the detection of malaria. We show that these targets (Pvr47 and Pfr364) exist in 14 to 41 copies and are more sensitive than 18S rRNA when utilized in a single-step PCR. Parasites are routinely detected at levels of 1 to 10 parasites/μl. The reaction can be multiplexed to detect both species in a single reaction. We have examined 7 P. falciparum strains and 91 P. falciparum clinical isolates from Tanzania and 10 P. vivax strains and 96 P. vivax clinical isolates from Venezuela, and we have verified a sensitivity and specificity of ∼100% for both targets compared with a nested 18S rRNA approach. We show that bioinformatics approaches can be successfully applied to identify novel diagnostic targets and improve molecular methods for pathogen detection. These novel targets provide a powerful alternative molecular diagnostic method for the detection of P. falciparum and P. vivax in conventional or multiplex PCR platforms. PMID:21525225

Leishmania infantum: Lipophosphoglycan intraspecific variation and interaction with vertebrate and invertebrate hosts.

PubMed

Coelho-Finamore, J M; Freitas, V C; Assis, R R; Melo, M N; Novozhilova, N; Secundino, N F; Pimenta, P F; Turco, S J; Soares, R P

2011-03-01

Interspecies variations in lipophosphoglycan (LPG) have been the focus of intense study over the years due its role in specificity during sand fly-Leishmania interaction. This cell surface glycoconjugate is highly polymorphic among species with variations in sugars that branch off the conserved Gal(β1,4)Man(α1)-PO(4) backbone of repeat units. However, the degree of intraspecies polymorphism in LPG of Leishmania infantum (syn. Leishmania chagasi) is not known. In this study, intraspecific variation in the repeat units of LPG was evaluated in 16 strains of L. infantum from Brazil, France, Algeria and Tunisia. The structural polymorphism in the L. infantum LPG repeat units was relatively slight and consisted of three types: type I does not have side chains; type II has one β-glucose residue that branches off the disaccharide-phosphate repeat units and type III has up to three glucose residues (oligo-glucosylated). The significance of these modifications was investigated during in vivo interaction of L. infantum with Lutzomyia longipalpis, and in vitro interaction of the parasites and respective LPGs with murine macrophages. There were no consequential differences in the parasite densities in sand fly midguts infected with Leishmania strains exhibiting type I, II and III LPGs. However, higher nitric oxide production was observed in macrophages exposed to glucosylated type II LPG. Copyright © 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Molecular diagnostic testing for primary biliary cholangitis.

PubMed

Gatselis, Nikolaos K; Dalekos, George N

2016-09-01

A reliable liver autoimmune serology for the diagnosis of primary biliary cholangitis (PBC) is of particular importance. Recognition of patients at early stages and prompt treatment initiation may alter the outcome, slow progression, delays liver failure, and improves survival. In this review, we summarize and discuss the published data obtained from literature searches from PubMed and The National Library of Medicine (USA) and our own experience on the current and potential molecular based approaches to the diagnosis of PBC. Expert commentary: Standardization of liver diagnostic serology and clinical governance are two major points as antimitochondrial antibodies are the diagnostic hallmark of the disease and PBC-specific antinuclear antibodies could assist in the diagnosis and estimation of prognosis. New biomarkers such as novel autoantibodies, genetic polymorphisms, metabolomic profiling, micro-RNA and epigenetics may assist to the understanding, diagnosis and management of the disease.

Molecular Detection of Two Potential Probiotic Lactobacilli Strains and Evaluation of Their Performance as Starter Adjuncts in Yogurt Production.

PubMed

Saxami, Georgia; Papadopoulou, Olga S; Chorianopoulos, Nikos; Kourkoutas, Yiannis; Tassou, Chrysoula C; Galanis, Alex

2016-05-04

A molecular method for efficient and accurate detection and identification of two potential probiotic lactobacilli strains isolated from fermented olives, namely Lactobacillus pentosus B281 and Lb. plantarum B282, was developed in the present study. Random Amplified Polymorphic DNA (RAPD) analysis was performed, and strain specific primers were designed and applied in a multiplex polymerase chain reaction (PCR) assay. The specificity of the assay was tested and successfully confirmed in 27 and 22 lactobacilli strains for Lb. pentosus B281 and Lb. plantarum B282, respectively. Moreover, the two strains were used as starter cultures in yogurt production. Cell enumeration followed by multiplex PCR analysis demonstrated that the two strains were present in yogurt samples at levels ≥6 log CFU/g even after 35 days of storage at 4 °C. Microbiological analysis showed that lactobacilli and streptococci were present within usual levels, whereas enterobacteriaceae and yeast/mold counts were not detected as expected. Although the pH values of the novel products were slightly lower than the control ones, the yogurt containing the probiotic cultures scored similar values compared to the control in a series of sensory tests. Overall, these results demonstrated the possible use of the two strains as starter adjuncts in the production of yogurt with potential probiotic properties.

Molecular Detection of Two Potential Probiotic Lactobacilli Strains and Evaluation of Their Performance as Starter Adjuncts in Yogurt Production

PubMed Central

Saxami, Georgia; Papadopoulou, Olga S.; Chorianopoulos, Nikos; Kourkoutas, Yiannis; Tassou, Chrysoula C.; Galanis, Alex

2016-01-01

A molecular method for efficient and accurate detection and identification of two potential probiotic lactobacilli strains isolated from fermented olives, namely Lactobacillus pentosus B281 and Lb. plantarum B282, was developed in the present study. Random Amplified Polymorphic DNA (RAPD) analysis was performed, and strain specific primers were designed and applied in a multiplex polymerase chain reaction (PCR) assay. The specificity of the assay was tested and successfully confirmed in 27 and 22 lactobacilli strains for Lb. pentosus B281 and Lb. plantarum B282, respectively. Moreover, the two strains were used as starter cultures in yogurt production. Cell enumeration followed by multiplex PCR analysis demonstrated that the two strains were present in yogurt samples at levels ≥6 log CFU/g even after 35 days of storage at 4 °C. Microbiological analysis showed that lactobacilli and streptococci were present within usual levels, whereas enterobacteriaceae and yeast/mold counts were not detected as expected. Although the pH values of the novel products were slightly lower than the control ones, the yogurt containing the probiotic cultures scored similar values compared to the control in a series of sensory tests. Overall, these results demonstrated the possible use of the two strains as starter adjuncts in the production of yogurt with potential probiotic properties. PMID:27153065

Peptide-specific recognition of human cytomegalovirus strains controls adaptive natural killer cells.

PubMed

Hammer, Quirin; Rückert, Timo; Borst, Eva Maria; Dunst, Josefine; Haubner, André; Durek, Pawel; Heinrich, Frederik; Gasparoni, Gilles; Babic, Marina; Tomic, Adriana; Pietra, Gabriella; Nienen, Mikalai; Blau, Igor Wolfgang; Hofmann, Jörg; Na, Il-Kang; Prinz, Immo; Koenecke, Christian; Hemmati, Philipp; Babel, Nina; Arnold, Renate; Walter, Jörn; Thurley, Kevin; Mashreghi, Mir-Farzin; Messerle, Martin; Romagnani, Chiara

2018-05-01

Natural killer (NK) cells are innate lymphocytes that lack antigen-specific rearranged receptors, a hallmark of adaptive lymphocytes. In some people infected with human cytomegalovirus (HCMV), an NK cell subset expressing the activating receptor NKG2C undergoes clonal-like expansion that partially resembles anti-viral adaptive responses. However, the viral ligand that drives the activation and differentiation of adaptive NKG2C + NK cells has remained unclear. Here we found that adaptive NKG2C + NK cells differentially recognized distinct HCMV strains encoding variable UL40 peptides that, in combination with pro-inflammatory signals, controlled the population expansion and differentiation of adaptive NKG2C + NK cells. Thus, we propose that polymorphic HCMV peptides contribute to shaping of the heterogeneity of adaptive NKG2C + NK cell populations among HCMV-seropositive people.

ABCG-like transporter of Trypanosoma cruzi involved in benznidazole resistance: gene polymorphisms disclose inter-strain intragenic recombination in hybrid isolates.

PubMed

Franco, Jaques; Ferreira, Renata C; Ienne, Susan; Zingales, Bianca

2015-04-01

Benznidazole (BZ) is one of the two drugs for Chagas disease treatment. In a previous study we showed that the Trypanosoma cruzi ABCG-like transporter gene, named TcABCG1, is over-expressed in parasite strains naturally resistant to BZ and that the gene of TcI BZ-resistant strains exhibited several single nucleotide polymorphisms (SNPs) as compared to the gene of CL Brener BZ-susceptible strain. Here we report the sequence of TcABCG1 gene of fourteen T. cruzi strains, with diverse degrees of BZ sensitivity and belonging to different discrete typing units (DTUs) and Tcbat group. Although DTU-specific SNPs and amino acid changes were identified, no direct correlation with BZ-resistance phenotype was found. Thus, it is plausible that the transporter abundance is a determinant factor for drug resistance, as pointed out above. Sequence data were used for Bayesian phylogenies and network genealogy analysis. The network showed a high degree of reticulation suggesting genetic exchange between the parasites. TcI and TcII clades were clearly separated. Tcbat sequences were close to TcI. A fourth clade clustered TcABCG1 haplotypes of TcV, TcVI and TcIII strains, with closer proximity to TcI. Analysis of the recombination patterns indicated that hybrid strains contain haplotypes that are mosaics most likely derived by intragenic recombination of parental sequences. The data confirm that TcII and TcIII as the parentals of TcV and TcVI DTUs. Since genetic fingerprint of TcI was found in TcIII, we sustain the previously proposed "Two Hybridization model" for the origin of hybrid strains. Among the twenty best BLASTP hits in databases, orthologues of TcABCG1 transporter were found in Leishmania spp. and African trypanosomes, though their function remains undescribed. Copyright © 2015 Elsevier B.V. All rights reserved.

Understanding Strain-Induced Phase Transformations in BiFeO3 Thin Films.

PubMed

Dixit, Hemant; Beekman, Christianne; Schlepütz, Christian M; Siemons, Wolter; Yang, Yongsoo; Senabulya, Nancy; Clarke, Roy; Chi, Miaofang; Christen, Hans M; Cooper, Valentino R

2015-08-01

Experiments demonstrate that under large epitaxial strain a coexisting striped phase emerges in BiFeO 3 thin films, which comprises a tetragonal-like ( T ') and an intermediate S ' polymorph. It exhibits a relatively large piezoelectric response when switching between the coexisting phase and a uniform T ' phase. This strain-induced phase transformation is investigated through a synergistic combination of first-principles theory and experiments. The results show that the S ' phase is energetically very close to the T ' phase, but is structurally similar to the bulk rhombohedral ( R ) phase. By fully characterizing the intermediate S ' polymorph, it is demonstrated that the flat energy landscape resulting in the absence of an energy barrier between the T ' and S ' phases fosters the above-mentioned reversible phase transformation. This ability to readily transform between the S ' and T ' polymorphs, which have very different octahedral rotation patterns and c / a ratios, is crucial to the enhanced piezoelectricity in strained BiFeO 3 films. Additionally, a blueshift in the band gap when moving from R to S ' to T ' is observed. These results emphasize the importance of strain engineering for tuning electromechanical responses or, creating unique energy harvesting photonic structures, in oxide thin film architectures.

Mouse strain differences in Gurmarin-sensitivity of sweet taste responses are not associated with polymorphisms of the sweet receptor gene, Tas1r3.

PubMed

Sanematsu, Keisuke; Yasumatsu, Keiko; Yoshida, Ryusuke; Shigemura, Noriatsu; Ninomiya, Yuzo

2005-07-01

Gurmarin (Gur) is a peptide that selectively inhibits responses of the chorda tympani (CT) nerve to sweet compounds in rodents. In mice, the sweet-suppressing effect of Gur differs among strains. The inhibitory effect of Gur is clearly observed in C57BL/6 mice, but only slightly, if at all, in BALB/c mice. These two mouse strains possess different alleles of the sweet receptor gene, Sac (Tas1r3) (taster genotype for C57BL/6 and non-taster genotype for BALB/c mice), suggesting that polymorphisms in the gene may account for differential sensitivity to Gur. To investigate this possibility, we examined the effect of Gur in another Tas1r3 non-taster strain, 129 X 1/Sv mice. The results indicated that unlike non-taster BALB/c mice but similar to taster C57BL/6 mice, 129 X 1/Sv mice exhibited significant inhibition of CT responses to various sweet compounds by Gur. This suggests that the mouse strain difference in the Gur inhibition of sweet responses of the CT nerve may not be associated with polymorphisms of Tas1r3.

AFLP fingerprinting: an efficient technique for detecting genetic variation of Xanthomonas axonopodis pv. manihotis.

PubMed

Restrepo, S; Duque, M; Tohme, J; Verdier, V

1999-01-01

Xanthomonas axonopodis pv. manihotis (Xam) is the causative agent of cassava bacterial blight (CBB), a worldwide disease that is particularly destructive in South America and Africa. CBB is controlled essentially through the use of resistant varieties. To develop an appropriate disease management strategy, the genetic diversity of the pathogen's populations must be assessed. Until now, the genetic diversity of Xam was characterized by RFLP analyses using ribotyping, and plasmid and genomic Xam probes. We used AFLP (amplified fragment length polymorphism), a novel PCR-based technique, to characterize the genetic diversity of Colombian Xam isolates. Six Xam strains were tested with 65 AFLP primer combinations to identify the best selective primers. Eight primer combinations were selected according to their reproducibility, number of polymorphic bands and polymorphism detected between Xam strains. Forty-seven Xam strains, originating from different Colombian ecozones, were analysed with the selected combinations. Results obtained with AFLP are consistent with those obtained with RFLP, using plasmid DNA as a probe. Some primer combinations differentiated Xam strains that were not distinguished by RFLP analyses, thus AFLP fingerprinting allowed a better definition of the genetic relationships between Xam strains.

Gut metagenomes of type 2 diabetic patients have characteristic single-nucleotide polymorphism distribution in Bacteroides coprocola.

PubMed

Chen, Yaowen; Li, Zongcheng; Hu, Shuofeng; Zhang, Jian; Wu, Jiaqi; Shao, Ningsheng; Bo, Xiaochen; Ni, Ming; Ying, Xiaomin

2017-02-01

Gut microbes play a critical role in human health and disease, and researchers have begun to characterize their genomes, the so-called gut metagenome. Thus far, metagenomics studies have focused on genus- or species-level composition and microbial gene sets, while strain-level composition and single-nucleotide polymorphism (SNP) have been overlooked. The gut metagenomes of type 2 diabetes (T2D) patients have been found to be enriched with butyrate-producing bacteria and sulfate reduction functions. However, it is not known whether the gut metagenomes of T2D patients have characteristic strain patterns or SNP distributions. We downloaded public gut metagenome datasets from 170 T2D patients and 174 healthy controls and performed a systematic comparative analysis of their metagenome SNPs. We found that Bacteroides coprocola, whose relative abundance did not differ between the groups, had a characteristic distribution of SNPs in the T2D patient group. We identified 65 genes, all in B. coprocola, that had remarkably different enrichment of SNPs. The first and sixth ranked genes encode glycosyl hydrolases (GenBank accession EDU99824.1 and EDV02301.1). Interestingly, alpha-glucosidase, which is also a glycosyl hydrolase located in the intestine, is an important drug target of T2D. These results suggest that different strains of B. coprocola may have different roles in human gut and a specific set of B. coprocola strains are correlated with T2D.

Intergenic Variable-Number Tandem-Repeat Polymorphism Upstream of rocA Alters Toxin Production and Enhances Virulence in Streptococcus pyogenes.

PubMed

Zhu, Luchang; Olsen, Randall J; Horstmann, Nicola; Shelburne, Samuel A; Fan, Jia; Hu, Ye; Musser, James M

2016-07-01

Variable-number tandem-repeat (VNTR) polymorphisms are ubiquitous in bacteria. However, only a small fraction of them has been functionally studied. Here, we report an intergenic VNTR polymorphism that confers an altered level of toxin production and increased virulence in Streptococcus pyogenes The nature of the polymorphism is a one-unit deletion in a three-tandem-repeat locus upstream of the rocA gene encoding a sensor kinase. S. pyogenes strains with this type of polymorphism cause human infection and produce significantly larger amounts of the secreted cytotoxins S. pyogenes NADase (SPN) and streptolysin O (SLO). Using isogenic mutant strains, we demonstrate that deleting one or more units of the tandem repeats abolished RocA production, reduced CovR phosphorylation, derepressed multiple CovR-regulated virulence factors (such as SPN and SLO), and increased virulence in a mouse model of necrotizing fasciitis. The phenotypic effect of the VNTR polymorphism was nearly the same as that of inactivating the rocA gene. In summary, we identified and characterized an intergenic VNTR polymorphism in S. pyogenes that affects toxin production and virulence. These new findings enhance understanding of rocA biology and the function of VNTR polymorphisms in S. pyogenes. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Genetic discovery in Xylella fastidiosa through sequence analysis of selected randomly amplified polymorphic DNAs.

PubMed

Chen, Jianchi; Civerolo, Edwin L; Jarret, Robert L; Van Sluys, Marie-Anne; de Oliveira, Mariana C

2005-02-01

Xylella fastidiosa causes many important plant diseases including Pierce's disease (PD) in grape and almond leaf scorch disease (ALSD). DNA-based methodologies, such as randomly amplified polymorphic DNA (RAPD) analysis, have been playing key roles in genetic information collection of the bacterium. This study further analyzed the nucleotide sequences of selected RAPDs from X. fastidiosa strains in conjunction with the available genome sequence databases and unveiled several previously unknown novel genetic traits. These include a sequence highly similar to those in the phage family of Podoviridae. Genome comparisons among X. fastidiosa strains suggested that the "phage" is currently active. Two other RAPDs were also related to horizontal gene transfer: one was part of a broadly distributed cryptic plasmid and the other was associated with conjugal transfer. One RAPD inferred a genomic rearrangement event among X. fastidiosa PD strains and another identified a single nucleotide polymorphism of evolutionary value.

Geographical distribution of genetic polymorphism of the pathogen Histoplasma capsulatum isolated from infected bats, captured in a central zone of Mexico.

PubMed

Taylor, Maria Lucia; Chávez-Tapia, Catalina B; Rojas-Martínez, Alberto; del Rocio Reyes-Montes, Maria; del Valle, Mirian Bobadilla; Zúñiga, Gerardo

2005-09-01

Fourteen Histoplasma capsulatum isolates recovered from infected bats captured in Mexican caves and two human H. capsulatum reference strains were analyzed using random amplification of polymorphic DNA PCR-based and partial DNA sequences of four genes. Cluster analysis of random amplification of polymorphic DNA-patterns revealed differences for two H. capsulatum isolates of one migratory bat Tadarida brasiliensis. Three groups were identified by distance and maximum-parsimony analyses of arf, H-anti, ole, and tub1 H. capsulatum genes. Group I included most isolates from infected bats and one clinical strain from central Mexico; group II included the two isolates from T. brasiliensis; the human G-217B reference strain from USA formed an independent group III. Isolates from group II showed diversity in relation to groups I and III, suggesting a different H. capsulatum population.

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

ITS1/5.8S/ITS2, a Good Marker for Initial Classification of Shiitake Culinary-Medicinal Lentinus edodes (Agaricomycetes) Strains in China.

PubMed

Song, Xiao-Xia; Zhao, Yan; Song, Chun-Yan; Li, Chuan-Hua; Song, Ying; Chen, Ming-Jie; Bao, Da-Peng; Tan, Qi

2018-01-01

China is home to rich wild and cultivated strains of Lentinus edodes, an important edible and medicinal mushroom. Artificial selection of L. edodes has a long history, and the widely cultivated strains belong to populations different from those of most wild strains. Internal transcribed spacer (ITS) regions have been used as good markers to identify L. edodes populations. But because ITS regions exhibit incomplete concerted evolution, the use of an ITS to identify L. edodes populations has been questioned. The objective of this study was to determine whether the ITS region is suitable for identifying L. edodes populations and which populations the widely cultivated strains and the most wild strains belong to by investigating intraindividual and differential ITS polymorphisms between 44 cultivars and 44 wild strains of L. edodes in China. Intraindividual ITS polymorphism is common in L. edodes strains, and most strains possessed 2 different ITS sequences, which came from their heterokaryons. The genetic polymorphisms of ITS1, 5.8S, and ITS2 in L. edodes strains are distinct. All strains were divided into one 5.8S type (5.8S-A), 2 ITS1 types (ITS1-A and ITS1-B), and 2 ITS2 types (ITS2-A and ITS2-B), which were subdivided into 2 branches (ITS2-A1 and ITS2-A2; ITS2-B1 and ITS2-B2). ITS1/5.8S/ITS2 could be used as a good marker in preliminary classification of L. edodes strains in China. It not only exhibited classified information of ITS1, 5.8S, and ITS2 for each strain at the same time, it also indicated whether the strain was heterozygous. The 44 cultivated strains were mainly the A/A/A1 type, and the 44 wild strains were mainly the A/A/A2 and other mixed types.

Streptomyces euryhalinus sp. nov., a new actinomycete isolated from a mangrove forest.

PubMed

Biswas, Kaushik; Choudhury, Jayanta D; Mahansaria, Riddhi; Saha, Malay; Mukherjee, Joydeep

2017-06-01

A Gram-positive, aerobic, non-motile actinomycete (strain MS 3/20 T ) was isolated from the sediment of the Sundarbans mangrove forest in India. On International Streptomyces Project (ISP) medium 2, the isolate produced yellowish brown to red aerial hyphae that carried spiny-surfaced spores in a retinaculum-apertum arrangement. Whole-cell hydrolysate of the strain contained LL-diaminopimelic acid and galactose. Predominant menaquinones were MK-9(H 8 ) and MK-9(H 6 ). Diagnostic polar lipids were glycolipid, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, unidentified phospholipid and unidentified amino lipid. The major fatty acids were anteiso-C 15:0 (17.53%), iso-C 16:0 (23.89%) and anteiso-C 17:0 (10.29%). The strain showed 100% 16S ribosomal RNA (rRNA) gene sequence similarity with Streptomyces variabilis NBRC 12825 T , Streptomyces erythrogriseus LMG 19406 T , Streptomyces griseoincarnatus LMG 19316 T and Streptomyces labedae NBRC 15864 T . However, strain MS 3/20 T could be distinguished from these and seven other closely related species based on low levels of DNA-DNA relatedness (27.2-53.8%), supported by the unique banding pattern obtained from random amplified polymorphic DNA-PCR amplification and the distinctive matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS) profile of whole-cell proteins acquired for strain MS 3/20 T in comparison with its phylogenetic relatives. Disparate morphological, physiological and chemotaxonomic features, principally growth in NaCl, further corroborated the distinction of strain MS 3/20 T from other phylogenetic relatives. Strain MS 3/20 T is therefore suggested to be a novel species of the genus Streptomyces, for which the name Streptomyces euryhalinus sp. nov. is proposed. The type strain is MS 3/20 T (=CICC 11032 T =DSM 103378 T ).

Isolation and characterization of ethanol tolerant yeast strains

PubMed Central

Tikka, Chiranjeevi; Osuru, Hari Prasad; Atluri, Navya; Raghavulu, Praveen Chakravarthi Veera; yellapu, Nanda Kumar; Mannur, Ismail Shaik; Prasad, Uppu Venkateswara; Aluru, Sudheer; K, Narasimha Varma; Bhaskar, Matcha

2013-01-01

Yeast strains are commonly associated with sugar rich environments. Various fruit samples were selected as source for isolating yeast cells. The isolated cultures were identified at Genus level by colony morphology, biochemical characteristics and cell morphological characters. An attempt has been made to check the viability of yeast cells under different concentrations of ethanol. Ethanol tolerance of each strain was studied by allowing the yeast to grow in liquid YEPD (Yeast Extract Peptone Dextrose) medium having different concentrations of ethanol. A total of fifteen yeast strains isolated from different samples were used for the study. Seven strains of Saccharomyces cerevisiae obtained from different fruit sources were screened for ethanol tolerance. The results obtained in this study show a range of tolerance levels between 7%-12% in all the stains. Further, the cluster analysis based on 22 RAPD (Random Amplified polymorphic DNA) bands revealed polymorphisms in these seven Saccharomyces strains. PMID:23750092

Genome-scale investigation of phenotypically distinct but nearly clonal Trichoderma strains

PubMed Central

Weld, Richard J.; Cox, Murray P.; Bradshaw, Rosie E.; McLean, Kirstin L.; Stewart, Alison; Steyaert, Johanna M.

2016-01-01

Biological control agents (BCA) are beneficial organisms that are applied to protect plants from pests. Many fungi of the genus Trichoderma are successful BCAs but the underlying mechanisms are not yet fully understood. Trichoderma cf. atroviride strain LU132 is a remarkably effective BCA compared to T. cf. atroviride strain LU140 but these strains were found to be highly similar at the DNA sequence level. This unusual combination of phenotypic variability and high DNA sequence similarity between separately isolated strains prompted us to undertake a genome comparison study in order to identify DNA polymorphisms. We further investigated if the polymorphisms had functional effects on the phenotypes. The two strains were clearly identified as individuals, exhibiting different growth rates, conidiation and metabolism. Superior pathogen control demonstrated by LU132 depended on its faster growth, which is a prerequisite for successful distribution and competition. Genome sequencing identified only one non-synonymous single nucleotide polymorphism (SNP) between the strains. Based on this SNP, we successfully designed and validated an RFLP protocol that can be used to differentiate LU132 from LU140 and other Trichoderma strains. This SNP changed the amino acid sequence of SERF, encoded by the previously undescribed single copy gene “small EDRK-rich factor” (serf). A deletion of serf in the two strains did not lead to identical phenotypes, suggesting that, in addition to the single functional SNP between the nearly clonal Trichoderma cf. atroviride strains, other non-genomic factors contribute to their phenotypic variation. This finding is significant as it shows that genomics is an extremely useful but not exhaustive tool for the study of biocontrol complexity and for strain typing. PMID:27190719

Effect of 5-HT2A Receptor Polymorphisms, Work Stressors, and Social Support on Job Strain among Petroleum Workers in Xinjiang, China

PubMed Central

Jiang, Yu; Tang, Jinhua; Li, Rong; Zhao, Junling; Song, Zhixin; Ge, Hua; Lian, Yulong; Liu, Jiwen

2016-01-01

Previous studies have shown that work stressors and social support influence job strain. However, few studies have examined the impact of individual differences on job strain. In Xinjiang, there are a large number of petroleum workers in arid deserts. The present study investigated the effects of work stressors, social support, and 5-hydroxytryptamine receptor (5-HTR2A) genotype on the etiology of job strain among petroleum workers in Xinjiang. A cross-sectional study was carried out between January and August 2013. A total of 700 workers were selected by a three-stage stratified sampling method. 5-HTR2A genotypes were determined with the SNaPshot single nucleotide polymorphism assay. Work stressors and job strain were evaluated with the Occupational Stress Inventory-Revised questionnaire. Social support was assessed with the Chinese Social Support Rating Scale. Work overload and responsibility were significantly associated with job strain. Low social support was associated with severe vocational and interpersonal strain. High social support was a protective factor against job strain (odds ratio (OR) = 0.32, 95% confidence interval (CI): 0.14–0.76). The CC genotype of rs6313 and the AA genotype of rs2070040 were linked to severe vocational strain. Ordinal logistic regression analysis revealed that the CC genotype of rs6313 was linked to higher risk of job strain than the TT genotype (OR = 1.88, 95% CI: 1.10–3.23). These data provide evidence that work stressors, low social support, and 5-HTR2A gene polymorphism contributes to the risk of job strain. PMID:27999378

Different Type 1 Fimbrial Genes and Tropisms of Commensal and Potentially Pathogenic Actinomyces spp. with Different Salivary Acidic Proline-Rich Protein and Statherin Ligand Specificities

PubMed Central

Li, Tong; Khah, Massoud Kheir; Slavnic, Snjezana; Johansson, Ingegerd; Strömberg, Nicklas

2001-01-01

Actinomyces spp. exhibit type 1 fimbria-mediated adhesion to salivary acidic proline-rich proteins (PRPs) and statherin ligands. Actinomyces spp. with different animal and tissue origins belong to three major adhesion types as relates to ligand specificity and type 1 fimbria genes. (i) In preferential acidic-PRP binding, strains of Actinomyces naeslundii genospecies 1 and 2 from human and monkey mouths displayed at least three ligand specificities characterized by preferential acidic-PRP binding. Slot blot DNA hybridization showed seven highly conserved type 1 fimbria genes (orf1- to -6 and fimP) in genospecies 1 and 2 strains, except that orf5 and orf3 were divergent in genospecies 1. (ii) In preferential statherin binding, oral Actinomyces viscosus strains of rat and hamster origin (and strain 19246 from a human case of actinomycosis) bound statherin preferentially. DNA hybridization and characterization of the type 1 fimbria genes from strain 19246 revealed a homologous gene cluster of four open reading frames (orfA to -C and fimP). Bioinformatics suggested sortase (orfB, orf4, and part of orf5), prepilin peptidase (orfC and orf6), fimbria subunit (fimP), and usher- and autotransporter-like (orfA and orf1 to -3) functions. Those gene regions corresponding to orf3 and orf5 were divergent, those corresponding to orf2, orf1, and fimP were moderately conserved, and those corresponding to orf4 and orf6 were highly conserved. Restriction fragment length polymorphism analyses using a fimP probe separated human and monkey and rat and hamster strains into phylogenetically different groups. (iii) In statherin-specific binding, strains of A. naeslundii genospecies 1 from septic and other human infections displayed a low-avidity binding to statherin. Only the orf4 and orf6 gene regions were highly conserved. Finally, rat saliva devoid of statherin bound bacterial strains avidly irrespective of ligand specificity, and specific antisera detected either type 1, type 2, or both types of fimbria on the investigated Actinomyces strains. PMID:11705891

The stage-specific in vitro efficacy of a malaria antigen cocktail provides valuable insights into the development of effective multi-stage vaccines.

PubMed

Spiegel, Holger; Boes, Alexander; Kastilan, Robin; Kapelski, Stephanie; Edgue, Güven; Beiss, Veronique; Chubodova, Ivana; Scheuermayer, Matthias; Pradel, Gabriele; Schillberg, Stefan; Reimann, Andreas; Fischer, Rainer

2015-10-01

Multicomponent vaccines targeting different stages of Plasmodium falciparum represent a promising, holistic concept towards better malaria vaccines. Additionally, an effective vaccine candidate should demonstrate cross-strain specificity because many antigens are polymorphic, which can reduce vaccine efficacy. A cocktail of recombinant fusion proteins (VAMAX-Mix) featuring three diversity-covering variants of the blood-stage antigen PfAMA1, each combined with the conserved sexual-stage antigen Pfs25 and one of the pre-erythrocytic-stage antigens PfCSP_TSR or PfCelTOS, or the additional blood-stage antigen PfMSP1_19, was produced in Pichia pastoris and used to immunize rabbits. The immune sera and purified IgG were used to perform various assays determining antigen specific titers and in vitro efficacy against different parasite stages and strains. In functional in vitro assays we observed robust inhibition of blood-stage (up to 90%), and sexual-stage parasites (up to 100%) and biased inhibition of pre-erythrocytic parasites (0-40%). Cross-strain blood-stage efficacy was observed in erythrocyte invasion assays using four different P. falciparum strains. The quantification of antigen-specific IgGs allowed the determination of specific IC50 values. The significant difference in antigen-specific IC50 requirements, the direct correlation between antigen-specific IgG and the relative quantitative representation of antigens within the cocktail, provide valuable implementations for future multi-stage, multi-component vaccine designs. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Predictive models for subtypes of autism spectrum disorder based on single-nucleotide polymorphisms and magnetic resonance imaging.

PubMed

Jiao, Y; Chen, R; Ke, X; Cheng, L; Chu, K; Lu, Z; Herskovits, E H

2011-01-01

Autism spectrum disorder (ASD) is a neurodevelopmental disorder, of which Asperger syndrome and high-functioning autism are subtypes. Our goal is: 1) to determine whether a diagnostic model based on single-nucleotide polymorphisms (SNPs), brain regional thickness measurements, or brain regional volume measurements can distinguish Asperger syndrome from high-functioning autism; and 2) to compare the SNP, thickness, and volume-based diagnostic models. Our study included 18 children with ASD: 13 subjects with high-functioning autism and 5 subjects with Asperger syndrome. For each child, we obtained 25 SNPs for 8 ASD-related genes; we also computed regional cortical thicknesses and volumes for 66 brain structures, based on structural magnetic resonance (MR) examination. To generate diagnostic models, we employed five machine-learning techniques: decision stump, alternating decision trees, multi-class alternating decision trees, logistic model trees, and support vector machines. For SNP-based classification, three decision-tree-based models performed better than the other two machine-learning models. The performance metrics for three decision-tree-based models were similar: decision stump was modestly better than the other two methods, with accuracy = 90%, sensitivity = 0.95 and specificity = 0.75. All thickness and volume-based diagnostic models performed poorly. The SNP-based diagnostic models were superior to those based on thickness and volume. For SNP-based classification, rs878960 in GABRB3 (gamma-aminobutyric acid A receptor, beta 3) was selected by all tree-based models. Our analysis demonstrated that SNP-based classification was more accurate than morphometry-based classification in ASD subtype classification. Also, we found that one SNP--rs878960 in GABRB3--distinguishes Asperger syndrome from high-functioning autism.

Elastography in the differential diagnosis of thyroid nodules in Hashimoto thyroiditis.

PubMed

Şahin, Mustafa; Çakal, Erman; Özbek, Mustafa; Güngünes, Aşkin; Arslan, Müyesser Sayki; Akkaymak, Esra Tutal; Uçan, Bekir; Ünsal, Ilknur Öztürk; Bozkurt, Nujen Çolak; Delibaşi, Tuncay

2014-08-01

Elastography is a method which assesses the risk of the malignancy and provides information about the degree of hardness in tissue. Hashimoto's thyroiditis, autoimmune lymphocytic infiltration and fibrosis, is considered to be a very common disease that is able to change the hardness of the tissue. The diagnostic value of elastography of this group of patients has not previously been reported. In our study, we aimed to determine the diagnostic value of elastography in 283 patients (255 female, 28 male) with Hashimoto's thyroiditis. Elastography score and index were measured with real-time ultrasound elastography (Hitachi(®) EUB 7000 HV machine with using 13 MHz linear transducer). The outcome of this measure shows that malignant nodules were with higher elastography scores (ES) and strain indexes (SI) values. ES ≥3 were observed in 16/20 malignant and 130/263 benign nodules, respectively. The area under the curve (AUC) for the elasto score (AUC) was 0.72 (p = 0.001), and AUC for the strain index was 0.77 (p < 0.0001). Accordingly, our study suggests that strain index reflects malignancy better than the elasto score. We conclude that elastography score is ≥3 providing 80 % sensitivity and 50 %, six specificity for diagnosing malignancy. For strain index, we found that 2.45 (72.2 % sensitivity and 70 % specificity) is a cut-off point. We have detected a lower cut-off point for SI in Hashimoto patients although sensitivity and specificity decreases in Hashimoto in this population.

[The characteristics of Vibrio cholerae isolated in Dagestan in 1994].

PubMed

Podosinnikova, L S; Mazrukho, B L; Lomov, Iu M; Khanum'ian, T A; Voronezhskaia, L G; Kudriakova, T A; Makedonova, L D; Mironova, A V

1995-01-01

A high degree of resistance to cholera diagnostic phages and carriership of prophages characteristic of V. cholerae eltor strains vct+ were shown to be the specific features V. cholerae isolated in Daghestan during the period of June-October 1994. Among the strains under study, isolated respectively in 12 and 18 out of 19 regions of Daghestan, a high proportion was found to have resistance to tetracycline (65%) and chloramphenicol (28.6%). Moreover, some strains were found to be resistant to furagin and erythromycin. Out of 242 strains resistant to antibacterial preparations, 163 strains were found to have multiple resistance. Gentamicin, cipropfloxacin and doxicycline were shown to have high in vitro activity with respect to the strains under study.

Evolution and genome specialization of Brucella suis biovar 2 Iberian lineages.

PubMed

Ferreira, Ana Cristina; Tenreiro, Rogério; de Sá, Maria Inácia Corrêa; Dias, Ricardo

2017-09-12

Swine brucellosis caused by B. suis biovar 2 is an emergent disease in domestic pigs in Europe. The emergence of this pathogen has been linked to the increase of extensive pig farms and the high density of infected wild boars (Sus scrofa). In Portugal and Spain, the majority of strains share specific molecular characteristics, which allowed establishing an Iberian clonal lineage. However, several strains isolated from wild boars in the North-East region of Spain are similar to strains isolated in different Central European countries. Comparative analysis of five newly fully sequenced B. suis biovar 2 strains belonging to the main circulating clones in Iberian Peninsula, with publicly available Brucella spp. genomes, revealed that strains from Iberian clonal lineage share 74% similarity with those reference genomes. Besides the 210 kb translocation event present in all biovar 2 strains, an inversion with 944 kb was presented in chromosome I of strains from the Iberian clone. At left and right crossover points, the inversion disrupted a TRAP dicarboxylate transporter, DctM subunit, and an integral membrane protein TerC. The gene dctM is well conserved in Brucella spp. except in strains from the Iberian clonal lineage. Intraspecies comparative analysis also exposed a number of biovar-, haplotype- and strain-specific insertion-deletion (INDELs) events and single nucleotide polymorphisms (SNPs) that could explain differences in virulence and host specificities. Most discriminative mutations were associated to membrane related molecules (29%) and enzymes involved in catabolism processes (20%). Molecular identification of both B. suis biovar 2 clonal lineages could be easily achieved using the target-PCR procedures established in this work for the evaluated INDELs. Whole-genome analyses supports that the B. suis biovar 2 Iberian clonal lineage evolved from the Central-European lineage and suggests that the genomic specialization of this pathogen in the Iberian Peninsula is independent of a specific genomic event(s), but instead driven by allopatric speciation, resulting in the establishment of a new ecovar.

Eimeria genomics: Where are we now and where are we going?

PubMed

Blake, Damer P

2015-08-15

The evolution of sequencing technologies, from Sanger to next generation (NGS) and now the emerging third generation, has prompted a radical frameshift moving genomics from the specialist to the mainstream. For parasitology, genomics has moved fastest for the protozoa with sequence assemblies becoming available for multiple genera including Babesia, Cryptosporidium, Eimeria, Giardia, Leishmania, Neospora, Plasmodium, Theileria, Toxoplasma and Trypanosoma. Progress has commonly been slower for parasites of animals which lack zoonotic potential, but the deficit is now being redressed with impact likely in the areas of drug and vaccine development, molecular diagnostics and population biology. Genomics studies with the apicomplexan Eimeria species clearly illustrate the approaches and opportunities available. Specifically, more than ten years after initiation of a genome sequencing project a sequence assembly was published for Eimeria tenella in 2014, complemented by assemblies for all other Eimeria species which infect the chicken and Eimeria falciformis, a parasite of the mouse. Public access to these and other coccidian genome assemblies through resources such as GeneDB and ToxoDB now promotes comparative analysis, encouraging better use of shared resources and enhancing opportunities for development of novel diagnostic and control strategies. In the short term genomics resources support development of targeted and genome-wide genetic markers such as single nucleotide polymorphisms (SNPs), with whole genome re-sequencing becoming viable in the near future. Experimental power will develop rapidly as additional species, strains and isolates are sampled with particular emphasis on population structure and allelic diversity. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

Diagnostic implications of magnetic resonance feature tracking derived myocardial strain parameters in acute myocarditis.

PubMed

Baeßler, Bettina; Schaarschmidt, Frank; Dick, Anastasia; Michels, Guido; Maintz, David; Bunck, Alexander C

2016-01-01

The present study aims to evaluate the diagnostic value of cardiac magnetic resonance (CMR) feature tracking (FT) derived strain-analysis of both ventricles in patients with acute myocarditis (ACM) in order to improve its currently still challenging non-invasive diagnosis. CMR cine data of 31 patients with clinically suspected ACM and confirmation of diagnosis by CMR according to the Lake Louise criteria as well as 14 patients with clinically diagnosed ACM but inconspicuous CMR were retrospectively analyzed. 20 healthy volunteers (HV) served as a control. Analysis of global longitudinal, circumferential and radial strain and strain rate of both ventricles was performed in one long-axis and three short-axis slices using a dedicated FT-software (TomTec Imaging Systems). Patients with ACM showed significantly reduced LV longitudinal strain (-12.7 ± 6.5 vs. -16.8 ± 5.9%, p=0.021) and LV circumferential strain (LVCirStrain; -22.9 ± 5.7 vs. -27.8 ± 4.4 %, p<0.001) compared to HV. Conversely, they showed improved basal RV circumferential strain rate (BasalRVCirSR; -0.70 ± 0.23 vs. -0.47 ± 0.31s(-1), p=0.009). In ACM patients with preserved EF, BasalRVCirSR was significantly increased compared to HV while LV strain was not significantly different between both groups. In multinominal logistic regression analysis, LVCirStrain and BasalRVCirSR proved to be the best independent predictors of ACM with preserved EF. A combined cut-off of -0.53s(-1) for BasalRVCirSR and of -29.0% for LVCirStrain allowed a classification of ACM patients with preserved EF with a sensitivity of 89% and a specificity of 80%. Also patients with clinical ACM but inconspicuous CMR showed a significantly improved BasalRVCirSR and a cut-off of -0.77s(-1) allowed a classification of ACM patients with a sensitivity of 70% and a specificity of 90%, while all other CMR parameters were normal. The defined cut-offs for LVCirStrain and BasalRVCirSR allow a prediction of ACM with high sensitivity and specificity, even in patients with preserved EF and in patients with otherwise completely inconspicuous CMR. Our results point to a discriminative power especially of RV strain analysis in the CMR-based diagnosis of ACM. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Sibling Rivalry in Myxococcus xanthus Is Mediated by Kin Recognition and a Polyploid Prophage.

PubMed

Dey, Arup; Vassallo, Christopher N; Conklin, Austin C; Pathak, Darshankumar T; Troselj, Vera; Wall, Daniel

2016-01-19

Myxobacteria form complex social communities that elicit multicellular behaviors. One such behavior is kin recognition, in which cells identify siblings via their polymorphic TraA cell surface receptor, to transiently fuse outer membranes and exchange their contents. In addition, outer membrane exchange (OME) regulates behaviors, such as inhibition of wild-type Myxococcus xanthus (DK1622) from swarming. Here we monitored the fate of motile cells and surprisingly found they were killed by nonmotile siblings. The kill phenotype required OME (i.e., was TraA dependent). The genetic basis of killing was traced to ancestral strains used to construct DK1622. Specifically, the kill phenotype mapped to a large "polyploid prophage," Mx alpha. Sensitive strains contained a 200-kb deletion that removed two of three Mx alpha units. To explain these results, we suggest that Mx alpha expresses a toxin-antitoxin cassette that uses the OME machinery of M. xanthus to transfer a toxin that makes the population "addicted" to Mx alpha. Thus, siblings that lost Mx alpha units (no immunity) are killed by cells that harbor the element. To test this, an Mx alpha-harboring laboratory strain was engineered (by traA allele swap) to recognize a closely related species, Myxococcus fulvus. As a result, M. fulvus, which lacks Mx alpha, was killed. These TraA-mediated antagonisms provide an explanation for how kin recognition specificity might have evolved in myxobacteria. That is, recognition specificity is determined by polymorphisms in traA, which we hypothesize were selected for because OME with non-kin leads to lethal outcomes. The transition from single cell to multicellular life is considered a major evolutionary event. Myxobacteria have successfully made this transition. For example, in response to starvation, individual cells aggregate into multicellular fruiting bodies wherein cells differentiate into spores. To build fruits, cells need to recognize their siblings, and in part, this is mediated by the TraA cell surface receptor. Surprisingly, we report that TraA recognition can also involve sibling killing. We show that killing originates from a prophage-like element that has apparently hijacked the TraA system to deliver a toxin to kin. We hypothesize that this killing system has imposed selective pressures on kin recognition, which in turn has resulted in TraA polymorphisms and hence many different recognition groups. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Sibling Rivalry in Myxococcus xanthus Is Mediated by Kin Recognition and a Polyploid Prophage

PubMed Central

Dey, Arup; Vassallo, Christopher N.; Conklin, Austin C.; Pathak, Darshankumar T.; Troselj, Vera

2016-01-01

ABSTRACT Myxobacteria form complex social communities that elicit multicellular behaviors. One such behavior is kin recognition, in which cells identify siblings via their polymorphic TraA cell surface receptor, to transiently fuse outer membranes and exchange their contents. In addition, outer membrane exchange (OME) regulates behaviors, such as inhibition of wild-type Myxococcus xanthus (DK1622) from swarming. Here we monitored the fate of motile cells and surprisingly found they were killed by nonmotile siblings. The kill phenotype required OME (i.e., was TraA dependent). The genetic basis of killing was traced to ancestral strains used to construct DK1622. Specifically, the kill phenotype mapped to a large “polyploid prophage,” Mx alpha. Sensitive strains contained a 200-kb deletion that removed two of three Mx alpha units. To explain these results, we suggest that Mx alpha expresses a toxin-antitoxin cassette that uses the OME machinery of M. xanthus to transfer a toxin that makes the population “addicted” to Mx alpha. Thus, siblings that lost Mx alpha units (no immunity) are killed by cells that harbor the element. To test this, an Mx alpha-harboring laboratory strain was engineered (by traA allele swap) to recognize a closely related species, Myxococcus fulvus. As a result, M. fulvus, which lacks Mx alpha, was killed. These TraA-mediated antagonisms provide an explanation for how kin recognition specificity might have evolved in myxobacteria. That is, recognition specificity is determined by polymorphisms in traA, which we hypothesize were selected for because OME with non-kin leads to lethal outcomes. IMPORTANCE The transition from single cell to multicellular life is considered a major evolutionary event. Myxobacteria have successfully made this transition. For example, in response to starvation, individual cells aggregate into multicellular fruiting bodies wherein cells differentiate into spores. To build fruits, cells need to recognize their siblings, and in part, this is mediated by the TraA cell surface receptor. Surprisingly, we report that TraA recognition can also involve sibling killing. We show that killing originates from a prophage-like element that has apparently hijacked the TraA system to deliver a toxin to kin. We hypothesize that this killing system has imposed selective pressures on kin recognition, which in turn has resulted in TraA polymorphisms and hence many different recognition groups. PMID:26787762

Genome Sequencing Identifies Two Nearly Unchanged Strains of Persistent Listeria monocytogenes Isolated at Two Different Fish Processing Plants Sampled 6 Years Apart

PubMed Central

Holch, Anne; Webb, Kristen; Lukjancenko, Oksana; Ussery, David; Rosenthal, Benjamin M.

2013-01-01

Listeria monocytogenes is a food-borne human-pathogenic bacterium that can cause infections with a high mortality rate. It has a remarkable ability to persist in food processing facilities. Here we report the genome sequences for two L. monocytogenes strains (N53-1 and La111) that were isolated 6 years apart from two different Danish fish processers. Both strains are of serotype 1/2a and belong to a highly persistent DNA subtype (random amplified polymorphic DNA [RAPD] type 9). We demonstrate using in silico analyses that both strains belong to the multilocus sequence typing (MLST) type ST121 that has been isolated as a persistent subtype in several European countries. The purpose of this study was to use genome analyses to identify genes or proteins that could contribute to persistence. In a genome comparison, the two persistent strains were extremely similar and collectively differed from the reference lineage II strain, EGD-e. Also, they differed markedly from a lineage I strain (F2365). On the proteome level, the two strains were almost identical, with a predicted protein homology of 99.94%, differing at only 2 proteins. No single-nucleotide polymorphism (SNP) differences were seen between the two strains; in contrast, N53-1 and La111 differed from the EGD-e reference strain by 3,942 and 3,471 SNPs, respectively. We included a persistent L. monocytogenes strain from the United States (F6854) in our comparisons. Compared to nonpersistent strains, all three persistent strains were distinguished by two genome deletions: one, of 2,472 bp, typically contains the gene for inlF, and the other, of 3,017 bp, includes three genes potentially related to bacteriocin production and transport (lmo2774, lmo2775, and the 3′-terminal part of lmo2776). Further studies of highly persistent strains are required to determine if the absence of these genes promotes persistence. While the genome comparison did not point to a clear physiological explanation of the persistent phenotype, the remarkable similarity between the two strains indicates that subtypes with specific traits are selected for in the food processing environment and that particular genetic and physiological factors are responsible for the persistent phenotype. PMID:23435887

Exome sequencing and arrayCGH detection of gene sequence and copy number variation between ILS and ISS mouse strains.

PubMed

Dumas, Laura; Dickens, C Michael; Anderson, Nathan; Davis, Jonathan; Bennett, Beth; Radcliffe, Richard A; Sikela, James M

2014-06-01

It has been well documented that genetic factors can influence predisposition to develop alcoholism. While the underlying genomic changes may be of several types, two of the most common and disease associated are copy number variations (CNVs) and sequence alterations of protein coding regions. The goal of this study was to identify CNVs and single-nucleotide polymorphisms that occur in gene coding regions that may play a role in influencing the risk of an individual developing alcoholism. Toward this end, two mouse strains were used that have been selectively bred based on their differential sensitivity to alcohol: the Inbred long sleep (ILS) and Inbred short sleep (ISS) mouse strains. Differences in initial response to alcohol have been linked to risk for alcoholism, and the ILS/ISS strains are used to investigate the genetics of initial sensitivity to alcohol. Array comparative genomic hybridization (arrayCGH) and exome sequencing were conducted to identify CNVs and gene coding sequence differences, respectively, between ILS and ISS mice. Mouse arrayCGH was performed using catalog Agilent 1 × 244 k mouse arrays. Subsequently, exome sequencing was carried out using an Illumina HiSeq 2000 instrument. ArrayCGH detected 74 CNVs that were strain-specific (38 ILS/36 ISS), including several ISS-specific deletions that contained genes implicated in brain function and neurotransmitter release. Among several interesting coding variations detected by exome sequencing was the gain of a premature stop codon in the alpha-amylase 2B (AMY2B) gene specifically in the ILS strain. In total, exome sequencing detected 2,597 and 1,768 strain-specific exonic gene variants in the ILS and ISS mice, respectively. This study represents the most comprehensive and detailed genomic comparison of ILS and ISS mouse strains to date. The two complementary genome-wide approaches identified strain-specific CNVs and gene coding sequence variations that should provide strong candidates to contribute to the alcohol-related phenotypic differences associated with these strains.

High resolution identity testing of inactivated poliovirus vaccines

PubMed Central

Mee, Edward T.; Minor, Philip D.; Martin, Javier

2015-01-01

Background Definitive identification of poliovirus strains in vaccines is essential for quality control, particularly where multiple wild-type and Sabin strains are produced in the same facility. Sequence-based identification provides the ultimate in identity testing and would offer several advantages over serological methods. Methods We employed random RT-PCR and high throughput sequencing to recover full-length genome sequences from monovalent and trivalent poliovirus vaccine products at various stages of the manufacturing process. Results All expected strains were detected in previously characterised products and the method permitted identification of strains comprising as little as 0.1% of sequence reads. Highly similar Mahoney and Sabin 1 strains were readily discriminated on the basis of specific variant positions. Analysis of a product known to contain incorrect strains demonstrated that the method correctly identified the contaminants. Conclusion Random RT-PCR and shotgun sequencing provided high resolution identification of vaccine components. In addition to the recovery of full-length genome sequences, the method could also be easily adapted to the characterisation of minor variant frequencies and distinction of closely related products on the basis of distinguishing consensus and low frequency polymorphisms. PMID:26049003

Genetic analysis of Saccharomyces cerevisiae strains isolated from palm wine in eastern Nigeria. Comparison with other African strains.

PubMed

Ezeronye, O U; Legras, J-L

2009-05-01

To study the yeast diversity of Nigerian palm wines by comparison with other African strains. Twenty-three Saccharomyces cerevisiae strains were obtained from palm wine samples collected at four locations in eastern Nigeria, and characterized using different molecular techniques: internal transcribed spacer restriction fragment length polymorphism and sequence analysis, pulsed field gel electrophoresis, inter delta typing and microsatellite multilocus analysis. These techniques revealed that palm wine yeasts represent a group of closely related strains that includes other West African isolates (CBS400, NCYC110, DVPG6044). Population analysis revealed an excess of homozygote strains and an allelic richness similar to wine suggestive of local domestication. Several other African yeast strains were not connected to this group. Ghana sorghum beer strains and other African strains (DBVPG1853 and MUCL28071) displayed strikingly high relatedness with European bread, beer or wine strains, and the genome of strain MUCL30909 contained African and wine-type alleles, indicating its hybrid origin. Nigerian palm wine yeast represents a local specific yeast flora, whereas a European origin or hybrid was suspected for several other Africa isolates. This study presents the first genetic characterization of an autochthonous African palm wine yeast population and confirms the idea that human intervention has favoured yeast migration.

Complete cDNA sequence and amino acid analysis of a bovine ribonuclease K6 gene.

PubMed

Pietrowski, D; Förster, M

2000-01-01

The complete cDNA sequence of a ribonuclease k6 gene of Bos Taurus has been determined. It codes for a protein with 154 amino acids and contains the invariant cysteine, histidine and lysine residues as well as the characteristic motifs specific to ribonuclease active sites. The deduced protein sequence is 27 residues longer than other known ribonucleases k6 and shows amino acids exchanges which could reflect a strain specificity or polymorphism within the bovine genome. Based on sequence similarity we have termed the identified gene bovine ribonuclease k6 b (brk6b).

Inconsistencies in the red blood cell membrane proteome analysis: generation of a database for research and diagnostic applications

PubMed Central

Hegedűs, Tamás; Chaubey, Pururawa Mayank; Várady, György; Szabó, Edit; Sarankó, Hajnalka; Hofstetter, Lia; Roschitzki, Bernd; Sarkadi, Balázs

2015-01-01

Based on recent results, the determination of the easily accessible red blood cell (RBC) membrane proteins may provide new diagnostic possibilities for assessing mutations, polymorphisms or regulatory alterations in diseases. However, the analysis of the current mass spectrometry-based proteomics datasets and other major databases indicates inconsistencies—the results show large scattering and only a limited overlap for the identified RBC membrane proteins. Here, we applied membrane-specific proteomics studies in human RBC, compared these results with the data in the literature, and generated a comprehensive and expandable database using all available data sources. The integrated web database now refers to proteomic, genetic and medical databases as well, and contains an unexpected large number of validated membrane proteins previously thought to be specific for other tissues and/or related to major human diseases. Since the determination of protein expression in RBC provides a method to indicate pathological alterations, our database should facilitate the development of RBC membrane biomarker platforms and provide a unique resource to aid related further research and diagnostics. Database URL: http://rbcc.hegelab.org PMID:26078478

Catalog of MicroRNA Seed Polymorphisms in Vertebrates

PubMed Central

Calin, George Adrian; Horvat, Simon; Jiang, Zhihua; Dovc, Peter; Kunej, Tanja

2012-01-01

MicroRNAs (miRNAs) are a class of non-coding RNA that plays an important role in posttranscriptional regulation of mRNA. Evidence has shown that miRNA gene variability might interfere with its function resulting in phenotypic variation and disease susceptibility. A major role in miRNA target recognition is ascribed to complementarity with the miRNA seed region that can be affected by polymorphisms. In the present study, we developed an online tool for the detection of miRNA polymorphisms (miRNA SNiPer) in vertebrates (http://www.integratomics-time.com/miRNA-SNiPer) and generated a catalog of miRNA seed region polymorphisms (miR-seed-SNPs) consisting of 149 SNPs in six species. Although a majority of detected polymorphisms were due to point mutations, two consecutive nucleotide substitutions (double nucleotide polymorphisms, DNPs) were also identified in nine miRNAs. We determined that miR-SNPs are frequently located within the quantitative trait loci (QTL), chromosome fragile sites, and cancer susceptibility loci, indicating their potential role in the genetic control of various complex traits. To test this further, we performed an association analysis between the mmu-miR-717 seed SNP rs30372501, which is polymorphic in a large number of standard inbred strains, and all phenotypic traits in these strains deposited in the Mouse Phenome Database. Analysis showed a significant association between the mmu-miR-717 seed SNP and a diverse array of traits including behavior, blood-clinical chemistry, body weight size and growth, and immune system suggesting that seed SNPs can indeed have major pleiotropic effects. The bioinformatics analyses, data and tools developed in the present study can serve researchers as a starting point in testing more targeted hypotheses and designing experiments using optimal species or strains for further mechanistic studies. PMID:22303453

DEVELOPMENT OF CODOMINANT MARKERS FOR IDENTIFYING SPECIES HYBRIDS

EPA Science Inventory

Herein we describe a simple method for developing species-diagnostic markers that would permit the rapid identification of hybrid individuals. Our method relies on amplified length polymorphism (AFLP) and single strand conformation polymorphism (SSCP) technologies, both of which...

Strategy for Sensitive and Specific Detection of Yersinia pestis in Skeletons of the Black Death Pandemic

PubMed Central

Seifert, Lisa; Harbeck, Michaela; Thomas, Astrid; Hoke, Nadja; Zöller, Lothar; Wiechmann, Ingrid; Grupe, Gisela; Scholz, Holger C.; Riehm, Julia M.

2013-01-01

Yersinia pestis has been identified as the causative agent of the Black Death pandemic in the 14th century. However, retrospective diagnostics in human skeletons after more than 600 years are critical. We describe a strategy following a modern diagnostic algorithm and working under strict ancient DNA regime for the identification of medieval human plague victims. An initial screening and DNA quantification assay detected the Y. pestis specific pla gene of the high copy number plasmid pPCP1. Results were confirmed by conventional PCR and sequence analysis targeting both Y. pestis specific virulence plasmids pPCP1 and pMT1. All assays were meticulously validated according to human clinical diagnostics requirements (ISO 15189) regarding efficiency, sensitivity, specificity, and limit of detection (LOD). Assay specificity was 100% tested on 41 clinically relevant bacteria and 29 Y. pseudotuberculosis strains as well as for DNA of 22 Y. pestis strains and 30 previously confirmed clinical human plague samples. The optimized LOD was down to 4 gene copies. 29 individuals from three different multiple inhumations were initially assessed as possible victims of the Black Death pandemic. 7 samples (24%) were positive in the pPCP1 specific screening assay. Confirmation through second target pMT1 specific PCR was successful for 4 of the positive individuals (14%). A maximum of 700 and 560 copies per µl aDNA were quantified in two of the samples. Those were positive in all assays including all repetitions, and are candidates for future continuative investigations such as whole genome sequencing. We discuss that all precautions taken here for the work with aDNA are sufficient to prevent external sample contamination and fulfill the criteria of authenticity. With regard to retrospective diagnostics of a human pathogen and the uniqueness of ancient material we strongly recommend using a careful strategy and validated assays as presented in our study. PMID:24069445

Strategy for sensitive and specific detection of Yersinia pestis in skeletons of the black death pandemic.

PubMed

Seifert, Lisa; Harbeck, Michaela; Thomas, Astrid; Hoke, Nadja; Zöller, Lothar; Wiechmann, Ingrid; Grupe, Gisela; Scholz, Holger C; Riehm, Julia M

2013-01-01

Yersinia pestis has been identified as the causative agent of the Black Death pandemic in the 14(th) century. However, retrospective diagnostics in human skeletons after more than 600 years are critical. We describe a strategy following a modern diagnostic algorithm and working under strict ancient DNA regime for the identification of medieval human plague victims. An initial screening and DNA quantification assay detected the Y. pestis specific pla gene of the high copy number plasmid pPCP1. Results were confirmed by conventional PCR and sequence analysis targeting both Y. pestis specific virulence plasmids pPCP1 and pMT1. All assays were meticulously validated according to human clinical diagnostics requirements (ISO 15189) regarding efficiency, sensitivity, specificity, and limit of detection (LOD). Assay specificity was 100% tested on 41 clinically relevant bacteria and 29 Y. pseudotuberculosis strains as well as for DNA of 22 Y. pestis strains and 30 previously confirmed clinical human plague samples. The optimized LOD was down to 4 gene copies. 29 individuals from three different multiple inhumations were initially assessed as possible victims of the Black Death pandemic. 7 samples (24%) were positive in the pPCP1 specific screening assay. Confirmation through second target pMT1 specific PCR was successful for 4 of the positive individuals (14%). A maximum of 700 and 560 copies per µl aDNA were quantified in two of the samples. Those were positive in all assays including all repetitions, and are candidates for future continuative investigations such as whole genome sequencing. We discuss that all precautions taken here for the work with aDNA are sufficient to prevent external sample contamination and fulfill the criteria of authenticity. With regard to retrospective diagnostics of a human pathogen and the uniqueness of ancient material we strongly recommend using a careful strategy and validated assays as presented in our study.

Genotype diversity in the honey bee parasite Nosema ceranae: multi-strain isolates, cryptic sex or both?

PubMed

Sagastume, Soledad; Martín-Hernández, Raquel; Higes, Mariano; Henriques-Gil, Nuno

2016-10-18

There is great controversy as to whether Microsporidia undergo a sexual cycle. In the paradigmatic case of Nosema ceranae, although there is no morphological evidence of sex, some meiosis-specific genes are present in its reduced genome and there is also high intraspecific variability, with incongruent phylogenies having been systematically obtained. The possibility of sexual recombination is important from an epidemiological standpoint, particularly as N. ceranae is considered to be a major factor in the current disquieting epidemic of widespread bee colony losses. This parasite apparently originated in oriental honey bees, spreading out of Asia and Australia to infect honey bees worldwide. This study had three main objectives: i) to obtain genetic markers that are not part of known multi-copy arrays for strain determination; ii) to shed light on the intraspecific variability and recombination of N. ceranae; and iii) to assess the variability in N. ceranae populations. The answers to these questions are critical to understand the capacity of adaptation of microsporidia. Biallelic polymorphisms were detected at a number of specific points in the five coding loci analyzed from European and Australian isolates of N. ceranae. Heterozygous genotypes were abundant and cloning experiments demonstrate that they reflect the existence of multiple alternative sequences in each isolate. The comparisons of different clones and genotypes clearly indicate that new haplotypes are generated by homologous recombination. The N. ceranae isolates from honey bees correspond to genotypically distinct populations, revealing that individual honey bees may not be infected by a particular clone but rather, a pool of different strains. Homologous recombination implies the existence of a cryptic sex cycle yet to be described in N. ceranae. There are no diagnostic alleles associated with Australian or European origins, nor are there differences between the two hosts, A. cerana and A. mellifera, supporting the absence of biological barriers for N. ceranae transmission. Diversity is high among microsporidia of both these origins, and the maintenance of a high heterozygosis in the recently invaded European populations, could hypothetically underlie the stronger virulence of N. ceranae observed in A. mellifera.

A Comprehensive Genetic Study of Streptococcal Immunoglobulin A1 Proteases: Evidence for Recombination within and between Species

PubMed Central

Poulsen, Knud; Reinholdt, Jesper; Jespersgaard, Christina; Boye, Kit; Brown, Thomas A.; Hauge, Majbritt; Kilian, Mogens

1998-01-01

An analysis of 13 immunoglobulin A1 (IgA1) protease genes (iga) of strains of Streptococcus pneumoniae, Streptococcus oralis, Streptococcus mitis, and Streptococcus sanguis was carried out to obtain information on the structure, polymorphism, and phylogeny of this specific protease, which enables bacteria to evade functions of the predominant Ig isotype on mucosal surfaces. The analysis included cloning and sequencing of iga genes from S. oralis and S. mitis biovar 1, sequencing of an additional seven iga genes from S. sanguis biovars 1 through 4, and restriction fragment length polymorphism (RFLP) analyses of iga genes of another 10 strains of S. mitis biovar 1 and 6 strains of S. oralis. All 13 genes sequenced had the potential of encoding proteins with molecular masses of approximately 200 kDa containing the sequence motif HEMTH and an E residue 20 amino acids downstream, which are characteristic of Zn metalloproteinases. In addition, all had a typical gram-positive cell wall anchor motif, LPNTG, which, in contrast to such motifs in other known streptococcal and staphylococcal proteins, was located in their N-terminal parts. Repeat structures showing variation in number and sequence were present in all strains and may be of relevance to the immunogenicities of the enzymes. Protease activities in cultures of the streptococcal strains were associated with species of different molecular masses ranging from 130 to 200 kDa, suggesting posttranslational processing possibly as a result of autoproteolysis at post-proline peptide bonds in the N-terminal parts of the molecules. Comparison of deduced amino acid sequences revealed a 94% similarity between S. oralis and S. mitis IgA1 proteases and a 75 to 79% similarity between IgA1 proteases of these species and those of S. pneumoniae and S. sanguis, respectively. Combined with the results of RFLP analyses using different iga gene fragments as probes, the results of nucleotide sequence comparisons provide evidence of horizontal transfer of iga gene sequences among individual strains of S. sanguis as well as among S. mitis and the two species S. pneumoniae and S. oralis. While iga genes of S. sanguis and S. oralis were highly homogeneous, the genes of S. pneumoniae and S. mitis showed extensive polymorphism reflected in different degrees of antigenic diversity. PMID:9423856

A characteristic of polymorphic membrane protein F of Chlamydia trachomatis isolated from male urogenital tracts in Japan.

PubMed

Yamazaki, Tomohiro; Matsuo, Junji; Takahashi, Satoshi; Kumagai, Shouta; Shimoda, Tomoko; Abe, Kiyotaka; Minami, Kunihiro; Yamaguchi, Hiroyuki

2015-12-01

Although sexually transmitted disease due to Chlamydia trachomatis occurs similarly in both men and women, the female urogenital tract differs from that of males anatomically and physiologically, possibly leading to specific polymorphisms of the bacterial surface molecules. In the present study, we therefore characterized polymorphic features in a high-definition phylogenetic marker, polymorphic outer membrane protein (Pmp) F of C. trachomatis strains isolated from male urogenital tracts in Japan (Category: Japan-males, n = 12), when compared with those isolated from female cervical ducts in Japan (Category: Japan-females, n = 11), female cervical ducts in the other country (Category: Ref-females, n = 12) or homosexual male rectums in the other country (Category: Ref-males, n = 7), by general bioinformatics analysis tool with MAFFT software. As a result, phylogenetic reconstruction of the PmpF amino acid sequences showing three distinct clusters revealed that the Japan-males were limited into cluster 1 and 2, although there were only four clusters even though including an outgroup. Meanwhile, the phylogenetic distance values of PmpF passenger domain without hinge region, but not its full-length sequence, showed that the Japan-males were more stable and displayed less diversity when compared with the other categories, supported by the sequence conservation features. Thus, PmpF passenger domain is a useful phylogenetic maker, and the phylogenic features indicate that C. trachomatis strains isolated from male urogenital tracts in Japan may be unique, suggesting an adaptation depending on selective pressure, such as the presence or absence of microbial flora, furthermore possibly connecting to sexual differentiation. Copyright © 2015 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Analysis of genetic variation in Ganoderma Lucidum after space flight

NASA Astrophysics Data System (ADS)

Qi, Jian-Jun; Ma, Rong-Cai; Chen, Xiang-Dong; Lan, Jin

A modified CTAB method was used in the extraction of total cellular DNA of Ganoderma lucidum. Four strains Cx, Ch, C3 and C4, and their counterparts, four space flown strains Sx, Xh, S3 and S4, were analysed by amplified fragment length polymorphism (AFLP) with several primer combinations. Polymorphic bands were detected between Sx and Cx, S3 and C3, respectively. Somatic incompatibility tests further confirmed their heterogeneity. However, no disparity between Sh and Ch, S4 and C4 was detectable. The results suggest that spaceflight may be used to accelerate breeding of Ganoderma lucidum strains for commercial cultivation.

Genomic landscapes of endogenous retroviruses unveil intricate genetics of conventional and genetically-engineered laboratory mouse strains.

PubMed

Lee, Kang-Hoon; Lim, Debora; Chiu, Sophia; Greenhalgh, David; Cho, Kiho

2016-04-01

Laboratory strains of mice, both conventional and genetically engineered, have been introduced as critical components of a broad range of studies investigating normal and disease biology. Currently, the genetic identity of laboratory mice is primarily confirmed by surveying polymorphisms in selected sets of "conventional" genes and/or microsatellites in the absence of a single completely sequenced mouse genome. First, we examined variations in the genomic landscapes of transposable repetitive elements, named the TREome, in conventional and genetically engineered mouse strains using murine leukemia virus-type endogenous retroviruses (MLV-ERVs) as a probe. A survey of the genomes from 56 conventional strains revealed strain-specific TREome landscapes, and certain families (e.g., C57BL) of strains were discernible with defined patterns. Interestingly, the TREome landscapes of C3H/HeJ (toll-like receptor-4 [TLR4] mutant) inbred mice were different from its control C3H/HeOuJ (TLR4 wild-type) strain. In addition, a CD14 knock-out strain had a distinct TREome landscape compared to its control/backcross C57BL/6J strain. Second, an examination of superantigen (SAg, a "TREome gene") coding sequences of mouse mammary tumor virus-type ERVs in the genomes of the 46 conventional strains revealed a high diversity, suggesting a potential role of SAgs in strain-specific immune phenotypes. The findings from this study indicate that unexplored and intricate genomic variations exist in laboratory mouse strains, both conventional and genetically engineered. The TREome-based high-resolution genetics surveillance system for laboratory mice would contribute to efficient study design with quality control and accurate data interpretation. This genetics system can be easily adapted to other species ranging from plants to humans. Copyright © 2016 Elsevier Inc. All rights reserved.

Comparative genomics of Bacillus anthracis from the wool industry highlights polymorphisms of lineage A.Br.Vollum.

PubMed

Derzelle, Sylviane; Aguilar-Bultet, Lisandra; Frey, Joachim

2016-12-01

With the advent of affordable next-generation sequencing (NGS) technologies, major progress has been made in the understanding of the population structure and evolution of the B. anthracis species. Here we report the use of whole genome sequencing and computer-based comparative analyses to characterize six strains belonging to the A.Br.Vollum lineage. These strains were isolated in Switzerland, in 1981, during iterative cases of anthrax involving workers in a textile plant processing cashmere wool from the Indian subcontinent. We took advantage of the hundreds of currently available B. anthracis genomes in public databases, to investigate the genetic diversity existing within the A.Br.Vollum lineage and to position the six Swiss isolates into the worldwide B. anthracis phylogeny. Thirty additional genomes related to the A.Br.Vollum group were identified by whole-genome single nucleotide polymorphism (SNP) analysis, including two strains forming a new evolutionary branch at the basis of the A.Br.Vollum lineage. This new phylogenetic lineage (termed A.Br.H9401) splits off the branch leading to the A.Br.Vollum group soon after its divergence to the other lineages of the major A clade (i.e. 6 SNPs). The available dataset of A.Br.Vollum genomes were resolved into 2 distinct groups. Isolates from the Swiss wool processing facility clustered together with two strains from Pakistan and one strain of unknown origin isolated from yarn. They were clearly differentiated (69 SNPs) from the twenty-five other A.Br.Vollum strains located on the branch leading to the terminal reference strain A0488 of the lineage. Novel analytic assays specific to these new subgroups were developed for the purpose of rapid molecular epidemiology. Whole genome SNP surveys greatly expand upon our knowledge on the sub-structure of the A.Br.Vollum lineage. Possible origin and route of spread of this lineage worldwide are discussed. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Comparison and Combination of Strain and Shear Wave Elastography of Breast Masses for Differentiation of Benign and Malignant Lesions by Quantitative Assessment: Preliminary Study.

PubMed

Seo, Mirinae; Ahn, Hye Shin; Park, Sung Hee; Lee, Jong Beum; Choi, Byung Ihn; Sohn, Yu-Mee; Shin, So Youn

2018-01-01

To compare the diagnostic performance of strain and shear wave elastography of breast masses for quantitative assessment in differentiating benign and malignant lesions and to evaluate the diagnostic accuracy of combined strain and shear wave elastography. Between January and February 2016, 37 women with 45 breast masses underwent both strain and shear wave ultrasound (US) elastographic examinations. The American College of Radiology Breast Imaging Reporting and Data System (BI-RADS) final assessment on B-mode US imaging was assessed. We calculated strain ratios for strain elastography and the mean elasticity value and elasticity ratio of the lesion to fat for shear wave elastography. Diagnostic performances were compared by using the area under the receiver operating characteristic curve (AUC). The 37 women had a mean age of 47.4 years (range, 20-79 years). Of the 45 lesions, 20 were malignant, and 25 were benign. The AUCs for elasticity values on strain and shear wave elastography showed no significant differences (strain ratio, 0.929; mean elasticity, 0.898; and elasticity ratio, 0.868; P > .05). After selectively downgrading BI-RADS category 4a lesions based on strain and shear wave elastographic cutoffs, the AUCs for the combined sets of B-mode US and elastography were improved (B-mode + strain, 0.940; B-mode + shear wave; 0.964; and B-mode, 0.724; P < .001). Combined strain and shear wave elastography showed significantly higher diagnostic accuracy than each individual elastographic modality (P = .031). These preliminary results showed that strain and shear wave elastography had similar diagnostic performance. The addition of strain and shear wave elastography to B-mode US improved diagnostic performance. The combination of strain and shear wave elastography results in a higher diagnostic yield than each individual elastographic modality. © 2017 by the American Institute of Ultrasound in Medicine.

Simple sequence repeats in Escherichia coli: abundance, distribution, composition, and polymorphism.

PubMed

Gur-Arie, R; Cohen, C J; Eitan, Y; Shelef, L; Hallerman, E M; Kashi, Y

2000-01-01

Computer-based genome-wide screening of the DNA sequence of Escherichia coli strain K12 revealed tens of thousands of tandem simple sequence repeat (SSR) tracts, with motifs ranging from 1 to 6 nucleotides. SSRs were well distributed throughout the genome. Mononucleotide SSRs were over-represented in noncoding regions and under-represented in open reading frames (ORFs). Nucleotide composition of mono- and dinucleotide SSRs, both in ORFs and in noncoding regions, differed from that of the genomic region in which they occurred, with 93% of all mononucleotide SSRs proving to be of A or T. Computer-based analysis of the fine position of every SSR locus in the noncoding portion of the genome relative to downstream ORFs showed SSRs located in areas that could affect gene regulation. DNA sequences at 14 arbitrarily chosen SSR tracts were compared among E. coli strains. Polymorphisms of SSR copy number were observed at four of seven mononucleotide SSR tracts screened, with all polymorphisms occurring in noncoding regions. SSR polymorphism could prove important as a genome-wide source of variation, both for practical applications (including rapid detection, strain identification, and detection of loci affecting key phenotypes) and for evolutionary adaptation of microbes.

Whole genome sequencing based characterization of extensively drug-resistant Mycobacterium tuberculosis isolates from Pakistan.

PubMed

Ali, Asho; Hasan, Zahra; McNerney, Ruth; Mallard, Kim; Hill-Cawthorne, Grant; Coll, Francesc; Nair, Mridul; Pain, Arnab; Clark, Taane G; Hasan, Rumina

2015-01-01

Improved molecular diagnostic methods for detection drug resistance in Mycobacterium tuberculosis (MTB) strains are required. Resistance to first- and second- line anti-tuberculous drugs has been associated with single nucleotide polymorphisms (SNPs) in particular genes. However, these SNPs can vary between MTB lineages therefore local data is required to describe different strain populations. We used whole genome sequencing (WGS) to characterize 37 extensively drug-resistant (XDR) MTB isolates from Pakistan and investigated 40 genes associated with drug resistance. Rifampicin resistance was attributable to SNPs in the rpoB hot-spot region. Isoniazid resistance was most commonly associated with the katG codon 315 (92%) mutation followed by inhA S94A (8%) however, one strain did not have SNPs in katG, inhA or oxyR-ahpC. All strains were pyrazimamide resistant but only 43% had pncA SNPs. Ethambutol resistant strains predominantly had embB codon 306 (62%) mutations, but additional SNPs at embB codons 406, 378 and 328 were also present. Fluoroquinolone resistance was associated with gyrA 91-94 codons in 81% of strains; four strains had only gyrB mutations, while others did not have SNPs in either gyrA or gyrB. Streptomycin resistant strains had mutations in ribosomal RNA genes; rpsL codon 43 (42%); rrs 500 region (16%), and gidB (34%) while six strains did not have mutations in any of these genes. Amikacin/kanamycin/capreomycin resistance was associated with SNPs in rrs at nt1401 (78%) and nt1484 (3%), except in seven (19%) strains. We estimate that if only the common hot-spot region targets of current commercial assays were used, the concordance between phenotypic and genotypic testing for these XDR strains would vary between rifampicin (100%), isoniazid (92%), flouroquinolones (81%), aminoglycoside (78%) and ethambutol (62%); while pncA sequencing would provide genotypic resistance in less than half the isolates. This work highlights the importance of expanded targets for drug resistance detection in MTB isolates.

Molecular Strain Typing of Clinical Isolates, Trichophyton rubrum using Non Transcribed Spacer (NTS) Region as a Molecular Marker

PubMed Central

Ramaraj, Vijayakumar; Vijayaraman, Rajyoganandh S; Elavarashi, Elangovan; Rangarajan, Sudha

2017-01-01

Introduction Dermatophytes are a group of fungi which infect keratinized tissues and causes superficial mycoses in humans and animals. The group comprises of three major genera, Trichophyton, Microsporum and Epidermophyton. Among them Trichophyton rubrum is a predominant anthropophilic fungi which causes chronic infections. Although, the infection is superficial and treatable, reinfection/coinfection causes inflation in the treatment cost. Identifying the source and mode of transmission is essential to prevent its transmission. Accurate discrimination is required to understand the clinical (relapse or reinfection) and epidemiological implications of the genetic heterogeneity of this species. Polymorphism in the Non Transcribed Spacer (NTS) region of ribosomal DNA (rDNA) clusters renders an effective way to discriminate strains among T. rubrum. Aim To carry out the strain typing of the clinical isolates, Trichophyton rubrum using NTS as a molecular marker. Materials and Methods Seventy T.rubrum clinical isolates obtained from April-2011-March 2013, from Sri Ramachandra Medical Centre, Chennai, Tamil Nadu, India, were identified by conventional phenotypic methods and included in this prospective study. The isolates were then subjected to Polymerase Chain Reaction (PCR) targeting two subrepeat elements (SREs), TRS-1 and TRS-2 of the NTS region. Results Strain-specific polymorphism was observed in both subrepeat loci. Total, nine different strains were obtained on combining both TRS-1 and TRS-2, SREs. Conclusion The outcome has given a strong representation for using NTS region amplification in discriminating the T. rubrum clinical isolates. The method can be adapted as a tool for conducting epidemiology and population based study in T. rubrum infections. This will help in future exploration of the epidemiology of T. rubrum. PMID:28658757

Molecular Strain Typing of Clinical Isolates, Trichophyton rubrum using Non Transcribed Spacer (NTS) Region as a Molecular Marker.

PubMed

Ramaraj, Vijayakumar; Vijayaraman, Rajyoganandh S; Elavarashi, Elangovan; Rangarajan, Sudha; Kindo, Anupma Jyoti

2017-05-01

Dermatophytes are a group of fungi which infect keratinized tissues and causes superficial mycoses in humans and animals. The group comprises of three major genera, Trichophyton , Microsporum and Epidermophyton . Among them Trichophyton rubrum is a predominant anthropophilic fungi which causes chronic infections. Although, the infection is superficial and treatable, reinfection/coinfection causes inflation in the treatment cost. Identifying the source and mode of transmission is essential to prevent its transmission. Accurate discrimination is required to understand the clinical (relapse or reinfection) and epidemiological implications of the genetic heterogeneity of this species. Polymorphism in the Non Transcribed Spacer (NTS) region of ribosomal DNA (rDNA) clusters renders an effective way to discriminate strains among T. rubrum . To carry out the strain typing of the clinical isolates, Trichophyton rubrum using NTS as a molecular marker. Seventy T.rubrum clinical isolates obtained from April-2011-March 2013, from Sri Ramachandra Medical Centre, Chennai, Tamil Nadu, India, were identified by conventional phenotypic methods and included in this prospective study. The isolates were then subjected to Polymerase Chain Reaction (PCR) targeting two subrepeat elements (SREs), TRS-1 and TRS-2 of the NTS region. Strain-specific polymorphism was observed in both subrepeat loci. Total, nine different strains were obtained on combining both TRS-1 and TRS-2, SREs. The outcome has given a strong representation for using NTS region amplification in discriminating the T. rubrum clinical isolates. The method can be adapted as a tool for conducting epidemiology and population based study in T. rubrum infections. This will help in future exploration of the epidemiology of T. rubrum .

CRISPR Diversity and Microevolution in Clostridium difficile

PubMed Central

Andersen, Joakim M.; Shoup, Madelyn; Robinson, Cathy; Britton, Robert; Olsen, Katharina E.P.; Barrangou, Rodolphe

2016-01-01

Abstract Virulent strains of Clostridium difficile have become a global health problem associated with morbidity and mortality. Traditional typing methods do not provide ideal resolution to track outbreak strains, ascertain genetic diversity between isolates, or monitor the phylogeny of this species on a global basis. Here, we investigate the occurrence and diversity of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (cas) in C. difficile to assess the potential of CRISPR-based phylogeny and high-resolution genotyping. A single Type-IB CRISPR-Cas system was identified in 217 analyzed genomes with cas gene clusters present at conserved chromosomal locations, suggesting vertical evolution of the system, assessing a total of 1,865 CRISPR arrays. The CRISPR arrays, markedly enriched (8.5 arrays/genome) compared with other species, occur both at conserved and variable locations across strains, and thus provide a basis for typing based on locus occurrence and spacer polymorphism. Clustering of strains by array composition correlated with sequence type (ST) analysis. Spacer content and polymorphism within conserved CRISPR arrays revealed phylogenetic relationship across clades and within ST. Spacer polymorphisms of conserved arrays were instrumental for differentiating closely related strains, e.g., ST1/RT027/B1 strains and pathogenicity locus encoding ST3/RT001 strains. CRISPR spacers showed sequence similarity to phage sequences, which is consistent with the native role of CRISPR-Cas as adaptive immune systems in bacteria. Overall, CRISPR-Cas sequences constitute a valuable basis for genotyping of C. difficile isolates, provide insights into the micro-evolutionary events that occur between closely related strains, and reflect the evolutionary trajectory of these genomes. PMID:27576538

Evaluation of the Specificity of BP3385 for Bordetella pertussis

USDA-ARS?s Scientific Manuscript database

BP3385 has been proposed as a diagnostic PCR target for discriminating between Bordetella pertussis and other Bordetella species that also infect humans. Our results demonstrate this gene is also present in some strains of Bordetella hinzii and Bordetella bronchiseptica....

Use of Restriction Fragment Length Polymorphisms to Investigate Strain Variation Within Neisseria Meningitidis.

NASA Astrophysics Data System (ADS)

Williams, Shelley Diane

Similarity within bacterial populations is difficult to assess due to the limited number of characters available for evaluation and the heterogeneity of bacterial species. Currently, the preferred method used to evaluate the structure of bacterial populations is multilocus enzyme electrophoresis. However, this method is extremely cumbersome and only offers an indirect measure of genetic similarities. The development of a more direct and less cumbersome method for this purpose is warranted. Restriction fragment length polymorphism analysis was evaluated as a tool for use in the study of bacterial population structures and in the epidemiology and surveillance of infectious disease. A collection of Neisseria meningitidis was available for use in the investigation of this technique. Neisseria meningitidis is the causative agent of epidemic cerebrospinal meningitis and septicemia as well as a variety of other clinical manifestations. Each isolate in the collection was defined in terms of serogroup specificity, clinical history, geographic source, and date of isolation. Forty -six strains were chosen for this study. The DNA from each strain was restricted with Pst1 and EcoR1 and electrophoresed on agarose gels. The DNA was transferred to nylon filters and hybridized with P ^{32} labeled DNA probes. Two randomly generated probes and a gene-specific probe were used to estimate the genetic similarities between and among the strains in the study population. A total of 28 different restriction fragment migration types were detected by the probes used. Data obtained from the RFLP analysis was analysed by cluster analysis and multivariate statistical methods. A total of 7 clones groups were detected. Two of these appear to be major clones that comprise 35% of the population. This analysis demonstrates the lack of structure within Neisseria meningitidis due primarily to a heterogenous population and the lack of geographic segregation. The potential utility of this technique as a tool in epidemiologic surveillance is addressed. Further work is needed in the evaluation of RFLP analysis in the taxonomy bacteria.

The uses of AFLP for detecting DNA polymorphism, genotype identification and genetic diversity between yeasts isolated from Mexican agave-distilled beverages and from grape musts.

PubMed

Flores Berrios, E P; Alba González, J F; Arrizon Gaviño, J P; Romano, P; Capece, A; Gschaedler Mathis, A

2005-01-01

The objectives were to determine the variability and to compare the genetic diversity obtained using amplified fragment length polymorphism (AFLP) markers in analyses of wine, tequila, mezcal, sotol and raicilla yeasts. A molecular characterization of yeasts isolated from Mexican agave musts, has been performed by AFLP marker analysis, using reference wine strains from Italian and South African regions. A direct co-relation between genetic profile, origin and fermentation process of strains was found especially in strains isolated from agave must. In addition, unique molecular markers were obtained for all the strains using six combination primers, confirming the discriminatory power of AFLP markers. This is the first report of molecular characterization between yeasts isolated from different Mexican traditional agave-distilled beverages, which shows high genetic differences with respect to wine strains.

Genotypic and technological diversity of Brevibacterium linens strains for use as adjunct starter cultures in 'Pecorino di Filiano' cheese ripened in two different environments.

PubMed

Bonomo, Maria Grazia; Cafaro, Caterina; Salzano, Giovanni

2015-01-01

Twenty-two Brevibacterium linens strains isolated from 'Pecorino di Filiano' cheese ripened in two different environments (natural cave and storeroom) were characterized and differentiated for features of technological interest and by genotypic methods, in order to select strains with specific features to be used as surface starter cultures. Results showed significant differences among strains on the basis of physiological and technological features, indicating heterogeneity within the species. A middle-low level of proteolytic activity was observed in 27.3 % of strains, while a small group (9.1 %) showed a high ability. Lipolytic activity was observed at three different temperatures and the highest value was detected at 20 °C with 13.6 % of strains, while an increase in temperature produced a slightly lower lipolysis in all strains. The evaluation of diacetyl production revealed that only 22.8 % of strains showed this ability, and most of them were isolated from product ripened in the natural cave. All strains exhibited only leu-aminopeptidase activity, with values more elevated in strains coming from the natural cave product. The combined analysis of genotypic results with the data obtained by the features of technological interest study established that the random amplified polymorphic DNA (RAPD) clusters obtained were composed not only of different genotypes but of different profiles based on technological properties too. This study demonstrated the importance of the ripening environment that affects the typical features of the artisanal product, leading to the selection of a specific surface microflora. Characterized strains could be associated within surface starters to standardize the production process of cheese, but preserving its typical organoleptic and sensory characteristics and improving the quality of the final product.

SEROLOGICAL TYPING OF STAPHYLOCOCCI BY MEANS OF FLUORESCENT ANTIBODIES I.

PubMed Central

Cohen, Jay O.; Oeding, Per

1962-01-01

Cohen, Jay O. (Communicable Disease Center, Atlanta, Ga.) and Per Oeding. Serological typing of staphylococci by means of fluorescent antibodies. I. Development of specific reagents for seven serological factors. J. Bacteriol. 84:735–741. 1962—Fluorescent antibody reagents for identifying seven antigenic factors of staphylococci have been prepared. The fluorescent staining reactions of these reagents were compared to the agglutination reactions with diagnostic cultures of coagulase-positive staphylococci. Correlation between the two serological tests was almost complete with factors a, b, i, and k. The c fluorescent antibody reagent had a somewhat broader spectrum of activity than the corresponding agglutination serum, whereas the m fluorescent antibody reagent stained fewer strains than were agglutinated in m serum. The fluorescent antibody reagent for h factor stained strains possessing h1 factor but not strains possessing only h2 factor. Fluorescent antibody reagents for specific staphylococcal factors did not stain strains of group A streptococci. PMID:14022057

Detection by hemi-nested reverse transcription polymerase chain reaction and genetic characterization of wild type strains of Canine distemper virus in suspected infected dogs.

PubMed

Di Francesco, Cristina E; Di Francesco, Daniela; Di Martino, Barbara; Speranza, Roberto; Santori, Domenico; Boari, Andrea; Marsilio, Fulvio

2012-01-01

A new highly sensitive and specific hemi-nested reverse transcription polymerase chain reaction (RT-PCR) assay was applied to detect nucleoprotein (NP) gene of Canine distemper virus (CDV) in samples collected from dogs showing respiratory, gastrointestinal, and neurological signs. Thirty-eight out of 86 samples were positive suggesting that despite the vaccination, canine distemper may still represent a high risk to the canine population. The 968 base pair (bp) fragments from the hemagglutinin (H) gene of 10 viral strains detected in positive samples were amplified and analyzed by restriction fragment length polymorphism (RFLP) using AluI and PsiI enzymes in order to differentiate among vaccine and wild-type CDV strains and to characterize the field viral strains. The products of the both enzymatic digestions allowed identification all viruses as wild strains of CDV. In addition, the RFLP analysis with AluI provided additional information about the identity level among the strains analyzed on the basis of the positions of the cleavage site in the nucleotide sequences of the H gene. The method could be a more useful and simpler method for molecular studies of CDV strains.

Molecular epidemiology and genetic diversity of Orientia tsutsugamushi from patients with scrub typhus in 3 regions of India.

PubMed

Varghese, George M; Janardhanan, Jeshina; Mahajan, Sanjay K; Tariang, David; Trowbridge, Paul; Prakash, John A J; David, Thambu; Sathendra, Sowmya; Abraham, O C

2015-01-01

Scrub typhus, an acute febrile illness that is widespread in the Asia-Pacific region, is caused by the bacterium Orientia tsutsugamushi, which displays high levels of antigenic variation. We conducted an investigation to identify the circulating genotypes of O. tsutsugamushi in 3 scrub typhus-endemic geographic regions of India: South India, Northern India, and Northeast India. Eschar samples collected during September 2010-August 2012 from patients with scrub typhus were subjected to 56-kDa type-specific PCR and sequencing to identify their genotypes. Kato-like strains predominated (61.5%), especially in the South and Northeast, followed by Karp-like strains (27.7%) and Gilliam and Ikeda strains (2.3% each). Neimeng-65 genotype strains were also observed in the Northeast. Clarifying the genotypic diversity of O. tsutsugamushi in India enhances knowledge of the regional diversity among circulating strains and provides potential resources for future region-specific diagnostic studies and vaccine development.

[Improvement of laboratory diagnostics of cholera due to genetically altered (hybrid) variants of cholera Vibrio biovar El Tor].

PubMed

Savel'eva, I V; Khatsukov, K X; Savel'eva, E I; Moskvitina, S I; Kovalev, D A; Savel'ev, V N; Kulichenko, A N; Antonenko, A D; Babenyshev, B V

2015-01-01

Improvement of laboratory diagnostics of cholera taking into the account appearance of hybrid variants of cholera vibrio El Tor biovar in the 1990s. Phenotypic and molecular-genetic properties of typical toxigenic (151 strains) and hybrid (102 strains) variants of El Tor biovar cholera vibrios, isolated in the Caucuses in 1970-1990 and 1993-1998, respectively, were studied. Toxigenicity gene DNA fragments, inherent to El Tor biovars or classic, were detected by using a reagent kit "Genes of Vibrio cholerae variant ctxB-rstR-rstC, REF" developed by us. Reagent kit "Genes of V. cholerae variant ctxB-rstR-rstC, REF" is proposed to be used for laboratory diagnostics of cholera during study of material from humans or environmental objects and for identification of V. cholerae 01 on genome level in PCR-analysis as a necessary addition to the classic scheme of bacteriological analysis. Laboratory diagnostics of cholera due to genetically altered (hybrid) variants of cholera vibrio El Tor biovar is based on a complex study of material from humans and environmental objects by routine bacteriologic and PCR-analysis methods with the aim of detection of gene DNA fragments in the studied material, that determine biovar (classic or El Tor), identification of V. cholerae O1 strains with differentiation of El Tor vibrios into typical and altered, as well as determination of enterotoxin, produced by the specific cholera vibrio strain (by the presence ctxB(El) or ctxB(Cl) gene DNA fragment, coding biosynthesis of CT-2 or CT-1, respectively).

Genotyping of ancient Mycobacterium tuberculosis strains reveals historic genetic diversity.

PubMed

Müller, Romy; Roberts, Charlotte A; Brown, Terence A

2014-04-22

The evolutionary history of the Mycobacterium tuberculosis complex (MTBC) has previously been studied by analysis of sequence diversity in extant strains, but not addressed by direct examination of strain genotypes in archaeological remains. Here, we use ancient DNA sequencing to type 11 single nucleotide polymorphisms and two large sequence polymorphisms in the MTBC strains present in 10 archaeological samples from skeletons from Britain and Europe dating to the second-nineteenth centuries AD. The results enable us to assign the strains to groupings and lineages recognized in the extant MTBC. We show that at least during the eighteenth-nineteenth centuries AD, strains of M. tuberculosis belonging to different genetic groups were present in Britain at the same time, possibly even at a single location, and we present evidence for a mixed infection in at least one individual. Our study shows that ancient DNA typing applied to multiple samples can provide sufficiently detailed information to contribute to both archaeological and evolutionary knowledge of the history of tuberculosis.

Revisiting the Evolution of Mycobacterium bovis

PubMed Central

Mostowy, Serge; Inwald, Jackie; Gordon, Steve; Martin, Carlos; Warren, Rob; Kremer, Kristin; Cousins, Debby; Behr, Marcel A.

2005-01-01

Though careful consideration has been placed towards genetic characterization of tubercle bacillus isolates causing disease in humans, those causing disease predominantly among wild and domesticated mammals have received less attention. In contrast to Mycobacterium tuberculosis, whose host range is largely specific to humans, M. bovis and “M bovis-like” organisms infect a broad range of animal species beyond their most prominent host in cattle. To determine whether strains of variable genomic content are associated with distinct distributions of disease, the DNA contents of M. bovis or M. bovis-like isolates from a variety of hosts were investigated via Affymetrix GeneChip. Consistent with previous genomic analysis of the M. tuberculosis complex (MTC), large sequence polymorphisms of putative diagnostic and biological consequence were able to unambiguously distinguish interrogated isolates. The distribution of deleted regions indicates organisms genomically removed from M. bovis and also points to structured genomic variability within M. bovis. Certain genomic profiles spanned a variety of hosts but were clustered by geography, while others associated primarily with host type. In contrast to the prevailing assumption that M. bovis has broad host capacity, genomic profiles suggest that distinct MTC lineages differentially infect a variety of mammals. From this, a phylogenetic stratification of genotypes offers a predictive framework upon which to base future genetic and phenotypic studies of the MTC. PMID:16159772

Genetic mutation underlying orthostatic intolerance and diagnostic and therapeutic methods relating thereto

NASA Technical Reports Server (NTRS)

Blakely, Randy D. (Inventor); Robertson, David (Inventor)

2006-01-01

Isolated polynucleotide molecules and peptides encoded by these molecules are used in the analysis of human norepinephrine (NE) transporter variants, as well as in diagnostic and therapeutic applications, relating to a human NE transporter polymorphism. By analyzing genomic DNA or amplified genomic DNA, or amplified cDNA derived from mRNA, it is possible to type a human NE transporter with regard to the human NE transporter polymorphism, for example, in the context of diagnosing and treating NE transport impairments, and disorders associated with NE transport impairments, such as orthostatic intolerance.

[Study on once sampling quantitation based on information entropy of ISSR amplified bands of Houttuynia cordata].

PubMed

Wang, Haiqin; Liu, Wenlong; He, Fuyuan; Chen, Zuohong; Zhang, Xili; Xie, Xianggui; Zeng, Jiaoli; Duan, Xiaopeng

2012-02-01

To explore the once sampling quantitation of Houttuynia cordata through its DNA polymorphic bands that carried information entropy, from other form that the expression of traditional Chinese medicine polymorphism, genetic polymorphism, of traditional Chinese medicine. The technique of inter simple sequence repeat (ISSR) was applied to analyze genetic polymorphism of H. cordata samples from the same GAP producing area, the DNA genetic bands were transformed its into the information entropy, and the minimum once sampling quantitation with the mathematical mode was measured. One hundred and thirty-four DNA bands were obtained by using 9 screened ISSR primers to amplify from 46 strains DNA samples of H. cordata from the same GAP, the information entropy was H=0.365 6-0.978 6, and RSD was 14.75%. The once sampling quantitation was W=11.22 kg (863 strains). The "once minimum sampling quantitation" were calculated from the angle of the genetic polymorphism of H. cordata, and a great differences between this volume and the amount from the angle of fingerprint were found.

Molecular Characteristics of the Mycobacterium tuberculosis LAM-RUS Family Prevalent in Central Russia▿ †

PubMed Central

Dubiley, Svetlana; Kirillov, Eugene; Ignatova, Anna; Stepanshina, Valentina; Shemyakin, Igor

2007-01-01

We analyzed IS6110-associated polymorphisms in the phospholipase C genes of 107 isolates of Mycobacterium tuberculosis selected to be representative of isolates circulating in central Russia. We found that the majority of Latin American-Mediterranean family strains contained an insertion in a unique position in the plcA gene, suggesting a common ancestor. This insertion can serve as a specific genetic marker for this group, which we designate the LAM-RUS family. PMID:17942651

Detection of virulent Escherichia coli O157 strains using multiplex PCR and single base sequencing for SNP characterization.

PubMed

Haugum, K; Brandal, L T; Løbersli, I; Kapperud, G; Lindstedt, B-A

2011-06-01

To compare 167 Norwegian human and nonhuman Escherichia coli O157:H7/NM (nonmotile) isolates with respect to an A/T single nucleotide polymorphism (SNP) in the tir gene and to detect specific SNPs that differentiate STEC O157 into distinct virulence clades (1-3 and 8). We developed a multiplex PCR followed by single base sequencing for detection of the SNPs, and examined the association among SNP genotype, virulence profile (stx and eae status), multilocus variable number of tandem repeats analysis (MLVA) profile and clinical outcome. We found an over-representation of the T allele among human strains compared to nonhuman strains, including 5/6 haemolytic-uraemic syndrome cases. Fourteen strains belonged to clade 8, followed by two clade 2 strains. No clade 1 nor 3 isolates were observed. stx1 in combination with either stx2(EDL933) or stx2c were frequently observed among human strains, whereas stx2c was dominating in nonhuman strains. MLVA indicated that only single cases or small outbreaks with E. coli O157 have been observed in Norway through the years 1993-2008. We observed that the tir-255 A/T SNP and the stx status were different between human and nonhuman O157 strains. No major outbreaks were observed, and only a few strains were differentiated into the virulence clades 2 and 8. The detection of virulence clade-specific SNPs enables the rapid designation of virulent E. coli O157 strains, especially in outbreak situations. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

Diagnostic and prognostic value of human prion detection in cerebrospinal fluid.

PubMed

Foutz, Aaron; Appleby, Brian S; Hamlin, Clive; Liu, Xiaoqin; Yang, Sheng; Cohen, Yvonne; Chen, Wei; Blevins, Janis; Fausett, Cameron; Wang, Han; Gambetti, Pierluigi; Zhang, Shulin; Hughson, Andrew; Tatsuoka, Curtis; Schonberger, Lawrence B; Cohen, Mark L; Caughey, Byron; Safar, Jiri G

2017-01-01

Several prion amplification systems have been proposed for detection of prions in cerebrospinal fluid (CSF), most recently, the measurements of prion seeding activity with second-generation real-time quaking-induced conversion (RT-QuIC). The objective of this study was to investigate the diagnostic performance of the RT-QuIC prion test in the broad phenotypic spectrum of prion diseases. We performed CSF RT-QuIC testing in 2,141 patients who had rapidly progressive neurological disorders, determined diagnostic sensitivity and specificity in 272 cases that were autopsied, and evaluated the impact of mutations and polymorphisms in the PRNP gene, and type 1 or type 2 human prions on diagnostic performance. The 98.5% diagnostic specificity and 92% sensitivity of CSF RT-QuIC in a blinded retrospective analysis matched the 100% specificity and 95% sensitivity of a blind prospective study. The CSF RT-QuIC differentiated 94% of cases of sporadic Creutzfeldt-Jakob disease (sCJD) MM1 from the sCJD MM2 phenotype, and 80% of sCJD VV2 from sCJD VV1. The mixed prion type 1-2 and cases heterozygous for codon 129 generated intermediate CSF RT-QuIC patterns, whereas genetic prion diseases revealed distinct profiles for each PRNP gene mutation. The diagnostic performance of the improved CSF RT-QuIC is superior to surrogate marker tests for prion diseases such as 14-3-3 and tau proteins, and together with PRNP gene sequencing the test allows the major prion subtypes to be differentiated in vivo. This differentiation facilitates prediction of the clinicopathological phenotype and duration of the disease-two important considerations for envisioned therapeutic interventions. ANN NEUROL 2017;81:79-92. © 2016 American Neurological Association.

Diagnostic and Prognostic Value of Human Prion Detection in Cerebrospinal Fluid

PubMed Central

Foutz, Aaron; Appleby, Brian S.; Hamlin, Clive; Liu, Xiaoqin; Yang, Sheng; Cohen, Yvonne; Chen, Wei; Blevins, Janis; Fausett, Cameron; Wang, Han; Gambetti, Pierluigi; Zhang, Shulin; Hughson, Andrew; Tatsuoka, Curtis; Schonberger, Lawrence B.; Cohen, Mark L.; Caughey, Byron; Safar, Jiri G.

2016-01-01

Objective Several prion amplification systems have been proposed for detection of prions in cerebrospinal fluid (CSF), most recently, the measurements of prion seeding activity with second-generation real-time quaking-induced conversion (RT-QuIC). The objective of this study was to investigate the diagnostic performance of the RT-QuIC prion test in the broad phenotypic spectrum of prion diseases. Methods We performed CSF RT-QuIC testing in 2,141 patients who had rapidly progressive neurological disorders, determined diagnostic sensitivity and specificity in 272 cases which were autopsied, and evaluated the impact of mutations and polymorphisms in the PRNP gene, and Type 1 or Type 2 of human prions on diagnostic performance. Results The 98.5% diagnostic specificity and 92% sensitivity of CSF RT-QuIC in a blinded retrospective analysis matched the 100% specificity and 95% sensitivity of a blind prospective study. The CSF RT-QuIC differentiated 94% of cases of sporadic Creutzfeldt-Jakob disease (sCJD) MM1 from the sCJD MM2 phenotype, and 80% of sCJD VV2 from sCJD VV1. The mixed prion type 1–2 and cases heterozygous for codon 129 generated intermediate CSF RT-QuIC patterns, while genetic prion diseases revealed distinct profiles for each PRNP gene mutation. Interpretation The diagnostic performance of the improved CSF RT-QuIC is superior to surrogate marker tests for prion diseases such as 14-3-3 and Tau proteins and together with PRNP gene sequencing, the test allows the major prion subtypes to be differentiated in vivo. This differentiation facilitates prediction of the clinicopathological phenotype and duration of the disease—two important considerations for envisioned therapeutic interventions. PMID:27893164

GETPrime 2.0: gene- and transcript-specific qPCR primers for 13 species including polymorphisms

PubMed Central

David, Fabrice P.A.; Rougemont, Jacques; Deplancke, Bart

2017-01-01

GETPrime (http://bbcftools.epfl.ch/getprime) is a database with a web frontend providing gene- and transcript-specific, pre-computed qPCR primer pairs. The primers have been optimized for genome-wide specificity and for allowing the selective amplification of one or several splice variants of most known genes. To ease selection, primers have also been ranked according to defined criteria such as genome-wide specificity (with BLAST), amplicon size, and isoform coverage. Here, we report a major upgrade (2.0) of the database: eight new species (yeast, chicken, macaque, chimpanzee, rat, platypus, pufferfish, and Anolis carolinensis) now complement the five already included in the previous version (human, mouse, zebrafish, fly, and worm). Furthermore, the genomic reference has been updated to Ensembl v81 (while keeping earlier versions for backward compatibility) as a result of re-designing the back-end database and automating the import of relevant sections of the Ensembl database in species-independent fashion. This also allowed us to map known polymorphisms to the primers (on average three per primer for human), with the aim of reducing experimental error when targeting specific strains or individuals. Another consequence is that the inclusion of future Ensembl releases and other species has now become a relatively straightforward task. PMID:28053161

Multi-locus genotyping of bottom fermenting yeasts by single nucleotide polymorphisms indicative of brewing characteristics.

PubMed

Ikushima, Shigehito; Tateishi, Yoshiyuki; Kanai, Keiko; Shimada, Emiko; Tanaka, Misa; Ishiguro, Tatsuji; Mizutani, Satoru; Kobayashi, Osamu

2012-04-01

Yeast plays a capital role in brewing fermentation and has a direct impact on flavor and aroma. For the evaluation of competent brewing strains during quality control or development of novel strains it is standard practice to perform fermentation tests, which are costly and time-consuming. Here, we have categorized DNA markers which enable to distinguish and to screen brewing strains more efficiently than ever before. Sequence analysis at 289 loci in the genomes of six bottom fermenting Saccharomyces pastorianus strains revealed that 30 loci contained single nucleotide polymorphisms (SNPs). By determining the nucleotide sequences at the SNP-loci in 26 other S. pastorianus strains and 20 strains of the top fermenting yeast Saccharomyces cerevisiae, almost all these strains could be discriminated solely on the basis of the SNPs. By comparing the fermentative phenotypes of these strains we found that some DNA markers showed a strong association with brewing characteristics, such as the production of ethyl acetate and hydrogen sulphide (H2S). Therefore, the DNA markers we identified will facilitate quality control and the efficient development of brewing yeast strains. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Specific Detection of Enteroaggregative Hemorrhagic Escherichia coli O104:H4 Strains by Use of the CRISPR Locus as a Target for a Diagnostic Real-Time PCR

PubMed Central

Delannoy, Sabine; Beutin, Lothar; Burgos, Ylanna

2012-01-01

In 2011, a large outbreak of an unusual bacterial strain occurred in Europe. This strain was characterized as a hybrid of an enteroaggregative Escherichia coli (EAEC) and a Shiga toxin-producing E. coli (STEC) strain of the serotype O104:H4. Here, we present a single PCR targeting the clustered regularly interspaced short palindromic repeats locus of E. coli O104:H4 (CRISPRO104:H4) for specific detection of EAEC STEC O104:H4 strains from different geographical locations and time periods. The specificity of the CRISPRO104:H4 PCR was investigated using 1,321 E. coli strains, including reference strains for E. coli O serogroups O1 to O186 and flagellar (H) types H1 to H56. The assay was compared for specificity using PCR assays targeting different O104 antigen-encoding genes (wbwCO104, wzxO104, and wzyO104). The PCR assays reacted with all types of E. coli O104 strains (O104:H2, O104:H4, O104:H7, and O104:H21) and with E. coli O8 and O9 strains carrying the K9 capsular antigen and were therefore not specific for detection of the EAEC STEC O104:H4 type. A single PCR developed for the CRISPRO104:H4 target was sufficient for specific identification and detection of the 48 tested EAEC STEC O104:H4 strains. The 35 E. coli O104 strains expressing H types other than H4 as well as 8 E. coli strains carrying a K9 capsular antigen tested all negative for the CRISPRO104:H4 locus. Only 12 (0.94%) of the 1,273 non-O104:H4 E. coli strains (serotypes Ont:H2, O43:H2, O141:H2, and O174:H2) reacted positive in the CRISPRO104:H4 PCR (99.06% specificity). PMID:22895033

Geographical markers for Saccharomyces cerevisiae strains with similar technological origins domesticated for rice-based ethnic fermented beverages production in North East India.

PubMed

Jeyaram, Kumaraswamy; Tamang, Jyoti Prakash; Capece, Angela; Romano, Patrizia

2011-11-01

Autochthonous strains of Saccharomyces cerevisiae from traditional starters used for the production of rice-based ethnic fermented beverage in North East India were examined for their genetic polymorphism using mitochondrial DNA-RFLP and electrophoretic karyotyping. Mitochondrial DNA-RFLP analysis of S. cerevisiae strains with similar technological origins from hamei starter of Manipur and marcha starter of Sikkim revealed widely separated clusters based on their geographical origin. Electrophoretic karyotyping showed high polymorphism amongst the hamei strains within similar mitochondrial DNA-RFLP cluster and one unique karyotype of marcha strain was widely distributed in the Sikkim-Himalayan region. We conceptualized the possibility of separate domestication events for hamei strains in Manipur (located in the Indo-Burma biodiversity hotspot) and marcha strains in Sikkim (located in Himalayan biodiversity hotspot), as a consequence of less homogeneity in the genomic structure between these two groups, their clear separation being based on geographical origin, but not on technological origin and low strain level diversity within each group. The molecular markers developed based on HinfI-mtDNA-RFLP profile and the chromosomal doublets in chromosome VIII position of Sikkim-Himalayan strains could be effectively used as geographical markers for authenticating the above starter strains and differentiating them from other commercial strains.

Group B Streptococcus Infections Caused by Improper Sourcing and Handling of Fish for Raw Consumption, Singapore, 2015-2016.

PubMed

Chau, Man L; Chen, Swaine L; Yap, Min; Hartantyo, Sri H P; Chiew, Paul K T; Fernandez, Charlene J; Wong, Wai K; Fong, Rockey K; Tan, Wei L; Tan, Brian Z Y; Ng, Youming; Aung, Kyaw T; Mehershahi, Kurosh S; Goh, Christopher; Kang, Joanne S L; Barkham, Timothy; Leong, Adeline O K; Gutiérrez, Ramona A; Ng, Lee C

2017-12-01

We assessed microbial safety and quality of raw fish sold in Singapore during 2015-2016 to complement epidemiologic findings for an outbreak of infection with group B Streptococcus serotype III sequence type (ST) 283 associated with raw fish consumption. Fish-associated group B Streptococcus ST283 strains included strains nearly identical (0-2 single-nucleotide polymorphisms) with the human outbreak strain, as well as strains in another distinct ST283 clade (57-71 single-nucleotide polymorphisms). Our investigations highlight the risk for contamination of freshwater fish (which are handled and distributed separately from saltwater fish sold as sashimi) and the need for improved hygienic handling of all fish for raw consumption. These results have led to updated policy and guidelines regarding the sale of ready-to-eat raw fish dishes in Singapore.

Strain Variation in the Transcriptome of the Dengue Fever Vector, Aedes aegypti.

PubMed

Bonizzoni, Mariangela; Dunn, W Augustine; Campbell, Corey L; Olson, Ken E; Marinotti, Osvaldo; James, Anthony A

2012-01-01

Studies of transcriptome dynamics provide a basis for understanding functional elements of the genome and the complexity of gene regulation. The dengue vector mosquito, Aedes aegypti, exhibits great adaptability to diverse ecological conditions, is phenotypically polymorphic, and shows variation in vectorial capacity to arboviruses. Previous genome sequencing showed richness in repetitive DNA and transposable elements that can contribute to genome plasticity. Population genetic studies revealed a varying degree of worldwide genetic polymorphism. However, the extent of functional genetic polymorphism across strains is unknown. The transcriptomes of three Ae. aegypti strains, Chetumal (CTM), Rexville D-Puerto Rico (Rex-D) and Liverpool (LVP), were compared. CTM is more susceptible than Rex- D to infection by dengue virus serotype 2. A total of 4188 transcripts exhibit either no or small variation (<2-fold) among sugar-fed samples of the three strains and between sugar- and blood-fed samples within each strain, corresponding most likely to genes encoding products necessary for vital functions. Transcripts enriched in blood-fed mosquitoes encode proteins associated with catalytic activities, molecular transport, metabolism of lipids, carbohydrates and amino acids, and functions related to blood digestion and the progression of the gonotropic cycle. Significant qualitative and quantitative differences were found in individual transcripts among strains including differential representation of paralogous gene products. The majority of immunity-associated transcripts decreased in accumulation after a bloodmeal and the results are discussed in relation to the different susceptibility of CTM and Rex-D mosquitoes to DENV2 infection.

Molecular Properties of Salmonella enterica Serotype Paratyphi B Distinguish between Its Systemic and Its Enteric Pathovars

PubMed Central

Prager, Rita; Rabsch, Wolfgang; Streckel, Wiebke; Voigt, Wolfgang; Tietze, Erhardt; Tschäpe, Helmut

2003-01-01

Salmonella enterica serotype O1,4,5,12:Hb:1,2, designated according to the current Kauffmann-White scheme as S. enterica serotype Paratyphi B, is a very diverse serotype with respect to its clinical and microbiological properties. PCR and blot techniques, which identify the presence, polymorphism, and expression of various effector protein genes, help to distinguish between strains with systemic and enteric outcomes of disease. All serotype Paratyphi B strains from systemic infections have been found to be somewhat genetically related with respect to the pattern of their virulence genes sopB, sopD, sopE1, avrA, and sptP as well as other molecular properties (multilocus enzyme electrophoresis type, pulsed-field gel electrophoresis [PFGE] type, ribotype, and IS200 type). They have been classified as members of the systemic pathovar (SPV). All these SPV strains possess a new sopE1-carrying bacteriophage (designated ΦSopE309) with high SopE1 protein expression but lack the commonly occurring avrA determinant. They exhibit normal SopB protein expression but lack SopD protein production. In contrast, strains from enteric infections classified as belonging to the enteric pathovar possess various combinations of the respective virulence genes, PFGE pattern, and ribotypes. We propose that the PCR technique for testing for the presence of the virulence genes sopE1 and avrA be used as a diagnostic tool for identifying both pathovars of S. enterica serotype Paratyphi B. This will be of great public health importance, since strains of serotype Paratyphi B have recently reemerged worldwide. PMID:12958256

CRISPR Diversity and Microevolution in Clostridium difficile.

PubMed

Andersen, Joakim M; Shoup, Madelyn; Robinson, Cathy; Britton, Robert; Olsen, Katharina E P; Barrangou, Rodolphe

2016-09-19

Virulent strains of Clostridium difficile have become a global health problem associated with morbidity and mortality. Traditional typing methods do not provide ideal resolution to track outbreak strains, ascertain genetic diversity between isolates, or monitor the phylogeny of this species on a global basis. Here, we investigate the occurrence and diversity of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (cas) in C. difficile to assess the potential of CRISPR-based phylogeny and high-resolution genotyping. A single Type-IB CRISPR-Cas system was identified in 217 analyzed genomes with cas gene clusters present at conserved chromosomal locations, suggesting vertical evolution of the system, assessing a total of 1,865 CRISPR arrays. The CRISPR arrays, markedly enriched (8.5 arrays/genome) compared with other species, occur both at conserved and variable locations across strains, and thus provide a basis for typing based on locus occurrence and spacer polymorphism. Clustering of strains by array composition correlated with sequence type (ST) analysis. Spacer content and polymorphism within conserved CRISPR arrays revealed phylogenetic relationship across clades and within ST. Spacer polymorphisms of conserved arrays were instrumental for differentiating closely related strains, e.g., ST1/RT027/B1 strains and pathogenicity locus encoding ST3/RT001 strains. CRISPR spacers showed sequence similarity to phage sequences, which is consistent with the native role of CRISPR-Cas as adaptive immune systems in bacteria. Overall, CRISPR-Cas sequences constitute a valuable basis for genotyping of C. difficile isolates, provide insights into the micro-evolutionary events that occur between closely related strains, and reflect the evolutionary trajectory of these genomes. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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.

Development of a molecular method for the typing of Brettanomyces bruxellensis (Dekkera bruxellensis) at the strain level.

PubMed

Miot-Sertier, C; Lonvaud-Funel, A

2007-02-01

In recent years, Brettanomyces/Dekkera bruxellensis has caused increasingly severe quality problems in the wine industry. A typing method at the strain level is needed for a better knowledge of the dispersion and the dynamics of these yeasts from grape to wine. Three molecular tools, namely random-amplified polymorphic DNA, PCR fingerprinting with microsatellite oligonucleotide primers and SAU-PCR, were explored for their relevance to typing strains of Brettanomyces bruxellensis. The results indicated that discrimination of each individual strain was not possible with a single PCR typing technique. We described a typing method for B. bruxellensis based on restriction enzyme analysis and pulse field gel electrophoresis (REA-PFGE). Results showed that electrophoretic profiles were reproducible and specific for each strain under study. Consequently, REA-PFGE should be considered for the discrimination of B. bruxellensis strains. This technique allowed a fine discrimination of B. bruxellensis, as strains were identified by a particular profile. This study constitutes a prerequisite for accurate and appropriate investigations on the diversity of strains throughout the winemaking and ageing process. Such studies will probably give clearer and more up-to-date information on the origin of the presence of Brettanomyces in wine after vinification when they are latent spoilage agents.

Specificity of monoclonal antibodies to strains of Dickeya sp. that cause bacterial heart rot of pineapple.

PubMed

Peckham, Gabriel D; Kaneshiro, Wendy S; Luu, Van; Berestecky, John M; Alvarez, Anne M

2010-10-01

During a severe outbreak of bacterial heart rot that occurred in pineapple plantations on Oahu, Hawaii, in 2003 and years following, 43 bacterial strains were isolated from diseased plants or irrigation water and identified as Erwinia chrysanthemi (now Dickeya sp.) by phenotypic, molecular, and pathogenicity assays. Rep-PCR fingerprint patterns grouped strains from pineapple plants and irrigation water into five genotypes (A-E) that differed from representatives of other Dickeya species, Pectobacterium carotovorum and other enteric saprophytes isolated from pineapple. Monoclonal antibodies produced following immunization of mice with virulent type C Dickeya sp. showed only two specificities. MAb Pine-1 (2D11G1, IgG1 with kappa light chain) reacted to all 43 pineapple/water strains and some reference strains (D. dianthicola, D. chrysanthemi, D. paradisiaca, some D. dadantii, and uncharacterized Dickeya sp.) but did not react to reference strains of D. dieffenbachiae, D. zeae, or one of the two Malaysian pineapple strains. MAb Pine-2 (2A7F2, IgG3 with kappa light chain) reacted to all type B, C, and D strains but not to any A or E strains or any reference strains except Dickeya sp. isolated from Malaysian pineapple. Pathogenicity tests showed that type C strains were more aggressive than type A strains when inoculated during cool months. Therefore, MAb Pine-2 distinguishes the more virulent type C strains from less virulent type A pineapple strains and type E water strains. MAbs with these two specificities enable development of rapid diagnostic tests that will distinguish the systemic heart rot pathogen from opportunistic bacteria associated with rotted tissues. Use of the two MAbs in field assays also permits the monitoring of a known subpopulation and provides additional decision tools for disease containment and management practices.

Infection and colonisation due to Scedosporium in Northern Spain. An in vitro antifungal susceptibility and molecular epidemiology study of 60 isolates.

PubMed

Lackner, M; Rezusta, A; Villuendas, M C; Palacian, M P; Meis, J F; Klaassen, C H

2011-10-01

Since the latest taxonomical changes in the genus Scedosporium by Gilgado et al. in 2010, no species-specific studies on epidemiology and antifungal susceptibility patterns (AFSP) have so far been published. This study aimed to provide qualitative epidemiological data of Scedosporium spp. isolated from cystic fibrosis (CF) patients and immunocompromised patients from Northern Spain. Isolates were identified by using amplified fragment length polymorphism (AFLP), and species-specific AFSP were generated for all currently available antifungal compounds. AFLP was a useful tool for identification to species-level and for the discrimination of inter- and intra-patient isolates. Scedosporium prolificans represents the most prevalent species in the respiratory tract of CF patients and immunocompromised patients in Northern-Spain, followed by Pseudallescheria boydii, P. apiosperma, and P. ellipsoidea. CF patients were exclusively colonised with either P. boydii or S. prolificans. Patients were colonised over years exclusively with isolates affiliated to one species, but some patients were colonised with multiple strains with different AFSP. The sum of those co-colonising strains in one patient, may appear in vitro and in vivo as a multi-resistant S. prolificans isolate, as strains are morphologically identical and might therefore be regarded as only one strain. A majority of Scedosporium strains (with exception of S. prolificans) were found susceptible for voriconazole and micafungin. © 2011 Blackwell Verlag GmbH.

Molecular characterization of Histoplasma capsulatum isolated from an outbreak in treasure hunters Histoplasma capsulatum in treasure hunters

PubMed Central

2010-01-01

Background In Mexico, primary pulmonary histoplasmosis is the most relevant clinical form of the disease. The geographical distribution of specific strains of Histoplasma capsulatum circulating in Mexico has not been fully established. Outbreaks must be reported in order to have current, updated information on this disease, identifying new endemic areas, manner of exposure to the fungi, and molecular characterization of the causative agents. We report a recent outbreak of histoplasmosis in treasure hunters and the molecular characterization of two isolates obtained from these patients. Methods Six patients admitted to the National Institute of Respiratory Diseases (INER) in Mexico City presented severe respiratory symptoms suggestive of histoplasmosis. They acquired the infection in the Veracruz (VZ) endemic zone. Diagnosis was made by X-ray and Computed tomography (CT), liver function, immunological techniques, and culture. Identification of H. capsulatum isolates was confirmed by using Polymerase chain reaction (PCR) was conducted with a probe from the M antigen, and the isolates were characterized by means of Random amplification of polymorphic DNA (RAPD)-PCR employed the 1253 oligonucleotide and a mixture of oligonucleotides 1281 and 1283. These were compared to eight reference strain isolates from neighboring areas. Results X-ray and CT revealed disseminated micronodular images throughout lung parenchyma, as well as bilateral retrocaval, prevascular, subcarinal, and hilar adenopathies, hepatosplenomegaly, and altered liver function tests. Five of the six patients developed disseminated histoplasmosis. Two H. capsulatum strains were isolated. The same band profile was detected in both strains, indicating that both isolates corresponded to the sole H. capsulatum strain. Molecular characterization of the isolates was similar in 100% with the EH-53 Hidalgo human (HG) strain (reference strain integrated into the LAm A clade described for Latin America). Conclusions The two isolates appeared to possess the same polymorphic pattern; they are indistinguishable from each other and from EH-53. It is important to remain updated on recent outbreaks of histoplasmosis, the manner of exposure to the fungi, as well as the molecular characterization of the isolates. The severity of cases indicates that this strain is highly virulent and that it is probably prevalent in Hidalgo and Veracruz states. PMID:20825675

Identification of Degenerate Nuclei and Development of a SCAR Marker for Flammulina velutipes

PubMed Central

Kim, Sun Young; Kim, Kyung-Hee; Im, Chak Han; Ali, Asjad; Lee, Chang Yun; Kong, Won-Sik; Ryu, Jae-San

2014-01-01

Flammulina velutipes is one of the major edible mushrooms in the world. Recently, abnormalities that have a negative impact on crop production have been reported in this mushroom. These symptoms include slow vegetative growth, a compact mycelial mat, and few or even no fruiting bodies. The morphologies and fruiting capabilities of monokaryons of wild-type and degenerate strains that arose through arthrospore formation were investigated through test crossing. Only one monokaryotic group of the degenerate strains and its hybrid strains showed abnormal phenotypes. Because the monokaryotic arthrospore has the same nucleus as the parent strain, these results indicated that only one aberrant nucleus of the two nuclei in the degenerate strain was responsible for the degeneracy. A sequence-characterized amplified region marker that is linked to the degenerate monokaryon was identified based on a polymorphic sequence that was generated using random primers. Comparative analyses revealed the presence of a degenerate-specific genomic region in a telomere, which arose via the transfer of a genomic fragment harboring a putative helicase gene. Our findings have narrowed down the potential molecular targets responsible for this phenotype for future studies and have provided a marker for the detection of degenerate strains. PMID:25221949

High resolution identity testing of inactivated poliovirus vaccines.

PubMed

Mee, Edward T; Minor, Philip D; Martin, Javier

2015-07-09

Definitive identification of poliovirus strains in vaccines is essential for quality control, particularly where multiple wild-type and Sabin strains are produced in the same facility. Sequence-based identification provides the ultimate in identity testing and would offer several advantages over serological methods. We employed random RT-PCR and high throughput sequencing to recover full-length genome sequences from monovalent and trivalent poliovirus vaccine products at various stages of the manufacturing process. All expected strains were detected in previously characterised products and the method permitted identification of strains comprising as little as 0.1% of sequence reads. Highly similar Mahoney and Sabin 1 strains were readily discriminated on the basis of specific variant positions. Analysis of a product known to contain incorrect strains demonstrated that the method correctly identified the contaminants. Random RT-PCR and shotgun sequencing provided high resolution identification of vaccine components. In addition to the recovery of full-length genome sequences, the method could also be easily adapted to the characterisation of minor variant frequencies and distinction of closely related products on the basis of distinguishing consensus and low frequency polymorphisms. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Genetic Characterization of Simian Foamy Viruses Infecting Humans

PubMed Central

Rua, Réjane; Betsem, Edouard; Calattini, Sara; Saib, Ali

2012-01-01

Simian foamy viruses (SFVs) are retroviruses that are widespread among nonhuman primates (NHPs). SFVs actively replicate in their oral cavity and can be transmitted to humans after NHP bites, giving rise to a persistent infection even decades after primary infection. Very few data on the genetic structure of such SFVs found in humans are available. In the framework of ongoing studies searching for SFV-infected humans in south Cameroon rainforest villages, we studied 38 SFV-infected hunters whose times of infection had presumably been determined. By long-term cocultures of peripheral blood mononuclear cells with BHK-21 cells, we isolated five new SFV strains and obtained complete genomes of SFV strains from chimpanzee (Pan troglodytes troglodytes; strains BAD327 and AG15), monkey (Cercopithecus nictitans; strain AG16), and gorilla (Gorilla gorilla; strains BAK74 and BAD468). These zoonotic strains share a very high degree of similarity with their NHP counterparts and have a high degree of conservation of the genetic elements important for viral replication. Interestingly, analysis of FV DNA sequences obtained before cultivation revealed variants with deletions in both the U3 region and tas that may correlate with in vivo chronicity in humans. Genomic changes in bet (a premature stop codon) and gag were also observed. To determine if such changes were specific to zoonotic strains, we studied local SFV-infected chimpanzees and found the same genomic changes. Our study reveals that natural polymorphism of SFV strains does exist at both the intersubspecies level (gag, bet) and the intrasubspecies (U3, tas) levels but does not seem to reflect a viral adaptation specific to zoonotic SFV strains. PMID:23015714

A Real-Time PCR Assay to Identify and Discriminate Among Wild-Type and Vaccine Strains of Varicella-Zoster Virus and Herpes Simplex Virus in Clinical Specimens, and Comparison With the Clinical Diagnoses

PubMed Central

Harbecke, Ruth; Oxman, Michael N.; Arnold, Beth A.; Ip, Charlotte; Johnson, Gary R.; Levin, Myron J.; Gelb, Lawrence D.; Schmader, Kenneth E.; Straus, Stephen E.; Wang, Hui; Wright, Peter F.; Pachucki, Constance T.; Gershon, Anne A.; Arbeit, Robert D.; Davis, Larry E.; Simberkoff, Michael S.; Weinberg, Adriana; Williams, Heather M.; Cheney, Carol; Petrukhin, Luba; Abraham, Katalin G.; Shaw, Alan; Manoff, Susan; Antonello, Joseph M.; Green, Tina; Wang, Yue; Tan, Charles; Keller, Paul M.

2014-01-01

A real-time PCR assay was developed to identify varicella-zoster virus (VZV) and herpes simplex virus (HSV) DNA in clinical specimens from subjects with suspected herpes zoster (HZ; shingles). Three sets of primers and probes were used in separate PCR reactions to detect and discriminate among wild-type VZV (VZV-WT), Oka vaccine strain VZV (VZV-Oka), and HSV DNA, and the reaction for each virus DNA was multiplexed with primers and probe specific for the human β-globin gene to assess specimen adequacy. Discrimination of all VZV-WT strains, including Japanese isolates and the Oka parent strain, from VZV-Oka was based upon a single nucleotide polymorphism at position 106262 in ORF 62, resulting in preferential amplification by the homologous primer pair. The assay was highly sensitive and specific for the target virus DNA, and no cross-reactions were detected with any other infectious agent. With the PCR assay as the gold standard, the sensitivity of virus culture was 53% for VZV and 77% for HSV. There was 92% agreement between the clinical diagnosis of HZ by the Clinical Evaluation Committee and the PCR assay results. PMID:19475609

Genotypic intraspecies heterogeneity of Enterococcus italicus: data from dairy environments.

PubMed

Borgo, Francesca; Ferrario, Chiara; Ricci, Giovanni; Fortina, Maria Grazia

2013-01-01

The diversity of a collection of 19 Enterococcus italicus strains isolated from different dairy sources was explored using a molecular polyphasic approach, comprising random amplification of polymorphic DNA (RAPD-PCR), repetitive element PCR (REP-PCR), plasmid profiling and ribotyping. The data obtained showed a high-level of biodiversity, not always correlated to the niche of isolation. Particularly, REP-PCR with primer BOXA1R and plasmid profiling allowed the best discrimination at strain level. Exploiting the genome shotgun sequence of the type strain of the species, available in public database, genes related to insertion sequences present on enterococcal Pathogenic Islands (ISEf1, IS905), determinants related to virulence factors (codifying for hemolysin and cell wall surface proteins), exogenously DNA (conjugal transfer protein, replication plasmid protein, pheromone shutdown protein, phage integrase/recombinase) and penicillin binding proteins system were detected. The presence of most of these genes seemed a common genetic trait in the Enterococcus genus, sur gene (cell wall surface protein) was only detected in strains of E. italicus. To our knowledge, this is the first time that specific primers, with the expection of the species-specific probe targeted to 16S rRNA gene, have been designed for this species. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Insight into the Significance of Aspergillus fumigatus cyp51A Polymorphisms.

PubMed

Garcia-Rubio, Rocio; Alcazar-Fuoli, Laura; Monteiro, Maria Candida; Monzon, Sara; Cuesta, Isabel; Pelaez, Teresa; Mellado, Emilia

2018-06-01

Triazole antifungal compounds are the first treatment choice for invasive aspergillosis. However, in the last decade the rate of azole resistance among Aspergillus fumigatus strains has increased notoriously. The main resistance mechanisms are well defined and mostly related to point mutations of the azole target, 14-α sterol demethylase ( cyp51A ), with or without tandem repeat integrations in the cyp51A promoter. Furthermore, different combinations of five Cyp51A mutations (F46Y, M172V, N248T, D255E, and E427K) have been reported worldwide in about 10% of all A. fumigatus isolates tested. The azole susceptibility profile of these strains shows elevated azole MICs, although on the basis of the azole susceptibility breakpoints, these strains are not considered azole resistant. The purpose of the study was to determine whether these cyp51A polymorphisms (single nucleotide polymorphisms [SNPs]) are responsible for the azole susceptibility profile and whether they are reflected in a poorer azole treatment response in vivo that could compromise patient treatment and outcome. A mutant with a cyp51A deletion was generated and became fully susceptible to all azoles tested. Also, three cyp51A gene constructions with different combinations of SNPs were generated and reintroduced into an azole-susceptible wild-type (WT) strain (the Δ akuB KU80 strain). The alternative model host Galleria mellonella was used to compare the virulence and voriconazole response of G. mellonella larvae infected with A. fumigatus strains with WT cyp51A or cyp51A with SNPs. All strains were pathogenic in G. mellonella larvae, although they did not respond similarly to voriconazole therapeutic doses. Finally, the full genomes of these strains were sequenced and analyzed in comparison with those of A. fumigatus WT strains, revealing that they belong to different strain clusters or lineages. Copyright © 2018 American Society for Microbiology.

Novel PCRs for differential diagnosis of cestodes.

PubMed

Roelfsema, Jeroen H; Nozari, Nahid; Pinelli, Elena; Kortbeek, Laetitia M

2016-02-01

Cestodes or tapeworms belong to a diverse group of helminths. The adult Taenia saginata and Taenia solium tapeworm can infest the human gut and the larval stage of Echinococcus spp. and T. solium can infect tissues of the human body, causing serious disease. Molecular diagnostics can be performed on proglottids, eggs and on cyst fluids taken by biopsy. Detection of cestodes when a helminthic infection is suspected is of vital importance and species determination is required for appropriate patient care. For routine diagnostics a single test that is able to detect and type a range of cestodes is preferable. We sought to improve our diagnostic procedure that used to rely on PCR and subsequent sequencing of the Cox1 and Nad1 genes. We have compared these PCRs with novel PCRs on the 12S rRNA and Nad5 gene and established the sensitivity and specificity. A single PCR on the 12S gene proved to be very suitable for detection and specification of Taenia sp. and Echinococcus sp. Both targets harbour enough polymorphic sites to determine the various Echinococcus species. The 12S PCR was most sensitive of all tested. Copyright © 2015 Elsevier Inc. All rights reserved.

[An ophthalmologic diagnostic error leading to a rare systemic diagnosis: Erdheim-Chester disease].

PubMed

Gilles, M; Alberti, N; Seguy, C; Leger, F; Frulio, N; Pechmeja, J; Longueville, E; Korobelnik, J-F

2014-05-01

Erdheim-Chester disease is a rare systemic disease. The diagnosis is difficult due to significant clinical and morphological polymorphism. Orbital involvement is rare, but constitutes a classic means of detection. We report the case of a 60-year-old man, who consulted for evaluation of bilateral retro-orbital tumors. These tumors had been discovered on head CT two years previously during work-up of proptosis. Two biopsies were performed. The first one revealed polymorphous inflammatory tissue. The second one revealed intense granulomatous reaction, rich in non-specific foamy histiocytes. Thoracic-abdominal-pelvic CT scan detected peri-aortic and retroperitoneal infiltration. The association of these signs pointed to a diagnosis of Erdheim-Chester disease, confirmed by the re-examination of the histological samples. Erdheim-Chester disease is a rare non-Langerhans histiocytosis with a specific tropism for perivascular and fatty connective tissue. The cause is not known. The diagnosis of this systemic disease is histological. In the case of bilateral intra-orbital tumors, the diagnosis of Erdheim-Chester disease must be considered. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Real-time PCR for differential quantification of CVI988 vaccine virus and virulent strains of Marek’s disease virus

PubMed Central

Baigent, Susan J.; Nair, Venugopal K.; Le Galludec, Hervé

2016-01-01

CVI988/Rispens vaccine, the ‘gold standard’ vaccine against Marek’s disease in poultry, is not easily distinguishable from virulent strains of Marek’s disease herpesvirus (MDV). Accurate differential measurement of CVI988 and virulent MDV is commercially important to confirm successful vaccination, to diagnose Marek’s disease, and to investigate causes of vaccine failure. A real-time quantitative PCR assay to distinguish CVI988 and virulent MDV based on a consistent single nucleotide polymorphism in the pp38 gene, was developed, optimised and validated using common primers to amplify both viruses, but differential detection of PCR products using two short probes specific for either CVI988 or virulent MDV. Both probes showed perfect specificity for three commercial preparations of CVI988 and 12 virulent MDV strains. Validation against BAC-sequence-specific and US2-sequence-specific q-PCR, on spleen samples from experimental chickens co-infected with BAC-cloned pCVI988 and wild-type virulent MDV, demonstrated that CVI988 and virulent MDV could be quantified very accurately. The assay was then used to follow kinetics of replication of commercial CVI988 and virulent MDV in feather tips and blood of vaccinated and challenged experimental chickens. The assay is a great improvement in enabling accurate differential quantification of CVI988 and virulent MDV over a biologically relevant range of virus levels. PMID:26973285

Shock structures at ultrahigh strain rates: what can they tell us about material behavior on very fast time scales?

NASA Astrophysics Data System (ADS)

Crowhurst, Jonathan

2013-06-01

In recent years, techniques based on table-top laser systems have shown promise for investigating dynamic material behavior at high rates of both compressive and tensile strain. Common to these techniques is a laser pulse that is used in some manner to rapidly deliver energy to the sample; while the energy itself is often comparatively very small, the intensity can be made high by tightly focusing the pump light. In this way pressures or stresses can be obtained that are sufficiently large to have relevance to a wide range of basic and applied fields. Also, when combined with established ultrafast diagnostics these experiments provide very high time resolution which is particularly desirable when studying, for example shock waves, in which the time for the material to pass from undisturbed to fully compressed (the ``rise time'') can be extremely short (order 10 ps or less) even at fairly small peak stresses. Since much of the most interesting physics comes into play during this process it is important to be able to adequately resolve the shock rise. In this context I will discuss our measurements on aluminum and iron thin films and compare the results with known behavior observed at lower strain rates. Specifically, for aluminum, I will compare our assumed steady wave data at strain rates of up to 1010 s-1 to literature data up to ~107 s-1 and show that the well-known fourth power scaling relation of strain rate to shock stress is maintained even at these very high strain rates. For iron, I will show how we have used our nonsteady data (up to ~109 s-1) to infer a number of important properties of the alpha to epsilon polymorphic transition: 1. The transition can occur on the tens of ps time scale at sufficiently high strain rates and corresponding very large deviatoric stresses, and 2, most of the material appears to transform at a substantially higher stress than the nominal value usually inferred from shock wave experiments of about 13 GPa. This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344 with Laboratory directed Research and Development funding (12ERD042), as well as being based on work supported as part of the EFree, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Award No. DESC0001057.

Mycobacterium avium restriction fragment length polymorphism-IS IS1245 and the simple double repetitive element polymerase chain reaction typing method to screen genetic diversity in Brazilian strains.

PubMed

Sequeira, Patrícia Carvalho de; Fonseca, Leila de Souza; Silva, Marlei Gomes da; Saad, Maria Helena Féres

2005-11-01

Simple double repetitive element polymerase chain reaction (MaDRE-PCR) and Pvu II-IS1245 restriction fragment length polymorphism (RFLP) typing methods were used to type 41 Mycobacterium avium isolates obtained from 14 AIDS inpatients and 10 environment and animals specimens identified among 53 mycobacteria isolated from 237 food, chicken, and pig. All environmental and animals strains showed orphan patterns by both methods. By MaDRE-PCR four patients, with multiple isolates, showed different patterns, suggesting polyclonal infection that was confirmed by RFLP in two of them. This first evaluation of MaDRE-PCR on Brazilian M. avium strains demonstrated that the method seems to be useful as simple and less expensive typing method for screening genetic diversity in M. avium strains on selected epidemiological studies, although with limitation on analysis identical patterns except for one band.

Group B Streptococcus Infections Caused by Improper Sourcing and Handling of Fish for Raw Consumption, Singapore, 2015–2016

PubMed Central

Chau, Man L.; Chen, Swaine L.; Yap, Min; Hartantyo, Sri H.P.; Chiew, Paul K.T.; Fernandez, Charlene J.; Wong, Wai K.; Fong, Rockey K.; Tan, Wei L.; Tan, Brian Z.Y.; Ng, Youming; Aung, Kyaw T.; Mehershahi, Kurosh S.; Goh, Christopher; Kang, Joanne S.L.; Barkham, Timothy; Leong, Adeline O.K.; Gutiérrez, Ramona A.

2017-01-01

We assessed microbial safety and quality of raw fish sold in Singapore during 2015–2016 to complement epidemiologic findings for an outbreak of infection with group B Streptococcus serotype III sequence type (ST) 283 associated with raw fish consumption. Fish-associated group B Streptococcus ST283 strains included strains nearly identical (0–2 single-nucleotide polymorphisms) with the human outbreak strain, as well as strains in another distinct ST283 clade (57–71 single-nucleotide polymorphisms). Our investigations highlight the risk for contamination of freshwater fish (which are handled and distributed separately from saltwater fish sold as sashimi) and the need for improved hygienic handling of all fish for raw consumption. These results have led to updated policy and guidelines regarding the sale of ready-to-eat raw fish dishes in Singapore. PMID:29148967

Rapid group-, serotype-, and vaccine strain-specific identification of poliovirus isolates by real-time reverse transcription-PCR using degenerate primers and probes containing deoxyinosine residues.

PubMed

Kilpatrick, David R; Yang, Chen-Fu; Ching, Karen; Vincent, Annelet; Iber, Jane; Campagnoli, Ray; Mandelbaum, Mark; De, Lina; Yang, Su-Ju; Nix, Allan; Kew, Olen M

2009-06-01

We have adapted our previously described poliovirus diagnostic reverse transcription-PCR (RT-PCR) assays to a real-time RT-PCR (rRT-PCR) format. Our highly specific assays and rRT-PCR reagents are designed for use in the WHO Global Polio Laboratory Network for rapid and large-scale identification of poliovirus field isolates.

Identification of Echinococcus granulosus strains using polymerase chain reaction-restriction fragment length polymorphism amongst livestock in Moroto district, Uganda.

PubMed

Chamai, Martin; Omadang, Leonard; Erume, Joseph; Ocaido, Michael; Oba, Peter; Othieno, Emmanuel; Bonaventure, Straton; Kitibwa, Annah

2016-07-29

A descriptive study was conducted to identify the different strains of Echinococcus granulosus occurring in livestock in Moroto district, Uganda. Echinococcus cysts from 104 domestic animals, including cattle, sheep, goats and camels, were taken and examined by microscopy, polymerase chain reaction with restriction fragment length polymorphism and Sanger DNA sequencing. Echinococcus granulosus genotypes or strains were identified through use of Bioinformatics tools: BioEdit, BLAST and MEGA6. The major finding of this study was the existence of a limited number of E. granulosus genotypes from cattle, goats, sheep and camels. The most predominant genotype was G1 (96.05%), corresponding to the common sheep strain. To a limited extent (3.95%), the study revealed the existence of Echinococcus canadensis G6/7 in three (n = 3) of the E. granulosus-positive samples. No other strains of E. granulosus were identified. It was concluded that the common sheep strain of Echinococcus sensu stricto and G6/7 of E. canadensis were responsible for echinococcal disease in Moroto district, Uganda.

Evaluation of a reverse-hybridization StripAssay for the detection of genetic polymorphisms leading to acenocoumarol sensitivity.

PubMed

Gialeraki, Argyri; Markatos, Christos; Grouzi, Elisabeth; Merkouri, Efrosyni; Travlou, Anthi; Politou, Marianna

2010-04-01

Acenocoumarol is mainly catabolized by CYP2C9 isoform of cytochrome P450 (CYP) liver complex and exerts its anticoagulant effect through the inhibition of Vitamin K Epoxide Reductase (VKOR). The most important genetic polymorphisms which lead to an impaired enzymatic activity and therefore predispose to acenocoumarol sensitivity, are considered to be CYP2C9*2 (Arg144Cys), CYP2C9*3 (Ile359Leu) and VKORC1-1639G>A, respectively. In this study we compared the results of the PGXThrombo StripAssay kit (ViennaLab Diagnostics,Vienna, Austria) with direct DNA sequencing and in house Restriction Fragment Length Polymorphisms (RFLP) for the detection of the aforementioned Single Nucleotide Polymorphisms (SNPs). The reverse hybridization StripAssay was found to be equally effective with RFLP and direct DNA sequencing for the detection of CYP2C9*2 and CYP2C9*3 polymorphisms, respectively. The comparison of the RFLP reference method with the reverse hybridization StripAssay for the detection of VKORC1-1639 G>A polymorphism showed that the reverse hybridization StripAsssay might misclassify some A/A homozygotes as heterozygotes. Optimization of the hybridization procedures may eliminate the extra low signal band observed in some samples at the reverse hybridization StripAssay and improve its diagnostic value.

Applying the revised Chinese Job Content Questionnaire to assess psychosocial work conditions among Taiwan's hospital workers

PubMed Central

2011-01-01

Background For hospital accreditation and health promotion reasons, we examined whether the 22-item Job Content Questionnaire (JCQ) could be applied to evaluate job strain of individual hospital employees and to determine the number of factors extracted from JCQ. Additionally, we developed an Excel module of self-evaluation diagnostic system for consultation with experts. Methods To develop an Excel-based self-evaluation diagnostic system for consultation to experts to make job strain assessment easier and quicker than ever, Rasch rating scale model was used to analyze data from 1,644 hospital employees who enrolled in 2008 for a job strain survey. We determined whether the 22-item Job Content Questionnaire (JCQ) could evaluate job strain of individual employees in work sites. The respective item responding to specific groups' occupational hazards causing job stress was investigated by using skewness coefficient with its 95% CI through item-by-item analyses. Results Each of those 22 items on the questionnaire was examined to have five factors. The prevalence rate of Chinese hospital workers with high job strain was 16.5%. Conclusions Graphical representations of four quadrants, item-by-item bar chart plots and skewness 95% CI comparison generated in Excel can help employers and consultants of an organization focusing on a small number of key areas of concern for each worker in job strain. PMID:21682912

Applying the revised Chinese Job Content Questionnaire to assess psychosocial work conditions among Taiwan's hospital workers.

PubMed

Chien, Tsair-Wei; Lai, Wen-Pin; Wang, Hsien-Yi; Hsu, Sen-Yen; Castillo, Roberto Vasquez; Guo, How-Ran; Chen, Shih-Chung; Su, Shih-Bin

2011-06-18

For hospital accreditation and health promotion reasons, we examined whether the 22-item Job Content Questionnaire (JCQ) could be applied to evaluate job strain of individual hospital employees and to determine the number of factors extracted from JCQ. Additionally, we developed an Excel module of self-evaluation diagnostic system for consultation with experts. To develop an Excel-based self-evaluation diagnostic system for consultation to experts to make job strain assessment easier and quicker than ever, Rasch rating scale model was used to analyze data from 1,644 hospital employees who enrolled in 2008 for a job strain survey. We determined whether the 22-item Job Content Questionnaire (JCQ) could evaluate job strain of individual employees in work sites. The respective item responding to specific groups' occupational hazards causing job stress was investigated by using skewness coefficient with its 95% CI through item-by-item analyses. Each of those 22 items on the questionnaire was examined to have five factors. The prevalence rate of Chinese hospital workers with high job strain was 16.5%. Graphical representations of four quadrants, item-by-item bar chart plots and skewness 95% CI comparison generated in Excel can help employers and consultants of an organization focusing on a small number of key areas of concern for each worker in job strain.

Polymorphisms of blood forms and in vitro metacyclogenesis of Trypanosoma cruzi I, II, and IV.

PubMed

Abegg, Camila Piva; Abreu, Ana Paula de; Silva, Juliane Lopes da; Araújo, Silvana Marques de; Gomes, Mônica Lúcia; Ferreira, Érika Cristina; Toledo, Max Jean de Ornelas

2017-05-01

Trypanosoma cruzi is the etiologic agent of American trypanosomiasis has broad biological and genetic diversity. Remaining to be studied are polymorphisms of the blood forms and metacyclogenesis of different T. cruzi discrete typing units (DTUs). Our goal was to evaluate the relationship between T. cruzi DTUs, the morphology of blood trypomastigotes, and in vitro metacyclogenesis. T. cruzi strains that pertained to DTUs TcI, TcII, and TcIV from different Brazilian states were used. Parameters that were related to the morphology of eight strains were assessed in thin blood smears that were obtained from mice that were inoculated with blood or culture forms, depending on strain. The metacyclogenesis of 12 strains was measured using smears with Liver Infusion Tryptose culture medium and M16 culture medium (which is poor in nutrients and has a low pH) at the exponential phase of growth, both stained with Giemsa. The morphological pattern of TcII strains was consistent with broad forms of the parasite. In TcIV strains, slender forms predominated. The Y strain (TcII) was morphologically more similar to TcIV. Significant differences in polymorphisms were observed between DTUs. Metacyclogenesis parameters, although displaying large standard deviations, differed between the DTUs, with the following descending rank order: TcII > TcI > TcIV. The mean numbers of metacyclic trypomastigotes for TcII were significantly higher than the other DTUs. Although the DTUs presented overlapping characteristics, the general pattern was that different DTUs exhibited significantly different morphologies and metacyclogenesis, suggesting that the genetic diversity of T. cruzi could be related to parameters that are associated with the evolution of infection in mammalian hosts and its ability to disperse in nature. Copyright © 2017 Elsevier Inc. All rights reserved.

Variable Virulence and Efficacy of BCG Vaccine Strains in Mice and Correlation With Genome Polymorphisms

PubMed Central

Zhang, Lu; Ru, Huan-wei; Chen, Fu-zeng; Jin, Chun-yan; Sun, Rui-feng; Fan, Xiao-yong; Guo, Ming; Mai, Jun-tao; Xu, Wen-xi; Lin, Qing-xia; Liu, Jun

2016-01-01

Bacille Calmette–Guérin (BCG), an attenuated strain of Mycobacterium bovis, is the only vaccine available for tuberculosis (TB) control. However, BCG is not an ideal vaccine and has two major limitations: BCG exhibits highly variable effectiveness against the development of TB both in pediatric and adult populations and can cause disseminated BCG disease in immunocompromised individuals. BCG comprises a number of substrains that are genetically distinct. Whether and how these genetic differences affect BCG efficacy remains largely unknown. In this study, we performed comparative analyses of the virulence and efficacy of 13 BCG strains, representing different genetic lineages, in SCID and BALB/c mice. Our results show that BCG strains of the DU2 group IV (BCG-Phipps, BCG-Frappier, BCG-Pasteur, and BCG-Tice) exhibit the highest levels of virulence, and BCG strains of the DU2 group II (BCG-Sweden, BCG-Birkhaug) are among the least virulent group. These distinct levels of virulence may be explained by strain-specific duplications and deletions of genomic DNA. There appears to be a general trend that more virulent BCG strains are also more effective in protection against Mycobacterium tuberculosis challenge. Our findings have important implications for current BCG vaccine programs and for future TB vaccine development. PMID:26643797

Variable Virulence and Efficacy of BCG Vaccine Strains in Mice and Correlation With Genome Polymorphisms.

PubMed

Zhang, Lu; Ru, Huan-Wei; Chen, Fu-Zeng; Jin, Chun-Yan; Sun, Rui-Feng; Fan, Xiao-Yong; Guo, Ming; Mai, Jun-Tao; Xu, Wen-Xi; Lin, Qing-Xia; Liu, Jun

2016-02-01

Bacille Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis, is the only vaccine available for tuberculosis (TB) control. However, BCG is not an ideal vaccine and has two major limitations: BCG exhibits highly variable effectiveness against the development of TB both in pediatric and adult populations and can cause disseminated BCG disease in immunocompromised individuals. BCG comprises a number of substrains that are genetically distinct. Whether and how these genetic differences affect BCG efficacy remains largely unknown. In this study, we performed comparative analyses of the virulence and efficacy of 13 BCG strains, representing different genetic lineages, in SCID and BALB/c mice. Our results show that BCG strains of the DU2 group IV (BCG-Phipps, BCG-Frappier, BCG-Pasteur, and BCG-Tice) exhibit the highest levels of virulence, and BCG strains of the DU2 group II (BCG-Sweden, BCG-Birkhaug) are among the least virulent group. These distinct levels of virulence may be explained by strain-specific duplications and deletions of genomic DNA. There appears to be a general trend that more virulent BCG strains are also more effective in protection against Mycobacterium tuberculosis challenge. Our findings have important implications for current BCG vaccine programs and for future TB vaccine development.

Genetic variation within and between strains of outbred Swiss mice.

PubMed

Cui, S; Chesson, C; Hope, R

1993-04-01

The aim of this survey was to measure levels of genetic variation within and between 5 different strains of outbred Swiss mice. Ten to 15 animals from each strain (NIH, Q(S), ARC, IMVS and STUD) were typed, using allozyme electrophoresis, at 10 gene loci: Mod-1, Idh-1, Gpi-I, Es-1, Es-3, Hbb, Pep-3, Gr-1, Got-2 and Pgm-1. Polymorphic variation in at least one of the 5 strains was detected at all 10 loci. The proportion of polymorphic loci ranged from 0.3 (NIH) to 0.8 (IMVS) with a mean of 0.52. Average expected heterozygosities ranged from 0.08 (NIH) to 0.37 (IMVS) with a mean of 0.21. The inbred strain SWR was, as expected, homozygous at all 10 loci. The amount of allelic substitution between pairs of strains was quantified using Nei's genetic distance, and a dendrogram based on these genetic distances showed a close overall similarity in its branching pattern to the known genealogy of the strains. This survey showed that a considerable degree of genetic variation persists in the 5 strains examined, a level of variation similar to that previously detected by Rice and O'Brien (1980) in 3 other outbred Swiss strains.

A novel HRM assay for differentiating classical strains and highly pathogenic strains of type 2 porcine reproductive and respiratory syndrome virus.

PubMed

Sun, Junying; Bingga, Gali; Liu, Zhicheng; Zhang, Chunhong; Shen, Haiyan; Guo, Pengju; Zhang, Jianfeng

2018-06-01

Differentiation of classical strains and highly pathogenic strains of porcine reproductive and respiratory syndrome virus is crucial for effective vaccination programs and epidemiological studies. We used nested PCR and high resolution melting curve analysis with unlabeled probe to distinguish between the classical and the highly pathogenic strains of this virus. Two sets of primers and a 20 bp unlabeled probe were designed from the NSP3 gene. The unlabeled probe included two mutations specific for the classical and highly pathogenic strains of the virus. An additional primer set from the NSP2 gene of the highly pathogenic vaccine strain JXA1-R was used to detect its exclusive single nucleotide polymorphism. We tested 107 clinical samples, 21 clinical samples were positive for PRRSV (consistent with conventional PCR assay), among them four were positive for the classical strain with the remainder 17 for the highly pathogenic strain. Around 10 °C difference between probe melting temperatures showed the high discriminatory power of this method. Among highly pathogenic positive samples, three samples were determined as positive for JXA1-R vaccine-related strain with a 95% genotype confidence percentage. All these genotyping results using the high resolution melting curve assay were confirmed with DNA sequencing. This unlabeled probe method provides an alternative means to differentiate the classical strains from the highly pathogenic porcine reproductive and respiratory syndrome virus strains rapidly and accurately. Copyright © 2018. Published by Elsevier Ltd.

Clostridial Strain-Specific Characteristics Associated with Necrotizing Enterocolitis.

PubMed

Schönherr-Hellec, Sophia; Klein, Geraldine L; Delannoy, Johanne; Ferraris, Laurent; Rozé, Jean Christophe; Butel, Marie José; Aires, Julio

2018-04-01

We aimed at identifying potential bacterial factors linking clostridia with necrotizing enterocolitis (NEC). We compared the phenotypic traits, stress responses, cellular cytotoxicity, and inflammatory capabilities of the largest collection of Clostridium butyricum and Clostridium neonatale strains isolated from fecal samples of NEC preterm neonates (PN) and control PNs. When strain characteristics were used as explanatory variables, a statistical discriminant analysis allowed the separation of NEC and control strains into separate groups. Strains isolated from NEC PN were characterized by a higher viability at 30°C ( P = 0.03) and higher aerotolerance ( P = 0.01), suggesting that NEC strains may have a competitive and/or survival advantage in the environmental gastrointestinal tract conditions of NEC PN. Heat-treated NEC bacteria induced higher production of interleukin-8 in Caco-2 cells ( P = 0.03), suggesting proinflammatory activity. In vitro , bacteria, bacterial components, and fecal filtrates showed variable cytotoxic effects affecting the cellular network and/or cell viability, without specific association with NEC or control samples. Altogether, our data support the existence of a specific clostridial strain signature associated with NEC. IMPORTANCE Clostridia are part of the commensal microbiota in preterm neonates (PN). However, microbiota analyses by culture and metagenomics have linked necrotizing enterocolitis (NEC) and intestinal colonization with clostridial species. Nevertheless, little is known about the specific characteristics that may be shared by clostridia associated with NEC compared to commensal clostridia. Therefore, our goal was to identify specific bacterial factors linking clostridial strains with NEC. We report the existence of a specific bacterial signature associated with NEC and propose that activation of the innate immune response may be a unifying causative mechanism for the development of NEC independent of a specific pathogenic organism. The present study provides new insights into NEC pathophysiology that are needed for better diagnostics and strategies for implementing prevention of the disease. Copyright © 2018 American Society for Microbiology.

DNApod: DNA polymorphism annotation database from next-generation sequence read archives.

PubMed

Mochizuki, Takako; Tanizawa, Yasuhiro; Fujisawa, Takatomo; Ohta, Tazro; Nikoh, Naruo; Shimizu, Tokurou; Toyoda, Atsushi; Fujiyama, Asao; Kurata, Nori; Nagasaki, Hideki; Kaminuma, Eli; Nakamura, Yasukazu

2017-01-01

With the rapid advances in next-generation sequencing (NGS), datasets for DNA polymorphisms among various species and strains have been produced, stored, and distributed. However, reliability varies among these datasets because the experimental and analytical conditions used differ among assays. Furthermore, such datasets have been frequently distributed from the websites of individual sequencing projects. It is desirable to integrate DNA polymorphism data into one database featuring uniform quality control that is distributed from a single platform at a single place. DNA polymorphism annotation database (DNApod; http://tga.nig.ac.jp/dnapod/) is an integrated database that stores genome-wide DNA polymorphism datasets acquired under uniform analytical conditions, and this includes uniformity in the quality of the raw data, the reference genome version, and evaluation algorithms. DNApod genotypic data are re-analyzed whole-genome shotgun datasets extracted from sequence read archives, and DNApod distributes genome-wide DNA polymorphism datasets and known-gene annotations for each DNA polymorphism. This new database was developed for storing genome-wide DNA polymorphism datasets of plants, with crops being the first priority. Here, we describe our analyzed data for 679, 404, and 66 strains of rice, maize, and sorghum, respectively. The analytical methods are available as a DNApod workflow in an NGS annotation system of the DNA Data Bank of Japan and a virtual machine image. Furthermore, DNApod provides tables of links of identifiers between DNApod genotypic data and public phenotypic data. To advance the sharing of organism knowledge, DNApod offers basic and ubiquitous functions for multiple alignment and phylogenetic tree construction by using orthologous gene information.

DNApod: DNA polymorphism annotation database from next-generation sequence read archives

PubMed Central

Mochizuki, Takako; Tanizawa, Yasuhiro; Fujisawa, Takatomo; Ohta, Tazro; Nikoh, Naruo; Shimizu, Tokurou; Toyoda, Atsushi; Fujiyama, Asao; Kurata, Nori; Nagasaki, Hideki; Kaminuma, Eli; Nakamura, Yasukazu

2017-01-01

With the rapid advances in next-generation sequencing (NGS), datasets for DNA polymorphisms among various species and strains have been produced, stored, and distributed. However, reliability varies among these datasets because the experimental and analytical conditions used differ among assays. Furthermore, such datasets have been frequently distributed from the websites of individual sequencing projects. It is desirable to integrate DNA polymorphism data into one database featuring uniform quality control that is distributed from a single platform at a single place. DNA polymorphism annotation database (DNApod; http://tga.nig.ac.jp/dnapod/) is an integrated database that stores genome-wide DNA polymorphism datasets acquired under uniform analytical conditions, and this includes uniformity in the quality of the raw data, the reference genome version, and evaluation algorithms. DNApod genotypic data are re-analyzed whole-genome shotgun datasets extracted from sequence read archives, and DNApod distributes genome-wide DNA polymorphism datasets and known-gene annotations for each DNA polymorphism. This new database was developed for storing genome-wide DNA polymorphism datasets of plants, with crops being the first priority. Here, we describe our analyzed data for 679, 404, and 66 strains of rice, maize, and sorghum, respectively. The analytical methods are available as a DNApod workflow in an NGS annotation system of the DNA Data Bank of Japan and a virtual machine image. Furthermore, DNApod provides tables of links of identifiers between DNApod genotypic data and public phenotypic data. To advance the sharing of organism knowledge, DNApod offers basic and ubiquitous functions for multiple alignment and phylogenetic tree construction by using orthologous gene information. PMID:28234924

Biogeography of Oenococcus oeni Reveals Distinctive but Nonspecific Populations in Wine-Producing Regions

PubMed Central

El Khoury, Mariette; Campbell-Sills, Hugo; Salin, Franck; Guichoux, Erwan; Claisse, Olivier

2016-01-01

ABSTRACT Understanding the mechanisms behind the typicity of regional wines inevitably brings attention to microorganisms associated with their production. Oenococcus oeni is the main bacterial species involved in wine and cider making. It develops after the yeast-driven alcoholic fermentation and performs the malolactic fermentation, which improves the taste and aromatic complexity of most wines. Here, we have evaluated the diversity and specificity of O. oeni strains in six regions. A total of 235 wines and ciders were collected during spontaneous malolactic fermentations and used to isolate 3,212 bacterial colonies. They were typed by multilocus variable analysis, which disclosed a total of 514 O. oeni strains. Their phylogenetic relationships were evaluated by a second typing method based on single nucleotide polymorphism (SNP) analysis. Taken together, the results indicate that each region holds a high diversity of strains that constitute a unique population. However, strains present in each region belong to diverse phylogenetic groups, and the same groups can be detected in different regions, indicating that strains are not genetically adapted to regions. In contrast, greater strain identity was seen for cider, white wine, or red wine of Burgundy, suggesting that genetic adaptation to these products occurred. IMPORTANCE This study reports the isolation, genotyping, and geographic distribution analysis of the largest collection of O. oeni strains performed to date. It reveals that there is very high diversity of strains in each region, the majority of them being detected in a single region. The study also reports the development of an SNP genotyping method that is useful for analyzing the distribution of O. oeni phylogroups. The results show that strains are not genetically adapted to regions but to specific types of wines. They reveal new phylogroups of strains, particularly two phylogroups associated with white wines and red wines of Burgundy. Taken together, the results shed light on the diversity and specificity of wild strains of O. oeni, which is crucial for understanding their real contribution to the unique properties of wines. PMID:27864168

Biogeography of Oenococcus oeni Reveals Distinctive but Nonspecific Populations in Wine-Producing Regions.

PubMed

El Khoury, Mariette; Campbell-Sills, Hugo; Salin, Franck; Guichoux, Erwan; Claisse, Olivier; Lucas, Patrick M

2017-02-01

Understanding the mechanisms behind the typicity of regional wines inevitably brings attention to microorganisms associated with their production. Oenococcus oeni is the main bacterial species involved in wine and cider making. It develops after the yeast-driven alcoholic fermentation and performs the malolactic fermentation, which improves the taste and aromatic complexity of most wines. Here, we have evaluated the diversity and specificity of O. oeni strains in six regions. A total of 235 wines and ciders were collected during spontaneous malolactic fermentations and used to isolate 3,212 bacterial colonies. They were typed by multilocus variable analysis, which disclosed a total of 514 O. oeni strains. Their phylogenetic relationships were evaluated by a second typing method based on single nucleotide polymorphism (SNP) analysis. Taken together, the results indicate that each region holds a high diversity of strains that constitute a unique population. However, strains present in each region belong to diverse phylogenetic groups, and the same groups can be detected in different regions, indicating that strains are not genetically adapted to regions. In contrast, greater strain identity was seen for cider, white wine, or red wine of Burgundy, suggesting that genetic adaptation to these products occurred. This study reports the isolation, genotyping, and geographic distribution analysis of the largest collection of O. oeni strains performed to date. It reveals that there is very high diversity of strains in each region, the majority of them being detected in a single region. The study also reports the development of an SNP genotyping method that is useful for analyzing the distribution of O. oeni phylogroups. The results show that strains are not genetically adapted to regions but to specific types of wines. They reveal new phylogroups of strains, particularly two phylogroups associated with white wines and red wines of Burgundy. Taken together, the results shed light on the diversity and specificity of wild strains of O. oeni, which is crucial for understanding their real contribution to the unique properties of wines. Copyright © 2017 American Society for Microbiology.

A PCR marker linked to a THCA synthase polymorphism is a reliable tool to discriminate potentially THC-rich plants of Cannabis sativa L.

PubMed

Staginnus, Christina; Zörntlein, Siegfried; de Meijer, Etienne

2014-07-01

Neither absolute THC content nor morphology allows the unequivocal discrimination of fiber cultivars and drug strains of Cannabis sativa L. unequivocally. However, the CBD/THC ratio remains constant throughout the plant's life cycle, is independent of environmental factors, and considered to be controlled by a single locus (B) with two codominant alleles (B(T) and B(D)). The homozygous B(T)/B(T) genotype underlies the THC-predominant phenotype, B(D)/B(D) is CBD predominant, and an intermediate phenotype is induced by the heterozygous state (B(T)/B(D)). Using PCR-based markers in two segregating populations, we proved that the THCA synthase gene represents the postulated B locus and that specific sequence polymorphisms are absolutely linked either to the THC-predominant or the THC-intermediate chemotype. The absolute linkage provides an excellent reliability of the marker signal in forensic casework. For validation, the species-specific marker system was applied to a large number of casework samples and fiber hemp cultivars. © 2014 American Academy of Forensic Sciences.

Genomic diversity of Mycobacterium tuberculosis Beijing strains isolated in Tuscany, Italy, based on large sequence deletions, SNPs in putative DNA repair genes and MIRU-VNTR polymorphisms.

PubMed

Garzelli, Carlo; Lari, Nicoletta; Rindi, Laura

2016-03-01

The Beijing genotype of Mycobacterium tuberculosis is cause of global concern as it is rapidly spreading worldwide, is considered hypervirulent, and is most often associated to massive spread of MDR/XDR TB, although these epidemiological or pathological properties have not been confirmed for all strains and in all geographic settings. In this paper, to gain new insights into the biogeographical heterogeneity of the Beijing family, we investigated a global sample of Beijing strains (22% from Italian-born, 78% from foreign-born patients) by determining large sequence polymorphism of regions RD105, RD181, RD150 and RD142, single nucleotide polymorphism of putative DNA repair genes mutT4 and mutT2 and MIRU-VNTR profiles based on 11 discriminative loci. We found that, although our sample of Beijing strains showed a considerable genomic heterogeneity, yielding both ancient and recent phylogenetic strains, the prevalent successful Beijing subsets were characterized by deletions of RD105 and RD181 and by one nucleotide substitution in one or both mutT genes. MIRU-VNTR analysis revealed 47 unique patterns and 9 clusters including a total of 33 isolates (41% of total isolates); the relatively high proportion of Italian-born Beijing TB patients, often occurring in mixed clusters, supports the possibility of an ongoing cross-transmission of the Beijing genotype to autochthonous population. High rates of extra-pulmonary localization and drug-resistance, particularly MDR, frequently reported for Beijing strains in other settings, were not observed in our survey. Copyright © 2015 Elsevier Ltd. All rights reserved.

High resolution melting (HRM) analysis as a new tool for rapid identification of Salmonella enterica serovar Gallinarum biovars Pullorum and Gallinarum.

PubMed

Ren, Xingxing; Fu, Ying; Xu, Chenggang; Feng, Zhou; Li, Miao; Zhang, Lina; Zhang, Jianmin; Liao, Ming

2017-05-01

Salmonella enterica serovar Gallinarum biovars Pullorum and Gallinarum represent the most common causative agents of chicken salmonellosis, which result in high mortality and morbidity throughout the world. It is difficult and laborious to discriminate these diseases based on biochemical or phenotypic methods. Herein, we report the development of a single nucleotide polymorphism (SNP) PCR-high resolution melt (PCR-HRM) assay for the detection and discrimination of both S. Pullorum and S. Gallinarun. The gene rfbS, which encodes a factor involved in the biosynthesis of ADP paratose in serogroup D of Salmonella, has been identified as a robust genetic marker for the identification of S. Pullorum and S. Gallinarun based on polymorphisms at positions 237 and 598. Therefore, PCR-HRM analyses were used to characterize this gene. A total of 15 reference and 33 clinical isolates of Salmonella and related Gram-negative bacteria were detected using 2 sets of primers. Our PCR-HRM assay could distinguish S. Pullorum from S. Gallinarun and other strains using the primer pair SP-237F/237R. Similarly, S. Gallinarun could be distinguished from S. Pullorum and other strains using primer set SG-598F/598R. These 2 assays showed high specificity (100%) for both S. Pullorum and S. Gallinarun; the sensitivity of these 2 assays was at least 100-fold greater than that of the allele-specific PCR assay. This present study demonstrated that HRM analysis represents a potent, simple, and economic tool for the rapid, specific, and sensitive detection of S. Pullorum and S. Gallinarun. Our approach also may aid efforts for purification of Avian Salmonella disease. © 2016 Poultry Science Association Inc.

Characterization of Mycobacterium leprae Genotypes in China--Identification of a New Polymorphism C251T in the 16S rRNA Gene.

PubMed

Yuan, Youhua; Wen, Yan; You, Yuangang; Xing, Yan; Li, Huanying; Weng, Xiaoman; Wu, Nan; Liu, Shuang; Zhang, Shanshan; Zhang, Wenhong; Zhang, Ying

2015-01-01

Leprosy continues to be prevalent in some mountainous regions of China, and genotypes of leprosy strains endemic to the country are not known. Mycobacterium lepromatosis is a new species that was discovered in Mexico in 2008, and it remains unclear whether this species exists in China. Here, we conducted PCR- restriction fragment length polymorphism (RFLP) analysis to classify genotypes of 85 DNA samples collected from patients from 18 different provinces. All 171 DNA samples from skin biopsies of leprosy patients were tested for the presence of Mycobacterium leprae and Mycobacterium lepromatosis by amplifying the 16S rRNA gene using nested PCR, followed by DNA sequencing. The new species M. lepromatosis was not found among the 171 specimens from leprosy patients in 22 provinces in China. However, we found three SNP genotypes among 85 leprosy patients. A mutation at C251T in the 16S rRNA gene was found in 76% of the strains. We also found that the strains that showed the 16S rRNA C251T mutation belonged to SNP type 3, whereas strains without the point mutation belonged to SNP type 1. The SNP type 3 leprosy strains were observed in patients from both the inner and coastal regions of China, but the SNP type 1 strains were focused only in the coastal region. This indicated that the SNP type 3 leprosy strains were more prevalent than the SNP type 1 strains in China. In addition, the 16S rRNA gene sequence mutation at C251T also indicated a difference in the geographical distribution of the strains. To our knowledge, this is the first report of a new polymorphism in 16S rRNA gene in M. leprae in China. Our findings shed light on the prevalent genotypes and provide insight about leprosy transmission that are important for leprosy control in China.

Two approaches to biological decontamination of groundwater and soil polluted by aromatics-characterization of microbial populations.

PubMed

Demnerová, Katerina; Mackova, Martina; Spevákova, Veronika; Beranova, Katarina; Kochánková, Lucie; Lovecká, Petra; Ryslavá, Edita; Macek, Tomas

2005-09-01

As part of the EU project MULTIBARRIERS, six new endogenous aerobic bacterial isolates able to grow in the presence of BTmX (benzene, toluene, m-xylene) were characterized with respect to their growth specificities. Preliminary analysis included restriction fragment length polymorphism profiles and 16S rDNA sequencing. The diversity of these strains was confirmed by denaturing gradient gel electrophoresis. Additional aerobic bacterial strains were isolated from the rhizospheres of plants grown in polychlorinated biphenyl (PCB)-contaminated soils. Pot experiments were designed to show the beneficial effect of plants on the bacterial degradation of PCBs. The effect of PCB removal from soil was evaluated and bacteria isolated from three different plant species were examined for the presence of the bph operon.

Simultaneous Differentiation and Typing of Entamoeba histolytica and Entamoeba dispar

PubMed Central

Zaki, Mehreen; Meelu, Parool; Sun, Wei; Clark, C. Graham

2002-01-01

Sequences corresponding to some of the polymorphic loci previously reported from Entamoeba histolytica have been detected in Entamoeba dispar. Comparison of nucleotide sequences of two loci between E. dispar strain SAW760 and E. histolytica strain HM-1:IMSS revealed significant differences in both repeat and flanking regions. The tandem repeat units varied not only in sequence but also in number and arrangement between the two species at both the loci. Using the sequences obtained, primer pairs aimed at amplifying species-specific products were designed and tested on a variety of E. histolytica and E. dispar samples. Amplification results were in complete agreement with the original species classification in all cases, and the PCR products displayed discernible size and pattern variations among the isolates. PMID:11923344

The biological and practical significance of antigenic variability in protective T cell responses against Theileria parva.

PubMed

Morrison, W I

2007-08-19

The evolution of antigenically distinct pathogen strains that fail to cross-protect is well documented for pathogens controlled primarily by humoral immune responses. Unlike antibodies, which recognise native proteins, protective T cells can potentially recognise epitopes in a variety of proteins that are not necessarily displayed on the pathogen surface. Moreover, individual hosts of different MHC genotypes generally respond to different sets of epitopes. It is therefore less easy to envisage how strain restricted immunity can arise for pathogens controlled by T cell responses, particularly in antigenically complex parasites. Nevertheless, strain restricted immunity is clearly a feature of a number of parasitic infections, where immunity is known to be mediated by T cell responses. One such parasite is Theileria parva which induces potent CD8 T cell responses that play an important role in immunity. CD8 T cells specific for parasitized lymphoblasts exhibit strain specificity, which appears to correlate with the ability of parasite strains to cross-protect. Studies using recently identified T. parva antigens recognised by CD8 T cells have shown that the strain restricted nature of immunity is a consequence of the CD8 T cell response in individual animals being focused on a limited number of dominant polymorphic antigenic determinants. Responses in animals of different MHC genotypes are often directed to different parasite antigens, indicating that, at the host population level, a larger number of parasite proteins can serve as targets for the protective T cell response. Nevertheless, the finding that parasite strains show overlapping antigenic profiles, probably as a consequence of sexual recombination, suggests that induction of responses to an extended but limited set of antigens in individual animals may overcome the strain restricted nature of immunity.

Epidemiology, Relative Invasive Ability, Molecular Characterization, and Competitive Performance of Campylobacter jejuni Strains in the Chicken Gut▿

PubMed Central

Pope, Christopher; Wilson, Janet; Taboada, Eduardo N.; MacKinnon, Joanne; Felipe Alves, Cristiano A.; Nash, John H. E.; Rahn, Kris; Tannock, Gerald W.

2007-01-01

One hundred forty-one Campylobacter jejuni isolates from humans with diarrhea and 100 isolates from retailed poultry meat were differentiated by flaA typing. The bacteria were isolated in a specific geographical area (Dunedin) in New Zealand over a common time period. Twenty nine flaA types were detected, one of which (flaA restriction fragment length polymorphism type 15 [flaA-15]) predominated among isolates from humans (∼30% of isolates). This strain was of low prevalence (5% of isolates) among poultry isolates. flaA-15 strains were five to six times more invasive of HEp2 cells in an in vitro assay than a flaA type (flaA-3) that was commonly encountered on poultry meat (23% of isolates) but was seldom associated with human illness (5%). Competitive-exclusion experiments with chickens, utilizing real-time quantitative PCR to measure the population sizes of specific strains representing flaA-15 (T1016) and flaA-3 (Pstau) in digesta, were carried out. These experiments showed that T1016 always outcompeted Pstau in the chicken intestine. Genomic comparisons of T1016 and Pstau were made using DNA microarrays representing the genome of C. jejuni NCTC 11168. These comparisons revealed differences between the strains in the gene content of the Cj1417c-to-Cj1442c region of the genome, which is associated with the formation of capsular polysaccharide. The strains differed in Penner type (T1016, O42; Pstau, O53). It was concluded that poultry meat was at least one source of human infection with C. jejuni, that some Campylobacter strains detected in poultry meat are of higher virulence for humans than others, and that bacterial attributes affecting strain virulence and commensal colonization ability may be linked. PMID:17921281

Molecular and antimicrobial susceptibility characterization of Globicatella sulfidifaciens isolated from sow's urinary tract infection.

PubMed

Matajira, Carlos E C; Moreno, Luisa Z; Gomes, Vasco T M; Silva, Ana Paula S; Mesquita, Renan E; Amigo, Cristina R; Christ, Ana Paula G; Sato, Maria Inês Z; Moreno, Andrea M

2017-12-01

The Globicatella genus comprises Gram-positive, facultative anaerobic, α-hemolytic and catalase negative cocci morphologically and phenotypically very similar to Streptococcus and Aerococcus genus which can lead to misidentification and underestimation of this pathogen. Globicatella species have already been isolated from human and animals with heart and brain disorders. Their clinical relevance in animals, and its zoonotic potential, remains unknown due to the difficulty in their identification. To present the isolation, phenotypic and molecular characterization of G. sulfidifaciens from urinary tract infection in sows. Urine samples from 140 sows of two swine herds located in São Paulo State (Brazil) yielded the isolation of three presumptive G. sulfidifaciens strains. Identification and species confirmation were done by MALDI-TOF MS and 16S rRNA sequencing. Strains were further characterized by single enzyme amplified fragments length polymorphism (SE-AFLP) and broth microdilution techniques. All three isolates were confirmed as G. sulfidifaciens. The SE-AFLP genotyping resulted in distinct fingerprint patterns for each strain. All isolates presented high MIC values to tetracycline, sulphonamides, aminoglycosides and tylosin tartrate, which present high usage in human and animal medicine. Globicatella sulfidifaciens could be related to sporadic urinary tract infections in swine and appear to present alarming antimicrobial susceptibility profile. It is necessary to differentiate Streptococcus-like microorganisms in routine laboratory diagnostics for the correct identification of underestimated species potentially pathogenic to animals.

Specific Primers for Rapid Detection of Microsporum audouinii by PCR in Clinical Samples▿

PubMed Central

Roque, H. D.; Vieira, R.; Rato, S.; Luz-Martins, M.

2006-01-01

This report describes application of PCR fingerprinting to identify common species of dermatophytes using the microsatellite primers M13, (GACA)4, and (GTG)5. The initial PCR analysis rendered a specific DNA fragment for Microsporum audouinii, which was cloned and sequenced. Based on the sequencing data of this fragment, forward (MA_1F) and reverse (MA_1R) primers were designed and verified by PCR to establish their reliability in the diagnosis of M. audouinii. These primers produced a singular PCR band of 431 bp specific only to strains and isolates of M. audouinii, based on a global test of 182 strains/isolates belonging to 11 species of dermatophytes. These findings indicate these primers are reliable for diagnostic purposes, and we recommend their use in laboratory analysis. PMID:17005755

Specific primers for rapid detection of Microsporum audouinii by PCR in clinical samples.

PubMed

Roque, H D; Vieira, R; Rato, S; Luz-Martins, M

2006-12-01

This report describes application of PCR fingerprinting to identify common species of dermatophytes using the microsatellite primers M13, (GACA)4, and (GTG)5. The initial PCR analysis rendered a specific DNA fragment for Microsporum audouinii, which was cloned and sequenced. Based on the sequencing data of this fragment, forward (MA_1F) and reverse (MA_1R) primers were designed and verified by PCR to establish their reliability in the diagnosis of M. audouinii. These primers produced a singular PCR band of 431 bp specific only to strains and isolates of M. audouinii, based on a global test of 182 strains/isolates belonging to 11 species of dermatophytes. These findings indicate these primers are reliable for diagnostic purposes, and we recommend their use in laboratory analysis.

Serotonin transporter gene polymorphism and psychiatric disorders: Is there a link?

PubMed Central

Margoob, Mushtaq A.; Mushtaq, Dhuha

2011-01-01

Though still in infancy, the field of psychiatric genetics holds great potential to contribute to the development of new diagnostic and therapeutic options to treat these disorders. Among a large number of existing neurotransmitter systems, the serotonin system dysfunction has been implicated in many psychiatric disorders and therapeutic efficacy of many drugs is also thought to be based on modulation of serotonin. Serotonin transporter gene polymorphism is one of the most extensively studied polymorphisms in psychiatric behavioral genetics. In this article, we review the status of evidence for association between the serotonin gene polymorphism and some common mental disorders like affective disorders, post-traumatic stress disorder, obsessive-compulsive disorder, suicide, autism, and other anxiety and personality disorders. Going beyond traditional association studies, gene-environment interaction, currently gaining momentum, is also discussed in the review. While the existing information of psychiatric genetics is inadequate for putting into practice genetic testing in the diagnostic work-up of the psychiatric patient, if consistent in future research attempts, such results can be of great help to improve the clinical care of a vast majority of patients suffering from such disorders. PMID:22303036

Evidence for a purifying selection acting on the β-lactamase locus in epidemic clones of methicillin-resistant Staphylococcus aureus.

PubMed

Milheiriço, Catarina; Portelinha, Ana; Krippahl, Ludwig; de Lencastre, Hermínia; Oliveira, Duarte C

2011-04-15

The β-lactamase (bla) locus, which confers resistance to penicillins only, may control the transcription of mecA, the central element of methicillin resistance, which is embedded in a polymorphic heterelogous chromosomal cassette (the SCCmec element). In order to assess the eventual correlation between bla allotypes and genetic lineages, SCCmec types and/or β-lactam resistance phenotypes, the allelic variation on the bla locus was evaluated in a representative collection of 54 international epidemic methicillin-resistant Staphylococcus aureus (MRSA) clinical strains and, for comparative purposes, also in 24 diverse methicillin-susceptible S. aureus (MSSA) strains. Internal fragments of blaZ (the β-lactamase structural gene) were sequenced for all strains. A subset of strains, representative of blaZ allotypes, was further characterized by sequencing of internal fragments of the blaZ transcriptional regulators, blaI and blaR1. Thirteen allotypes for blaZ, nine for blaI and 12 for blaR1 were found. In a total of 121 unique single-nucleotide polymorphisms (SNP) detected, no frameshift mutations were identified and only one nonsense mutation within blaZ was found in a MRSA strain. On average, blaZ alleles were more polymorphic among MSSA than in MRSA (14.7 vs 11.4 SNP/allele). Overall, blaR1 was the most polymorphic gene with an average of 24.8 SNP/allele. No correlation could be established between bla allotypes and genetic lineages, SCCmec types and/or β-lactam resistance phenotypes. In order to estimate the selection pressure acting on the bla locus, the average dN/dS values were computed. In the three genes and in both collections dN/dS ratios were significantly below 1. The data strongly suggests the existence of a purifying selection to maintain the bla locus fully functional even on MRSA strains. Although, this is in agreement with the notion that in most clinical MRSA strains mecA gene is under the control of the bla regulatory genes, these findings also suggest that the apparently redundant function of blaZ gene for the MRSA resistant phenotype is still important for these strains. In addition, the data shows that the sensor-inducer blaR1 is the primary target for the accumulation of mutations in the bla locus, presumably to modulate the response to the presence of β-lactam antibiotic.

Rapid identification of genes controlling virulence and immunity in malaria parasites

PubMed Central

Xangsayarath, Phonepadith; Tang, Jianxia; Yahata, Kazuhide; Zoungrana, Augustin; Mitaka, Hayato; Acharjee, Arita; Datta, Partha P.; Hunt, Paul; Carter, Richard; Kaneko, Osamu; Mustonen, Ville; Pain, Arnab

2017-01-01

Identifying the genetic determinants of phenotypes that impact disease severity is of fundamental importance for the design of new interventions against malaria. Here we present a rapid genome-wide approach capable of identifying multiple genetic drivers of medically relevant phenotypes within malaria parasites via a single experiment at single gene or allele resolution. In a proof of principle study, we found that a previously undescribed single nucleotide polymorphism in the binding domain of the erythrocyte binding like protein (EBL) conferred a dramatic change in red blood cell invasion in mutant rodent malaria parasites Plasmodium yoelii. In the same experiment, we implicated merozoite surface protein 1 (MSP1) and other polymorphic proteins, as the major targets of strain-specific immunity. Using allelic replacement, we provide functional validation of the substitution in the EBL gene controlling the growth rate in the blood stages of the parasites. PMID:28704525

A Simple Sequence Repeat- and Single-Nucleotide Polymorphism-Based Genetic Linkage Map of the Brown Planthopper, Nilaparvata lugens

PubMed Central

Jairin, Jirapong; Kobayashi, Tetsuya; Yamagata, Yoshiyuki; Sanada-Morimura, Sachiyo; Mori, Kazuki; Tashiro, Kosuke; Kuhara, Satoru; Kuwazaki, Seigo; Urio, Masahiro; Suetsugu, Yoshitaka; Yamamoto, Kimiko; Matsumura, Masaya; Yasui, Hideshi

2013-01-01

In this study, we developed the first genetic linkage map for the major rice insect pest, the brown planthopper (BPH, Nilaparvata lugens). The linkage map was constructed by integrating linkage data from two backcross populations derived from three inbred BPH strains. The consensus map consists of 474 simple sequence repeats, 43 single-nucleotide polymorphisms, and 1 sequence-tagged site, for a total of 518 markers at 472 unique positions in 17 linkage groups. The linkage groups cover 1093.9 cM, with an average distance of 2.3 cM between loci. The average number of marker loci per linkage group was 27.8. The sex-linkage group was identified by exploiting X-linked and Y-specific markers. Our linkage map and the newly developed markers used to create it constitute an essential resource and a useful framework for future genetic analyses in BPH. PMID:23204257

Pneumonic Plague Outbreak, Northern Madagascar, 2011

PubMed Central

Richard, Vincent; Herindrainy, Perlinot; Soanandrasana, Rahelinirina; Ratsitoharina, Maherisoa; Rakotomanana, Fanjasoa; Andrianalimanana, Samuel; Scholz, Holger C.; Rajerison, Minoarisoa

2015-01-01

Yersinia pestis, the causative agent of plague, is endemic to Madagascar, particularly to the central highlands. Although plague has not been previously reported in northern Madagascar, an outbreak of pneumonic plague occurred in this remote area in 2011. Over a 27-day period, 17 suspected, 2 presumptive, and 3 confirmed human cases were identified, and all 15 untreated 20 patients died. Molecular typing of Y. pestis isolated from 2 survivors and 5 Rattus rattus rat samples identified the Madagascar-specific 1.ORI3-k single-nucleotide polymorphism genotype and 4 clustered regularly interspaced short palindromic repeat patterns. This outbreak had a case-fatality rate of 100% for nontreated patients. The Y. pestis 1.ORI3-k single-nucleotide polymorphism genotype might cause larger epidemics. Multidrug-resistant strains and persistence of the pathogen in natural foci near human settlements pose severe risks to populations in plague-endemic regions and require outbreak response strategies. PMID:25530466

Genomic comparison of the closely-related Salmonella enterica serovars enteritidis, dublin and gallinarum

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

Matthews, T. David; Schmieder, Robert; Silva, Genivaldo G. Z.

The Salmonella enterica serovars Enteritidis, Dublin, and Gallinarum are closely related but differ in virulence and host range. To identify the genetic elements responsible for these differences and to better understand how these serovars are evolving, we sequenced the genomes of Enteritidis strain LK5 and Dublin strain SARB12 and compared these genomes to the publicly available Enteritidis P125109, Dublin CT 02021853 and Dublin SD3246 genome sequences. We also compared the publicly available Gallinarum genome sequences from biotype Gallinarum 287/91 and Pullorum RKS5078. Using bioinformatic approaches, we identified single nucleotide polymorphisms, insertions, deletions, and differences in prophage and pseudogene content betweenmore » strains belonging to the same serovar. Through our analysis we also identified several prophage cargo genes and pseudogenes that affect virulence and may contribute to a host-specific, systemic lifestyle. These results strongly argue that the Enteritidis, Dublin and Gallinarum serovars of Salmonella enterica evolve by acquiring new genes through horizontal gene transfer, followed by the formation of pseudogenes. As a result, the loss of genes necessary for a gastrointestinal lifestyle ultimately leads to a systemic lifestyle and niche exclusion in the host-specific serovars.« less

Genomic comparison of the closely-related Salmonella enterica serovars enteritidis, dublin and gallinarum

DOE PAGES

Matthews, T. David; Schmieder, Robert; Silva, Genivaldo G. Z.; ...

2015-06-03

The Salmonella enterica serovars Enteritidis, Dublin, and Gallinarum are closely related but differ in virulence and host range. To identify the genetic elements responsible for these differences and to better understand how these serovars are evolving, we sequenced the genomes of Enteritidis strain LK5 and Dublin strain SARB12 and compared these genomes to the publicly available Enteritidis P125109, Dublin CT 02021853 and Dublin SD3246 genome sequences. We also compared the publicly available Gallinarum genome sequences from biotype Gallinarum 287/91 and Pullorum RKS5078. Using bioinformatic approaches, we identified single nucleotide polymorphisms, insertions, deletions, and differences in prophage and pseudogene content betweenmore » strains belonging to the same serovar. Through our analysis we also identified several prophage cargo genes and pseudogenes that affect virulence and may contribute to a host-specific, systemic lifestyle. These results strongly argue that the Enteritidis, Dublin and Gallinarum serovars of Salmonella enterica evolve by acquiring new genes through horizontal gene transfer, followed by the formation of pseudogenes. As a result, the loss of genes necessary for a gastrointestinal lifestyle ultimately leads to a systemic lifestyle and niche exclusion in the host-specific serovars.« less

Genomic Comparison of the Closely-Related Salmonella enterica Serovars Enteritidis, Dublin and Gallinarum

PubMed Central

Matthews, T. David; Schmieder, Robert; Silva, Genivaldo G. Z.; Busch, Julia; Cassman, Noriko; Dutilh, Bas E.; Green, Dawn; Matlock, Brian; Heffernan, Brian; Olsen, Gary J.; Farris Hanna, Leigh; Schifferli, Dieter M.; Maloy, Stanley; Dinsdale, Elizabeth A.; Edwards, Robert A.

2015-01-01

The Salmonella enterica serovars Enteritidis, Dublin, and Gallinarum are closely related but differ in virulence and host range. To identify the genetic elements responsible for these differences and to better understand how these serovars are evolving, we sequenced the genomes of Enteritidis strain LK5 and Dublin strain SARB12 and compared these genomes to the publicly available Enteritidis P125109, Dublin CT 02021853 and Dublin SD3246 genome sequences. We also compared the publicly available Gallinarum genome sequences from biotype Gallinarum 287/91 and Pullorum RKS5078. Using bioinformatic approaches, we identified single nucleotide polymorphisms, insertions, deletions, and differences in prophage and pseudogene content between strains belonging to the same serovar. Through our analysis we also identified several prophage cargo genes and pseudogenes that affect virulence and may contribute to a host-specific, systemic lifestyle. These results strongly argue that the Enteritidis, Dublin and Gallinarum serovars of Salmonella enterica evolve by acquiring new genes through horizontal gene transfer, followed by the formation of pseudogenes. The loss of genes necessary for a gastrointestinal lifestyle ultimately leads to a systemic lifestyle and niche exclusion in the host-specific serovars. PMID:26039056

Simple Sequence Repeats in Escherichia coli: Abundance, Distribution, Composition, and Polymorphism

PubMed Central

Gur-Arie, Riva; Cohen, Cyril J.; Eitan, Yuval; Shelef, Leora; Hallerman, Eric M.; Kashi, Yechezkel

2000-01-01

Computer-based genome-wide screening of the DNA sequence of Escherichia coli strain K12 revealed tens of thousands of tandem simple sequence repeat (SSR) tracts, with motifs ranging from 1 to 6 nucleotides. SSRs were well distributed throughout the genome. Mononucleotide SSRs were over-represented in noncoding regions and under-represented in open reading frames (ORFs). Nucleotide composition of mono- and dinucleotide SSRs, both in ORFs and in noncoding regions, differed from that of the genomic region in which they occurred, with 93% of all mononucleotide SSRs proving to be of A or T. Computer-based analysis of the fine position of every SSR locus in the noncoding portion of the genome relative to downstream ORFs showed SSRs located in areas that could affect gene regulation. DNA sequences at 14 arbitrarily chosen SSR tracts were compared among E. coli strains. Polymorphisms of SSR copy number were observed at four of seven mononucleotide SSR tracts screened, with all polymorphisms occurring in noncoding regions. SSR polymorphism could prove important as a genome-wide source of variation, both for practical applications (including rapid detection, strain identification, and detection of loci affecting key phenotypes) and for evolutionary adaptation of microbes.[The sequence data described in this paper have been submitted to the GenBank data library under accession numbers AF209020–209030 and AF209508–209518.] PMID:10645951

Genomic variant representation in a Chlamydia population is dynamic and adaptive with dependence on in vitro and in vivo passage.

PubMed

Jasper, Deana K; Sigar, Ira M; Schripsema, Justin H; Sainvil, Carlyn K; Smith, Christopher L; Yeruva, Laxmi; Rank, Roger G; Murthy, Ashlesh K; Widder, Jared R; Ramsey, Kyle H

2015-02-01

We have previously shown that Chlamydia muridarum has multiple genomic variants that concomitantly vary in their in vitro and in vivo phenotype. Herein, we used real-time polymerase chain reaction-based genotyping assays to query plaque-cloned isolates of C. muridarum for the frequency of eight selected polymorphisms. These strains had no history of passage in vivo since their original isolation from laboratory mice. There was significant variance in the frequency of two of the eight polymorphisms assessed with the remaining exhibiting a low rate of variance. To determine if any of these polymorphisms were more favorable for in vivo conditions, we blindly passaged non-clonal C. muridarum three times at 7-day intervals through the urogenital tract of mice. Seven of the eight polymorphisms varied in frequency following in vivo passage and four of these varied between C. muridarum strains. Selected isolates displayed variable growth rates and cytopathic effect in vitro. We conclude that multiple genotypic variants are present within the existing known C. muridarum strains and that the frequency of these variants changes upon introduction into the mouse host. These findings lend support to the concept that genotypic proportional representation in a chlamydial population is dynamic and adaptive. © The Author 2015. Published by Oxford University Press on behalf of on behalf of Federation of European Microbiological Society.

[Identification of mycobacteria by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry--using reference strains and clinical isolates of Mycobacterium].

PubMed

Niitsuma, Katsunao; Saito, Miwako; Koshiba, Shizuko; Kaneko, Michiyo

2014-05-01

Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) method is being played an important role for the inspection of clinical microorganism as a rapid and the price reduction. Mass spectra obtained by measuring become points of identification whether the peak pattern match any species mass spectral pattern. We currently use MALDI-TOF MS for rapid and accurate diagnosis of inactivated reference and clinical isolates of Mycobacterium because of the improved pretreatment techniques compared with former inspection methods that pose a higher risk of infection to the operator. The identification matching rate of score value (SV) peak pattern spectra was compared with that of conventional methods such as strain diffusion/amplification. Also, cultures were examined after a fixed number of days. Compared with the initial inspection technique, the pretreatment stage of current MALDI-TOF MS inspection techniques can improve the analysis of inactivated acid-fast bacteria that are often used as inspection criteria strains of clinical isolates. Next, we compared the concordance rate for identification between MALDI-TOF MS and conventional methods such as diffusion/amplification by comparison of peak pattern spectra and evaluated SV spectra to identify differences in the culture media after the retention period. In examination of 158 strains of clinical isolated Mycobacterium tuberculosis complex (MTC), the identification coincidence rate in the genus level in a matching pattern was 99.4%, when the species level was included 94.9%. About 37 strains of nontuberculous mycobacteria (NTM), the identification coincidence rate in the genus level was 94.6%. M. bovis BCG (Tokyo strain) in the reference strain was judged by the matching pattern to be MTC, and it suggested that they are M. tuberculosis and affinity species with high DNA homology. Nontuberculous mycobacterial M. gordonae strain JATA 33-01 shared peak pattern spectra, excluding the isolates, with each clinically isolated strain. However, the mass spectra of six M. gordonae clinical isolates suggested polymorphisms with similar mass-to-charge ratios compared with those of the reference strains. The peak pattern spectra of the clinical isolates and reference strains, excluding the NTM M. gordonae strain JATA33-01, were consistent with the peak pattern characteristics of each isolate. However, a comparison between the peak patterns of the reference strains and those of the six clinically isolated M. gordonae strains revealed a similar mass-to-charge ratio, which may indicate few polymorphisms. The SV spectrum of the improved inspection technique showed no fidelity, but it was acceptable after days of culture as indicated by the decrease in SV (0.3 degree). Also, the reproducibility of this method was good, but no difference was observed from the SV of the improved inspection technique, which decreased by approximately 0.3 because of the number of days of culture storage. In addition, expansion of the database and dissemination of regional specificity by genotype analysis of clinical isolates was relevant to the accumulated data, as expected. In future studies, the relevance and regional specificity of clinical isolates by genotype analysis can be determined by stacking the solid media and database penetration.

Rapid bacterial diagnostics via surface enhanced Raman microscopy.

PubMed

Premasiri, W R; Sauer-Budge, A F; Lee, J C; Klapperich, C M; Ziegler, L D

2012-06-01

There is a continuing need to develop new techniques for the rapid and specific identification of bacterial pathogens in human body fluids especially given the increasing prevalence of drug resistant strains. Efforts to develop a surface enhanced Raman spectroscopy (SERS) based approach, which encompasses sample preparation, SERS substrates, portable Raman microscopy instrumentation and novel identification software, are described. The progress made in each of these areas in our laboratory is summarized and illustrated by a spiked infectious sample for urinary tract infection (UTI) diagnostics. SERS bacterial spectra exhibit both enhanced sensitivity and specificity allowing the development of an easy to use, portable, optical platform for pathogen detection and identification. SERS of bacterial cells is shown to offer not only reproducible molecular spectroscopic signatures for analytical applications in clinical diagnostics, but also is a new tool for studying biochemical activity in real time at the outer layers of these organisms.

Diagnostic Assay for Rickettsia japonica

PubMed Central

Hanaoka, Nozomu; Matsutani, Minenosuke; Kawabata, Hiroki; Yamamoto, Seigo; Fujita, Hiromi; Sakata, Akiko; Azuma, Yoshinao; Ogawa, Motohiko; Takano, Ai; Watanabe, Haruo; Kishimoto, Toshio; Shirai, Mutsunori; Kurane, Ichiro

2009-01-01

We developed a specific and rapid detection system for Rickettsia japonica and R. heilongjiangensis, the causative agents of spotted fever, using a TaqMan minor groove binder probe for a particular open reading frame (ORF) identified by the R. japonica genome project. The target ORF was present only in R. japonica–related strains. PMID:19961684

Genetic evolution of Mycoplasma capricolum subsp. capripneumoniae strains and molecular epidemiology of contagious caprine pleuropneumonia by sequencing of locus H2.

PubMed

Lorenzon, S; Wesonga, H; Ygesu, Laikemariam; Tekleghiorgis, Tesfaalem; Maikano, Y; Angaya, M; Hendrikx, P; Thiaucourt, F

2002-03-01

Contagious caprine pleuropneumonia (CCPP) is a major threat to goat farming in developing countries. Its exact distribution is not well known, despite the fact that new diagnostic tools such as PCR and competitive ELISA are now available. The authors developed a study of the molecular epidemiology of the disease, based on the amplification of a 2400 bp long fragment containing two duplicated gene coding for a putative membrane protein. The sequence of this fragment, obtained on 19 Mycoplasma capricolum subsp. capripneumoniae (Mccp) strains from various geographical locations, gave 11 polymorphic positions. The three mutations found on gene H2prim were silent and did not appear to induce any amino acid modifications in the putative translated protein. The second gene may be a pseudogene not translated in vivo, as it bore a deletion of the ATG codon found in the other members of the "Mycoplasma mycoides cluster" and as the six mutations evidenced in the Mccp strains would induce modifications in the translated amino acids. In addition, an Mccp strain isolated in the United Arab Emirates showed a deletion of the whole pseudogene, a further indication that this gene is not compulsory for mycoplasma growth. Four lineages were defined, based on the nucleotide sequence. These correlated relatively well with the geographical origin of the strains: North, Central or East Africa. The strain of Turkish origin had a sequence similar to that found in North African strains, while strains isolated in Oman had sequences similar to those of North or East African strains. The latter is possibly due to the regular import of goats of various origins. Similar molecular epidemiology tools have been developed by sequencing the two operons of the 16S rRNA gene or by AFLP. All these various techniques give complementary results. One (16S rRNA) offers the likelihood of a finer identification of strains circulating in a region, another (H2) of determining the geographical origin of the strains. These tools can make a very useful contribution to understanding the epidemiology of CCPP.

Whole Genome Sequencing Based Characterization of Extensively Drug-Resistant Mycobacterium tuberculosis Isolates from Pakistan

PubMed Central

Ali, Asho; Hasan, Zahra; McNerney, Ruth; Mallard, Kim; Hill-Cawthorne, Grant; Coll, Francesc; Nair, Mridul; Pain, Arnab; Clark, Taane G.; Hasan, Rumina

2015-01-01

Improved molecular diagnostic methods for detection drug resistance in Mycobacterium tuberculosis (MTB) strains are required. Resistance to first- and second- line anti-tuberculous drugs has been associated with single nucleotide polymorphisms (SNPs) in particular genes. However, these SNPs can vary between MTB lineages therefore local data is required to describe different strain populations. We used whole genome sequencing (WGS) to characterize 37 extensively drug-resistant (XDR) MTB isolates from Pakistan and investigated 40 genes associated with drug resistance. Rifampicin resistance was attributable to SNPs in the rpoB hot-spot region. Isoniazid resistance was most commonly associated with the katG codon 315 (92%) mutation followed by inhA S94A (8%) however, one strain did not have SNPs in katG, inhA or oxyR-ahpC. All strains were pyrazimamide resistant but only 43% had pncA SNPs. Ethambutol resistant strains predominantly had embB codon 306 (62%) mutations, but additional SNPs at embB codons 406, 378 and 328 were also present. Fluoroquinolone resistance was associated with gyrA 91–94 codons in 81% of strains; four strains had only gyrB mutations, while others did not have SNPs in either gyrA or gyrB. Streptomycin resistant strains had mutations in ribosomal RNA genes; rpsL codon 43 (42%); rrs 500 region (16%), and gidB (34%) while six strains did not have mutations in any of these genes. Amikacin/kanamycin/capreomycin resistance was associated with SNPs in rrs at nt1401 (78%) and nt1484 (3%), except in seven (19%) strains. We estimate that if only the common hot-spot region targets of current commercial assays were used, the concordance between phenotypic and genotypic testing for these XDR strains would vary between rifampicin (100%), isoniazid (92%), flouroquinolones (81%), aminoglycoside (78%) and ethambutol (62%); while pncA sequencing would provide genotypic resistance in less than half the isolates. This work highlights the importance of expanded targets for drug resistance detection in MTB isolates. PMID:25719196

GETPrime 2.0: gene- and transcript-specific qPCR primers for 13 species including polymorphisms.

PubMed

David, Fabrice P A; Rougemont, Jacques; Deplancke, Bart

2017-01-04

GETPrime (http://bbcftools.epfl.ch/getprime) is a database with a web frontend providing gene- and transcript-specific, pre-computed qPCR primer pairs. The primers have been optimized for genome-wide specificity and for allowing the selective amplification of one or several splice variants of most known genes. To ease selection, primers have also been ranked according to defined criteria such as genome-wide specificity (with BLAST), amplicon size, and isoform coverage. Here, we report a major upgrade (2.0) of the database: eight new species (yeast, chicken, macaque, chimpanzee, rat, platypus, pufferfish, and Anolis carolinensis) now complement the five already included in the previous version (human, mouse, zebrafish, fly, and worm). Furthermore, the genomic reference has been updated to Ensembl v81 (while keeping earlier versions for backward compatibility) as a result of re-designing the back-end database and automating the import of relevant sections of the Ensembl database in species-independent fashion. This also allowed us to map known polymorphisms to the primers (on average three per primer for human), with the aim of reducing experimental error when targeting specific strains or individuals. Another consequence is that the inclusion of future Ensembl releases and other species has now become a relatively straightforward task. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Development and evaluation of an anti-rabies virus phosphoprotein-specific monoclonal antibody for detection of rabies neutralizing antibodies using RFFIT

PubMed Central

Chun, Byung Chul; Lee, Yeong Seon; Hwang, Kyu Jam; Yang, Dong-Kun

2017-01-01

Background Rabies is a major public health problem with a fatality rate close to 100%; however, complete prevention can be achieved through pre- or post-exposure prophylaxis. The rapid fluorescent focus inhibition test (RFFIT) is one of the recommended testing methods to determine the production of neutralizing antibodies after vaccination. Here, we report the development of a new monoclonal antibody (mAb) designed to react specifically with Rabies virus (RABV) phosphoprotein (P protein), and the evaluation of its applicability to the RFFIT and its effectiveness as a diagnostic reagent for human rabies. Methodology/principal findings The mAb KGH P 16B8 was produced to target the P protein of the Korean KGH RABV strain. An indirect immunofluorescence assay (IFA) was conducted to detect various strains of RABV in various cell lines. Alexa-conjugated KGH P 16B8 (16B8-Alexa) was developed for the RFFIT. The IFA test could detect RABV up to a 1:2,500 dilution, with a detection limit comparable to that of a commercial diagnostic reagent. The sensitivity, specificity, positive predictive value, and negative predictive value of the RFFIT using 16B8-Alexa in 414 clinical specimens were 98.67%, 99.47%, 99.55%, and 98.42%, respectively. The results of the RFFIT with 16B8-Alexa were strongly correlated with those obtained using an existing commercial diagnostic reagent (r = 0.995, p<0.001). Conclusions/significance The mAb developed in this study shows high sensitivity and specificity, confirming its clinical utility with the RFFIT to measure the rabies neutralizing antibody titer and establish a diagnosis in human. Thus, 16B8-Alexa is expected to serve as an alternative diagnostic reagent that is widely accessible, with potentially broad applications beyond those of the RFFIT in Korea. Further studies with 16B8-Alexa should provide insight into the immunological mechanism of the P protein of Korean RABV. PMID:29267277

Development and evaluation of an anti-rabies virus phosphoprotein-specific monoclonal antibody for detection of rabies neutralizing antibodies using RFFIT.

PubMed

Um, Jihye; Chun, Byung Chul; Lee, Yeong Seon; Hwang, Kyu Jam; Yang, Dong-Kun; Park, Jun-Sun; Kim, Su Yeon

2017-12-01

Rabies is a major public health problem with a fatality rate close to 100%; however, complete prevention can be achieved through pre- or post-exposure prophylaxis. The rapid fluorescent focus inhibition test (RFFIT) is one of the recommended testing methods to determine the production of neutralizing antibodies after vaccination. Here, we report the development of a new monoclonal antibody (mAb) designed to react specifically with Rabies virus (RABV) phosphoprotein (P protein), and the evaluation of its applicability to the RFFIT and its effectiveness as a diagnostic reagent for human rabies. The mAb KGH P 16B8 was produced to target the P protein of the Korean KGH RABV strain. An indirect immunofluorescence assay (IFA) was conducted to detect various strains of RABV in various cell lines. Alexa-conjugated KGH P 16B8 (16B8-Alexa) was developed for the RFFIT. The IFA test could detect RABV up to a 1:2,500 dilution, with a detection limit comparable to that of a commercial diagnostic reagent. The sensitivity, specificity, positive predictive value, and negative predictive value of the RFFIT using 16B8-Alexa in 414 clinical specimens were 98.67%, 99.47%, 99.55%, and 98.42%, respectively. The results of the RFFIT with 16B8-Alexa were strongly correlated with those obtained using an existing commercial diagnostic reagent (r = 0.995, p<0.001). The mAb developed in this study shows high sensitivity and specificity, confirming its clinical utility with the RFFIT to measure the rabies neutralizing antibody titer and establish a diagnosis in human. Thus, 16B8-Alexa is expected to serve as an alternative diagnostic reagent that is widely accessible, with potentially broad applications beyond those of the RFFIT in Korea. Further studies with 16B8-Alexa should provide insight into the immunological mechanism of the P protein of Korean RABV.

φX216, a P2-like bacteriophage with broad Burkholderia pseudomallei and B. mallei strain infectivity.

PubMed

Kvitko, Brian H; Cox, Christopher R; DeShazer, David; Johnson, Shannon L; Voorhees, Kent J; Schweizer, Herbert P

2012-12-07

Burkholderia pseudomallei and B. mallei are closely related Category B Select Agents of bioterrorism and the causative agents of the diseases melioidosis and glanders, respectively. Rapid phage-based diagnostic tools would greatly benefit early recognition and treatment of these diseases. There is extensive strain-to-strain variation in B. pseudomallei genome content due in part to the presence or absence of integrated prophages. Several phages have previously been isolated from B. pseudomallei lysogens, for example φK96243, φ1026b and φ52237. We have isolated a P2-like bacteriophage, φX216, which infects 78% of all B. pseudomallei strains tested. φX216 also infects B. mallei, but not other Burkholderia species, including the closely related B. thailandensis and B. oklahomensis. The nature of the φX216 host receptor remains unclear but evidence indicates that in B. mallei φX216 uses lipopolysaccharide O-antigen but a different receptor in B. pseudomallei. The 37,637 bp genome of φX216 encodes 47 predicted open reading frames and shares 99.8% pairwise identity and an identical strain host range with bacteriophage φ52237. Closely related P2-like prophages appear to be widely distributed among B. pseudomallei strains but both φX216 and φ52237 readily infect prophage carrying strains. The broad strain infectivity and high specificity for B. pseudomallei and B. mallei indicate that φX216 will provide a good platform for the development of phage-based diagnostics for these bacteria.

Single nucleotide polymorphism analysis of the enterocin P structural gene of Enterococcus faecium strains isolated from nonfermented animal foods.

PubMed

Arlindo, Samuel; Calo, Pilar; Franco, Carlos; Prado, Marta; Cepeda, Alberto; Barros-Velázquez, Jorge

2006-12-01

The bacteriocins produced by two lactic acid bacteria isolated from nonfermented fresh meat and fish, respectively, and exhibiting a remarkable antilisterial activity, were characterized. Bacteriocinogenic strains were identified as Enterococcus faecium and the maximum bacteriocin production by both strains was detected in the stationary phase of growth. The activity against Listeria monocytogenes was maintained in pH range of 3-7 and was stable in both strains after heating at 100 or 121 degrees C. The genes coding for enterocin P were detected, isolated, and sequenced in both E. faecium strains. They exhibited DNA/DNA homology in the 87.1-97.2% range with respect to the other four enterocin P genes reported so far. Three single nucleotide polymorphism events, silent at the amino acid level, were detected at nucleotide positions 45 (G/A), 75 (A/G), and 90 (T/C) in E. faecium LHICA 28-4 and may explain the differences reported for those loci in other enterocin P-producing E. faecium strains. This work provides the first description of enterocin P-producing E. faecium strains in nonfermented foodstuffs and, in the case of E. faecium LHICA 51, the first report of an enterocin P-producing strain isolated from fish so far.

Comparison of Quantitative Wall Motion Analysis and Strain For Detection Of Coronary Stenosis With Three-Dimensional Dobutamine Stress Echocardiography

PubMed Central

Parker, Katherine M.; Clark, Alexander P.; Goodman, Norman C.; Glover, David K.; Holmes, Jeffrey W.

2015-01-01

Background Quantitative analysis of wall motion from three-dimensional (3D) dobutamine stress echocardiography (DSE) could provide additional diagnostic information not available from qualitative analysis. In this study we compare the effectiveness of 3D fractional shortening (3DFS), a measure of wall motion computed from 3D echocardiography (3DE), to strain and strain rate measured with sonomicrometry for detecting critical stenoses during DSE. Methods Eleven open-chest dogs underwent DSE both with and without a critical stenosis. 3DFS was measured from 3DE images acquired at peak stress. 3DFS was normalized by subtracting average 3DFS during control peak stress (Δ3DFS). Strains in the perfusion defect (PD) were measured from sonomicrometry, and PD size and location were measured with microspheres. Results A Δ3DFS abnormality indicated the presence of a critical stenosis with high sensitivity and specificity (88% and 100%, respectively), and Δ3DFS abnormality size correlated with PD size (R2=0.54). The sensitivity and specificity for Δ3DFS was similar to that for area strain (88%, 100%) and circumferential strain and strain rate (88%, 92% and 88%, 86%, respectively), while longitudinal strain and strain rate were less specific. Δ3DFS correlated significantly with both coronary flow reserve (R2=0.71) and PD size (R2=0.97), while area strain correlated with PD size only (R2=0.67), and other measures were not significantly correlated with flow reserve or PD size. Conclusion Quantitative wall motion analysis using Δ3DFS is effective for detecting critical stenoses during DSE, performing similarly to 3D strain, and provides potentially useful information on the size and location of a perfusion defect. PMID:24815588

A new approach to ultrasonic elasticity imaging

NASA Astrophysics Data System (ADS)

Hoerig, Cameron; Ghaboussi, Jamshid; Fatemi, Mostafa; Insana, Michael F.

2016-04-01

Biomechanical properties of soft tissues can provide information regarding the local health status. Often the cells in pathological tissues can be found to form a stiff extracellular environment, which is a sensitive, early diagnostic indicator of disease. Quasi-static ultrasonic elasticity imaging provides a way to image the mechanical properties of tissues. Strain images provide a map of the relative tissue stiffness, but ambiguities and artifacts limit its diagnostic value. Accurately mapping intrinsic mechanical parameters of a region may increase diagnostic specificity. However, the inverse problem, whereby force and displacement estimates are used to estimate a constitutive matrix, is ill conditioned. Our method avoids many of the issues involved with solving the inverse problem, such as unknown boundary conditions and incomplete information about the stress field, by building an empirical model directly from measured data. Surface force and volumetric displacement data gathered during imaging are used in conjunction with the AutoProgressive method to teach artificial neural networks the stress-strain relationship of tissues. The Autoprogressive algorithm has been successfully used in many civil engineering applications and to estimate ocular pressure and corneal stiffness; here, we are expanding its use to any tissues imaged ultrasonically. We show that force-displacement data recorded with an ultrasound probe and displacements estimated at a few points in the imaged region can be used to estimate the full stress and strain vectors throughout an entire model while only assuming conservation laws. We will also demonstrate methods to parameterize the mechanical properties based on the stress-strain response of trained neural networks. This method is a fundamentally new approach to medical elasticity imaging that for the first time provides full stress and strain vectors from one set of observation data.

Dopamine D4 receptor VNTR polymorphism associated with greater risk for substance abuse among adolescents with disruptive behavior disorders: Preliminary results.

PubMed

Mallard, Travis T; Doorley, James; Esposito-Smythers, Christianne L; McGeary, John E

2016-01-01

The variable number tandem repeats (VNTR) polymorphism of the dopamine D4 receptor gene (DRD4) has received considerable attention as a potential genetic contributor to addiction. However, is unclear whether the polymorphism is involved in developing general traits that lead to risky behavior or an intermediate phenotype more specific to substance use disorders. Association studies have produced equivocal results. To control for potential confounds, the present study examined whether the long variant of the DRD4 VNTR polymorphism (DRD4L) is associated with greater substance misuse in a homogenous clinical sample of youth with a disruptive behavior disorder (DBD). Fifty-one psychiatrically hospitalized adolescents (mean age = 14.86 years) with a DBD diagnosis were recruited as part of a larger study. Participants provided saliva samples for genotyping procedures after completing a diagnostic interview and an assessment battery. The odds of a substance use disorder diagnosis were significantly greater among DRD4L than DRD4S carriers (OR = 5.20, 95%CI:1.42-19.04, p = .01). Relative to DRD4S homozygotes, DRD4L carriers also reported greater marijuana use (t = -2.68, p = .01) and hard drug use (t = -2.26, p = .03). Although adolescents with DBDs are already at heightened risk for substance misuse, the present findings suggest that DRD4L further increases those odds. As differences persisted even among a psychiatrically homogenous sample of impulsive and risk-prone adolescents, the present findings suggest that DRD4L may be involved in the development of an intermediate phenotype specific to substance abuse (eg, cue-elicited craving). © American Academy of Addiction Psychiatry.

Molecular complexity of successive bacterial epidemics deconvoluted by comparative pathogenomics.

PubMed

Beres, Stephen B; Carroll, Ronan K; Shea, Patrick R; Sitkiewicz, Izabela; Martinez-Gutierrez, Juan Carlos; Low, Donald E; McGeer, Allison; Willey, Barbara M; Green, Karen; Tyrrell, Gregory J; Goldman, Thomas D; Feldgarden, Michael; Birren, Bruce W; Fofanov, Yuriy; Boos, John; Wheaton, William D; Honisch, Christiane; Musser, James M

2010-03-02

Understanding the fine-structure molecular architecture of bacterial epidemics has been a long-sought goal of infectious disease research. We used short-read-length DNA sequencing coupled with mass spectroscopy analysis of SNPs to study the molecular pathogenomics of three successive epidemics of invasive infections involving 344 serotype M3 group A Streptococcus in Ontario, Canada. Sequencing the genome of 95 strains from the three epidemics, coupled with analysis of 280 biallelic SNPs in all 344 strains, revealed an unexpectedly complex population structure composed of a dynamic mixture of distinct clonally related complexes. We discovered that each epidemic is dominated by micro- and macrobursts of multiple emergent clones, some with distinct strain genotype-patient phenotype relationships. On average, strains were differentiated from one another by only 49 SNPs and 11 insertion-deletion events (indels) in the core genome. Ten percent of SNPs are strain specific; that is, each strain has a unique genome sequence. We identified nonrandom temporal-spatial patterns of strain distribution within and between the epidemic peaks. The extensive full-genome data permitted us to identify genes with significantly increased rates of nonsynonymous (amino acid-altering) nucleotide polymorphisms, thereby providing clues about selective forces operative in the host. Comparative expression microarray analysis revealed that closely related strains differentiated by seemingly modest genetic changes can have significantly divergent transcriptomes. We conclude that enhanced understanding of bacterial epidemics requires a deep-sequencing, geographically centric, comparative pathogenomics strategy.

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.

Inheritance of partial resistance against Colletotrichum lindemuthianum in Phaseolus vulgaris and co-localization of quantitative trait loci with genes involved in specific resistance.

PubMed

Geffroy, V; Sévignac, M; De Oliveira, J C; Fouilloux, G; Skroch, P; Thoquet, P; Gepts, P; Langin, T; Dron, M

2000-03-01

Anthracnose, one of the most important diseases of common bean (Phaseolus vulgaris), is caused by the fungus Colletotrichum lindemuthianum. A "candidate gene" approach was used to map anthracnose resistance quantitative trait loci (QTL). Candidate genes included genes for both pathogen recognition (resistance genes and resistance gene analogs [RGAs]) and general plant defense (defense response genes). Two strains of C. lindemuthianum, identified in a world collection of 177 strains, displayed a reproducible and differential aggressiveness toward BAT93 and JaloEEP558, two parental lines of P. vulgaris representing the two major gene pools of this crop. A reliable test was developed to score partial resistance in aerial organs of the plant (stem, leaf, petiole) under controlled growth chamber conditions. BAT93 was more resistant than JaloEEP558 regardless of the organ or strain tested. With a recombinant inbred line (RIL) population derived from a cross between these two parental lines, 10 QTL were located on a genetic map harboring 143 markers, including known defense response genes, anthracnose-specific resistance genes, and RGAs. Eight of the QTL displayed isolate specificity. Two were co-localized with known defense genes (phenylalanine ammonia-lyase and hydroxyproline-rich glycoprotein) and three with anthracnose-specific resistance genes and/or RGAs. Interestingly, two QTL, with different allelic contribution, mapped on linkage group B4 in a 5.0 cM interval containing Andean and Mesoamerican specific resistance genes against C. lindemuthianum and 11 polymorphic fragments revealed with a RGA probe. The possible relationship between genes underlying specific and partial resistance is discussed.

Genetic Polymorphism in Wine Yeasts: Mechanisms and Methods for Its Detection

PubMed Central

Guillamón, José M.; Barrio, Eladio

2017-01-01

The processes of yeast selection for using as wine fermentation starters have revealed a great phenotypic diversity both at interspecific and intraspecific level, which is explained by a corresponding genetic variation among different yeast isolates. Thus, the mechanisms involved in promoting these genetic changes are the main engine generating yeast biodiversity. Currently, an important task to understand biodiversity, population structure and evolutionary history of wine yeasts is the study of the molecular mechanisms involved in yeast adaptation to wine fermentation, and on remodeling the genomic features of wine yeast, unconsciously selected since the advent of winemaking. Moreover, the availability of rapid and simple molecular techniques that show genetic polymorphisms at species and strain levels have enabled the study of yeast diversity during wine fermentation. This review will summarize the mechanisms involved in generating genetic polymorphisms in yeasts, the molecular methods used to unveil genetic variation, and the utility of these polymorphisms to differentiate strains, populations, and species in order to infer the evolutionary history and the adaptive evolution of wine yeasts, and to identify their influence on their biotechnological and sensorial properties. PMID:28522998

A Comprehensive Systems Biology Approach to Studying Zika Virus.

PubMed

May, Meghan; Relich, Ryan F

2016-01-01

Zika virus (ZIKV) is responsible for an ongoing and intensifying epidemic in the Western Hemisphere. We examined the complete predicted proteomes, glycomes, and selectomes of 33 ZIKV strains representing temporally diverse members of the African lineage, the Asian lineage, and the current outbreak in the Americas. Derivation of the complete selectome is an 'omics' approach to identify distinct evolutionary pressures acting on different features of an organism. Employment of the M8 model did not show evidence of global diversifying selection acting on the ZIKV polyprotein; however, a mixed effect model of evolution showed strong evidence (P<0.05) for episodic diversifying selection acting on specific sites. Single nucleotide polymorphisms (SNPs) were predictably frequent across strains relative to the derived consensus sequence. None of the 9 published detection procedures utilize targets that share 100% identity across the 33 strains examined, indicating that ZIKV escape from molecular detection is predictable. The predicted O-linked glycome showed marked diversity across strains; however, the N-linked glycome was highly stable. All Asian and American strains examined were predicted to include glycosylation of E protein ASN154, a modification proposed to mediate neurotropism, whereas the modification was not predicted for African strains. SNP diversity, episodic diversifying selection, and differential glycosylation, particularly of ASN154, may have major biological implications for ZIKV disease. Taken together, the systems biology perspective of ZIKV indicates: a.) The recently emergent Asian/American N-glycotype is mediating the new and emerging neuropathogenic potential of ZIKV; and b.) further divergence at specific sites is predictable as endemnicity is established in the Americas.

Assessing host-specificity of Escherichia coli using a supervised learning logic-regression-based analysis of single nucleotide polymorphisms in intergenic regions.

PubMed

Zhi, Shuai; Li, Qiaozhi; Yasui, Yutaka; Edge, Thomas; Topp, Edward; Neumann, Norman F

2015-11-01

Host specificity in E. coli is widely debated. Herein, we used supervised learning logic-regression-based analysis of intergenic DNA sequence variability in E. coli in an attempt to identify single nucleotide polymorphism (SNP) biomarkers of E. coli that are associated with natural selection and evolution toward host specificity. Seven-hundred and eighty strains of E. coli were isolated from 15 different animal hosts. We utilized logic regression for analyzing DNA sequence data of three intergenic regions (flanked by the genes uspC-flhDC, csgBAC-csgDEFG, and asnS-ompF) to identify genetic biomarkers that could potentially discriminate E. coli based on host sources. Across 15 different animal hosts, logic regression successfully discriminated E. coli based on animal host source with relatively high specificity (i.e., among the samples of the non-target animal host, the proportion that correctly did not have the host-specific marker pattern) and sensitivity (i.e., among the samples from a given animal host, the proportion that correctly had the host-specific marker pattern), even after fivefold cross validation. Permutation tests confirmed that for most animals, host specific intergenic biomarkers identified by logic regression in E. coli were significantly associated with animal host source. The highest level of biomarker sensitivity was observed in deer isolates, with 82% of all deer E. coli isolates displaying a unique SNP pattern that was 98% specific to deer. Fifty-three percent of human isolates displayed a unique biomarker pattern that was 98% specific to humans. Twenty-nine percent of cattle isolates displayed a unique biomarker that was 97% specific to cattle. Interestingly, even within a related host group (i.e., Family: Canidae [domestic dogs and coyotes]), highly specific SNP biomarkers (98% and 99% specificity for dog and coyotes, respectively) were observed, with 21% of dog E. coli isolates displaying a unique dog biomarker and 61% of coyote isolates displaying a unique coyote biomarker. Application of a supervised learning method, such as logic regression, to DNA sequence analysis at certain intergenic regions demonstrates that some E. coli strains may evolve to become host-specific. Copyright © 2015 Elsevier Inc. All rights reserved.

Cross-reactive acquired immunity influences transmission success of the Lyme disease pathogen, Borrelia afzelii.

PubMed

Jacquet, Maxime; Durand, Jonas; Rais, Olivier; Voordouw, Maarten J

2015-12-01

Cross-reactive acquired immunity in the vertebrate host induces indirect competition between strains of a given pathogen species and is critical for understanding the ecology of mixed infections. In vector-borne diseases, cross-reactive antibodies can reduce pathogen transmission at the vector-to-host and the host-to-vector lifecycle transition. The highly polymorphic, immunodominant, outer surface protein C (OspC) of the tick-borne spirochete bacterium Borrelia afzelii induces a strong antibody response in the vertebrate host. To test how cross-immunity in the vertebrate host influences tick-to-host and host-to-tick transmission, mice were immunized with one of two strain-specific recombinant OspC proteins (A3, A10), challenged via tick bite with one of the two B. afzelii ospC strains (A3, A10), and infested with xenodiagnostic ticks. Immunization with a given rOspC antigen protected mice against homologous strains carrying the same major ospC group allele but provided little or no cross-protection against heterologous strains carrying a different major ospC group allele. There were cross-immunity effects on the tick spirochete load but not on the probability of host-to-tick transmission. The spirochete load in ticks that had fed on mice with cross-immune experience was reduced by a factor of two compared to ticks that had fed on naive control mice. In addition, strain-specific differences in mouse spirochete load, host-to-tick transmission, tick spirochete load, and the OspC-specific IgG response revealed the mechanisms that determine variation in transmission success between strains of B. afzelii. This study shows that cross-immunity in infected vertebrate hosts can reduce pathogen load in the arthropod vector with potential consequences for vector-to-host pathogen transmission. Copyright © 2015 Elsevier B.V. All rights reserved.

Epstein-Barr virus latent gene sequences as geographical markers of viral origin: unique EBNA3 gene signatures identify Japanese viruses as distinct members of the Asian virus family.

PubMed

Sawada, Akihisa; Croom-Carter, Deborah; Kondo, Osamu; Yasui, Masahiro; Koyama-Sato, Maho; Inoue, Masami; Kawa, Keisei; Rickinson, Alan B; Tierney, Rosemary J

2011-05-01

Polymorphisms in Epstein-Barr virus (EBV) latent genes can identify virus strains from different human populations and individual strains within a population. An Asian EBV signature has been defined almost exclusively from Chinese viruses, with little information from other Asian countries. Here we sequenced polymorphic regions of the EBNA1, 2, 3A, 3B, 3C and LMP1 genes of 31 Japanese strains from control donors and EBV-associated T/NK-cell lymphoproliferative disease (T/NK-LPD) patients. Though identical to Chinese strains in their dominant EBNA1 and LMP1 alleles, Japanese viruses were subtly different at other loci. Thus, while Chinese viruses mainly fall into two families with strongly linked 'Wu' or 'Li' alleles at EBNA2 and EBNA3A/B/C, Japanese viruses all have the consensus Wu EBNA2 allele but fall into two families at EBNA3A/B/C. One family has variant Li-like sequences at EBNA3A and 3B and the consensus Li sequence at EBNA3C; the other family has variant Wu-like sequences at EBNA3A, variants of a low frequency Chinese allele 'Sp' at EBNA3B and a consensus Sp sequence at EBNA3C. Thus, EBNA3A/B/C allelotypes clearly distinguish Japanese from Chinese strains. Interestingly, most Japanese viruses also lack those immune-escape mutations in the HLA-A11 epitope-encoding region of EBNA3B that are so characteristic of viruses from the highly A11-positive Chinese population. Control donor-derived and T/NK-LPD-derived strains were similarly distributed across allelotypes and, by using allelic polymorphisms to track virus strains in patients pre- and post-haematopoietic stem-cell transplant, we show that a single strain can induce both T/NK-LPD and B-cell-lymphoproliferative disease in the same patient.

Exploring the symbiotic pangenome of the nitrogen-fixing bacterium Sinorhizobium meliloti

PubMed Central

2011-01-01

Background Sinorhizobium meliloti is a model system for the studies of symbiotic nitrogen fixation. An extensive polymorphism at the genetic and phenotypic level is present in natural populations of this species, especially in relation with symbiotic promotion of plant growth. AK83 and BL225C are two nodule-isolated strains with diverse symbiotic phenotypes; BL225C is more efficient in promoting growth of the Medicago sativa plants than strain AK83. In order to investigate the genetic determinants of the phenotypic diversification of S. meliloti strains AK83 and BL225C, we sequenced the complete genomes for these two strains. Results With sizes of 7.14 Mbp and 6.97 Mbp, respectively, the genomes of AK83 and BL225C are larger than the laboratory strain Rm1021. The core genome of Rm1021, AK83, BL225C strains included 5124 orthologous groups, while the accessory genome was composed by 2700 orthologous groups. While Rm1021 and BL225C have only three replicons (Chromosome, pSymA and pSymB), AK83 has also two plasmids, 260 and 70 Kbp long. We found 65 interesting orthologous groups of genes that were present only in the accessory genome, consequently responsible for phenotypic diversity and putatively involved in plant-bacterium interaction. Notably, the symbiosis inefficient AK83 lacked several genes required for microaerophilic growth inside nodules, while several genes for accessory functions related to competition, plant invasion and bacteroid tropism were identified only in AK83 and BL225C strains. Presence and extent of polymorphism in regulons of transcription factors involved in symbiotic interaction were also analyzed. Our results indicate that regulons are flexible, with a large number of accessory genes, suggesting that regulons polymorphism could also be a key determinant in the variability of symbiotic performances among the analyzed strains. Conclusions In conclusions, the extended comparative genomics approach revealed a variable subset of genes and regulons that may contribute to the symbiotic diversity. PMID:21569405

Exploring the symbiotic pangenome of the nitrogen-fixing bacterium Sinorhizobium meliloti

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

Galardini, Marco; Mengoni, Alessio; Brilli, Matteo

Background: Sinorhizobium meliloti is a model system for the studies of symbiotic nitrogen fixation. An extensive polymorphism at the genetic and phenotypic level is present in natural populations of this species, especially in relation with symbiotic promotion of plant growth. AK83 and BL225C are two nodule-isolated strains with diverse symbiotic phenotypes; BL225C is more efficient in promoting growth of the Medicago sativa plants than strain AK83. In order to investigate the genetic determinants of the phenotypic diversification of S. meliloti strains AK83 and BL225C, we sequenced the complete genomes for these two strains. Results: With sizes of 7.14 Mbp andmore » 6.97 Mbp, respectively, the genomes of AK83 and BL225C are larger than the laboratory strain Rm1021. The core genome of Rm1021, AK83, BL225C strains included 5124 orthologous groups, while the accessory genome was composed by 2700 orthologous groups. While Rm1021 and BL225C have only three replicons (Chromosome, pSymA and pSymB), AK83 has also two plasmids, 260 and 70 Kbp long. We found 65 interesting orthologous groups of genes that were present only in the accessory genome, consequently responsible for phenotypic diversity and putatively involved in plant-bacterium interaction. Notably, the symbiosis inefficient AK83 lacked several genes required for microaerophilic growth inside nodules, while several genes for accessory functions related to competition, plant invasion and bacteroid tropism were identified only in AK83 and BL225C strains. Presence and extent of polymorphism in regulons of transcription factors involved in symbiotic interaction were also analyzed. Our results indicate that regulons are flexible, with a large number of accessory genes, suggesting that regulons polymorphism could also be a key determinant in the variability of symbiotic performances among the analyzed strains.« less

Peptidase-3 (Pep-3), dipeptidase variant in the rat homologous to mouse pep-3 (Dip-1) and human PEP-c.

PubMed

Womack, J E; Cramer, D V

1980-10-01

Starch gel electrophoresis and histochemical staining with L-leucyl-L-tyrosine have revealed genetic variation for dipeptidase in Rattus norvegicus. The tissue distribution, substrate specificity, and heterozygous expression as a monmeric protein suggest homology of the variant peptidase to human PEP-C and mouse Pep-3 (Dip-1). We propose Peptidase-3 (Pep-3) as a name for this autosomal locus in the rat. The allele responsible for slower (less anodal) electrophoretic migration is designated Pep-3a and is characteristic of strain ACI/Pit. A faster (more anodal) electrophoretic mobility is the product of the Pep-3b allele in strain F344/Pit. Twenty-five additional inbred strains carry Pep-3a and 16 others carry Pep-3b. Wild rats trapped in Pittsburgh were polymorphic for this locus. Alleles at Pep-3 segregated independently of c (linkage group I), a (linkage group IV), RT2 and Es-1 (linkage group V), h (linkage group VI), and RTI (linkage group VIII).

Identification of amino acid substitutions with compensational effects in the attachment protein of canine distemper virus.

PubMed

Sattler, Ursula; Khosravi, Mojtaba; Avila, Mislay; Pilo, Paola; Langedijk, Johannes P; Ader-Ebert, Nadine; Alves, Lisa A; Plattet, Philippe; Origgi, Francesco C

2014-07-01

The hemagglutinin (H) gene of canine distemper virus (CDV) encodes the receptor-binding protein. This protein, together with the fusion (F) protein, is pivotal for infectivity since it contributes to the fusion of the viral envelope with the host cell membrane. Of the two receptors currently known for CDV (nectin-4 and the signaling lymphocyte activation molecule [SLAM]), SLAM is considered the most relevant for host susceptibility. To investigate how evolution might have impacted the host-CDV interaction, we examined the functional properties of a series of missense single nucleotide polymorphisms (SNPs) naturally accumulating within the H-gene sequences during the transition between two distinct but related strains. The two strains, a wild-type strain and a consensus strain, were part of a single continental outbreak in European wildlife and occurred in distinct geographical areas 2 years apart. The deduced amino acid sequence of the two H genes differed at 5 residues. A panel of mutants carrying all the combinations of the SNPs was obtained by site-directed mutagenesis. The selected mutant, wild type, and consensus H proteins were functionally evaluated according to their surface expression, SLAM binding, fusion protein interaction, and cell fusion efficiencies. The results highlight that the most detrimental functional effects are associated with specific sets of SNPs. Strikingly, an efficient compensational system driven by additional SNPs appears to come into play, virtually neutralizing the negative functional effects. This system seems to contribute to the maintenance of the tightly regulated function of the H-gene-encoded attachment protein. Importance: To investigate how evolution might have impacted the host-canine distemper virus (CDV) interaction, we examined the functional properties of naturally occurring single nucleotide polymorphisms (SNPs) in the hemagglutinin gene of two related but distinct strains of CDV. The hemagglutinin gene encodes the attachment protein, which is pivotal for infection. Our results show that few SNPs have a relevant detrimental impact and they generally appear in specific combinations (molecular signatures). These drastic negative changes are neutralized by compensatory mutations, which contribute to maintenance of an overall constant bioactivity of the attachment protein. This compensational mechanism might reflect the reaction of the CDV machinery to the changes occurring in the virus following antigenic variations critical for virulence. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Assessing the germplasm of Laminaria (phaeophyceae) with random amplified polymorphic DNA (RAPD) method

NASA Astrophysics Data System (ADS)

He, Yingjun; Zou, Yuping; Wang, Xiaodong; Zheng, Zhiguo; Zhang, Daming; Duan, Delin

2003-06-01

Eighteen gametophytes including L. japonica, L. ochotensis and L. longissima, were verified with random amplified polymorphic DNA (RAPD) technique. Eighteen ten-base primers were chosen from 100 primers selected for final amplification test. Among the total of 205 bands amplified, 181 (88.3%) were polymorphic. The genetic distance among different strains ranged from 0.072 to 0.391. The dendrogram constructed by unweighted pair-group method with arithmetic (UPGMA) method showed that the female and male gametophytes of the same cell lines could be grouped in pairs respectively. It indicated that RAPD analysis could be used not only to distinguish different strains of Laminaria, but also to distinguish male and female gametophyte within the same cell lines. There is ambiguous systematic relationship if judged merely by the present data. It seems that the use of RAPD marker is limited to elucidation of the phylogenetic relationship among the species of Laminaria.

Isolation and cross-familial amplification of 41 microsatellites for the brook charr (Salvelinus fontinalis)

USGS Publications Warehouse

Perry, G.M.L.; King, T.L.; St. -Cyr, J.; Valcourt, M.; Bernatchez, L.

2005-01-01

The brook charr (Salvelinus fontinalis; Osteichthyes: Salmonidae) is a phenotypically diverse fish species inhabiting much of North America. But relatively few genetic diagnostic resources are available for this fish species. We isolated 41 microsatellites from S. fontinalis polymorphic in one or more species of salmonid fish. Thirty-seven were polymorphic in brook charr, 15 in the congener Arctic charr (Salvelinus alpinus) and 14 in the lake charr (Salvelinus namaycush). Polymorphism was also relatively high in Oncorhynchus, where 21 loci were polymorphic in rainbow trout (Oncorhynchus mykiss) and 16 in cutthroat trout (Oncorhynchus clarkii) but only seven and four microsatellite loci were polymorphic in the more distantly related lake whitefish (Coregonus clupeaformis) and Atlantic salmon (Salmo salar), respectively. One duplicated locus (Sfo228Lav) was polymorphic at both duplicates in S. fontinalis. ?? 2005 Blackwell Publishing Ltd.

Cluster of cases of Salmonella enterica serotype Rissen infection in a general hospital, Italy, 2007.

PubMed

Boschi, T; Aquilini, D; Degl'Innocenti, R; Aleo, A; Romani, C; Nicoletti, P; Buonomini, M I; Marconi, P; Bilei, S; Mammina, C; Nastasi, A

2010-12-01

In 2007, three strains of Salmonella enterica serotype Rissen (S. Rissen) were isolated in the laboratory of diagnostic microbiology of the General Hospital of Prato, Tuscany, Italy, over a 1 month and half interval of time. The first isolate was recovered on January 26 from an outpatient with enteritis. Then, two strains were isolated on February 16 and March 11 respectively, from central venous catheters of patients who were being hospitalized in two departments of the Hospital. An epidemiologically linked cluster of cases of salmonellosis was suspected. The three strains were submitted to single enzyme-amplified fragment length polymorphism (SE-AFLP) and XbaI macrorestriction and pulsed-field gel electrophoresis (PFGE) that yielded undistinguishable profiles. Epidemiological investigations failed to identify a common source of infection within the Hospital. Moreover, the third patient had been exclusively total parenteral nutrition fed since his admission with a stomach cancer diagnosis. The first patient had a community-acquired infection, but the source of her illness was uncertain. Twenty-five further isolates identified in the years 2004-2007 in the same geographical area showed distinctly different PFGE and SE-AFLP patterns. The three patients seemed to represent a cluster of epidemiologically unrelated cases caused by a previously never recognized S. Rissen strain. Rapid subtyping of isolates is essential in the early investigation of potential outbreaks, but synthesis of conventional and molecular epidemiological investigation and availability of surveillance data is often critical to prevent the initiation of time-consuming, expensive and ineffective further investigations and control interventions. © 2009 Blackwell Verlag GmbH.

Genetic Diversity Among Botulinum Neurotoxin Producing Clostridial Strains

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

Hill, K K; Smith, T J; Helma, C H

2006-07-06

Clostridium botulinum is a taxonomic designation for many diverse anaerobic spore forming rod-shaped bacteria which have the common property of producing botulinum neurotoxins (BoNTs). The BoNTs are exoneurotoxins that can cause severe paralysis and even death in humans and various other animal species. A collection of 174 C. botulinum strains were examined by amplified fragment length polymorphism (AFLP) analysis and by sequencing of the 16S rRNA gene and BoNT genes to examine genetic diversity within this species. This collection contained representatives of each of the seven different serotypes of botulinum neurotoxins (BoNT A-G). Analysis of the16S rRNA sequences confirmed earliermore » reports of at least four distinct genomic backgrounds (Groups I-IV) each of which has independently acquired one or more BoNT serotypes through horizontal gene transfer. AFLP analysis provided higher resolution, and can be used to further subdivide the four groups into sub-groups. Sequencing of the BoNT genes from serotypes A, B and E in multiple strains confirmed significant sequence variation within each serotype. Four distinct lineages within each of the BoNT A and B serotypes, and five distinct lineages of serotype E strains were identified. The nucleotide sequences of the seven serotypes of BoNT were compared and show varying degrees of interrelatedness and recombination as has been previously noted for the NTNH gene which is linked to BoNT. These analyses contribute to the understanding of the evolution and phylogeny within this species and assist in the development of improved diagnostics and therapeutics for treatment of botulism.« less

Typing of Human Mycobacterium avium Isolates in Italy by IS1245-Based Restriction Fragment Length Polymorphism Analysis

PubMed Central

Lari, Nicoletta; Cavallini, Michela; Rindi, Laura; Iona, Elisabetta; Fattorini, Lanfranco; Garzelli, Carlo

1998-01-01

All but 2 of 63 Mycobacterium avium isolates from distinct geographic areas of Italy exhibited markedly polymorphic, multibanded IS1245 restriction fragment length polymorphism (RFLP) patterns; 2 isolates showed the low-number banding pattern typical of bird isolates. By computer analysis, 41 distinct IS1245 patterns and 10 clusters of essentially identical strains were detected; 40% of the 63 isolates showed genetic relatedness, suggesting the existence of a predominant AIDS-associated IS1245 RFLP pattern. PMID:9817900

Genetic Polymorphisms and Phenotypic Profiles of Sulfadiazine-Resistant and Sensitive Toxoplasma gondii Isolates Obtained from Newborns with Congenital Toxoplasmosis in Minas Gerais, Brazil.

PubMed

Silva, Letícia Azevedo; Reis-Cunha, João Luís; Bartholomeu, Daniella Castanheira; Vítor, Ricardo Wagner Almeida

2017-01-01

Previous Toxoplasma gondii studies revealed that mutations in the dhps (dihydropteroate synthase) gene are associated with resistance to sulfonamides. Although Brazilian strains are genotypically different, very limited data are available regarding the susceptibility of strains obtained from human to sulfonamides. The aim of this study was to evaluate the efficacy of sulfadiazine (SDZ) against Brazilian isolates of T. gondii and verify whether isolates present polymorphisms in the dhps gene. We also investigated whether the virulence-phenotype and/or genotype were associated with the profile of susceptibility to SDZ. Five T. gondii isolates obtained from newborns with congenital toxoplasmosis were used to verify susceptibility. Mice were infected with 104 tachyzoites and orally treated with different doses of SDZ. The mortality curve was evaluated by the Log-rank test. The presence of polymorphisms in the dhps gene was verified using sequencing. A descriptive analysis for 11 Brazilian isolates was used to assess the association between susceptibility, genotype, and virulence-phenotype. Statistical analysis showed that TgCTBr03, 07, 08, and 16 isolates were susceptible to SDZ, whereas TgCTBr11 isolate presented a profile of resistance to SDZ. Nineteen polymorphisms were identified in dhps exons. Seven polymorphisms corresponded to non-synonymous mutations, with four being new mutations, described for the first time in this study. No association was found between the profile of susceptibility and the virulence-phenotype or genotype of the parasite. There is a high variability in the susceptibilities of Brazilian T. gondii strains to SDZ, with evidence of drug resistance. Despite the large number of polymorphisms identified, the profile of susceptibility to SDZ was not associated with any of the dhps variants identified in this study. Other genetic factors, not yet determined, may be associated with the resistance to SDZ; thus, further studies are needed as a basis for a more adequate toxoplasmosis treatment.

Genetic Polymorphisms and Phenotypic Profiles of Sulfadiazine-Resistant and Sensitive Toxoplasma gondii Isolates Obtained from Newborns with Congenital Toxoplasmosis in Minas Gerais, Brazil

PubMed Central

Silva, Letícia Azevedo; Reis-Cunha, João Luís; Bartholomeu, Daniella Castanheira; Vítor, Ricardo Wagner Almeida

2017-01-01

Background Previous Toxoplasma gondii studies revealed that mutations in the dhps (dihydropteroate synthase) gene are associated with resistance to sulfonamides. Although Brazilian strains are genotypically different, very limited data are available regarding the susceptibility of strains obtained from human to sulfonamides. The aim of this study was to evaluate the efficacy of sulfadiazine (SDZ) against Brazilian isolates of T. gondii and verify whether isolates present polymorphisms in the dhps gene. We also investigated whether the virulence-phenotype and/or genotype were associated with the profile of susceptibility to SDZ. Methods Five T. gondii isolates obtained from newborns with congenital toxoplasmosis were used to verify susceptibility. Mice were infected with 104 tachyzoites and orally treated with different doses of SDZ. The mortality curve was evaluated by the Log-rank test. The presence of polymorphisms in the dhps gene was verified using sequencing. A descriptive analysis for 11 Brazilian isolates was used to assess the association between susceptibility, genotype, and virulence-phenotype. Results Statistical analysis showed that TgCTBr03, 07, 08, and 16 isolates were susceptible to SDZ, whereas TgCTBr11 isolate presented a profile of resistance to SDZ. Nineteen polymorphisms were identified in dhps exons. Seven polymorphisms corresponded to non-synonymous mutations, with four being new mutations, described for the first time in this study. No association was found between the profile of susceptibility and the virulence-phenotype or genotype of the parasite. Conclusions There is a high variability in the susceptibilities of Brazilian T. gondii strains to SDZ, with evidence of drug resistance. Despite the large number of polymorphisms identified, the profile of susceptibility to SDZ was not associated with any of the dhps variants identified in this study. Other genetic factors, not yet determined, may be associated with the resistance to SDZ; thus, further studies are needed as a basis for a more adequate toxoplasmosis treatment. PMID:28118394

Polymorphism in hybrid male sterility in wild-derived Mus musculus musculus strains on proximal chromosome 17.

PubMed

Vyskocilová, Martina; Prazanová, Gabriela; Piálek, Jaroslav

2009-02-01

The hybrid sterility-1 (Hst1) locus at Chr 17 causes male sterility in crosses between the house mouse subspecies Mus musculus domesticus (Mmd) and M. m. musculus (Mmm). This locus has been defined by its polymorphic variants in two laboratory strains (Mmd genome) when mated to PWD/Ph mice (Mmm genome): C57BL/10 (carrying the sterile allele) and C3H (fertile allele). The occurrence of sterile and/or fertile (wild Mmm x C57BL)F1 males is evidence that polymorphism for this trait also exists in natural populations of Mmm; however, the nature of this polymorphism remains unclear. Therefore, we derived two wild-origin Mmm strains, STUS and STUF, that produce sterile and fertile males, respectively, in crosses with C57BL mice. To determine the genetic basis underlying male fertility, the (STUS x STUF)F1 females were mated to C57BL/10 J males. About one-third of resulting hybrid males (33.8%) had a significantly smaller epididymis and testes than parental animals and lacked spermatozoa due to meiotic arrest. A further one-fifth of males (20.3%) also had anomalous reproductive traits but produced some spermatozoa. The remaining fertile males (45.9%) displayed no deviation from values found in parental individuals. QTL analysis of the progeny revealed strong associations of male fitness components with the proximal end of Chr 17, and a significant effect of the central section of Chr X on testes mass. The data suggest that genetic incompatibilities associated with male sterility have evolved independently at the proximal end of Chr 17 and are polymorphic within both Mmd and Mmm genomes.

Screening and Characterization of RAPD Markers in Viscerotropic Leishmania Parasites

PubMed Central

Mkada–Driss, Imen; Talbi, Chiraz; Guerbouj, Souheila; Driss, Mehdi; Elamine, Elwaleed M.; Cupolillo, Elisa; Mukhtar, Moawia M.; Guizani, Ikram

2014-01-01

Visceral leishmaniasis (VL) is mainly due to the Leishmania donovani complex. VL is endemic in many countries worldwide including East Africa and the Mediterranean region where the epidemiology is complex. Taxonomy of these pathogens is under controversy but there is a correlation between their genetic diversity and geographical origin. With steady increase in genome knowledge, RAPD is still a useful approach to identify and characterize novel DNA markers. Our aim was to identify and characterize polymorphic DNA markers in VL Leishmania parasites in diverse geographic regions using RAPD in order to constitute a pool of PCR targets having the potential to differentiate among the VL parasites. 100 different oligonucleotide decamers having arbitrary DNA sequences were screened for reproducible amplification and a selection of 28 was used to amplify DNA from 12 L. donovani, L. archibaldi and L. infantum strains having diverse origins. A total of 155 bands were amplified of which 60.65% appeared polymorphic. 7 out of 28 primers provided monomorphic patterns. Phenetic analysis allowed clustering the parasites according to their geographical origin. Differentially amplified bands were selected, among them 22 RAPD products were successfully cloned and sequenced. Bioinformatic analysis allowed mapping of the markers and sequences and priming sites analysis. This study was complemented with Southern-blot to confirm assignment of markers to the kDNA. The bioinformatic analysis identified 16 nuclear and 3 minicircle markers. Analysis of these markers highlighted polymorphisms at RAPD priming sites with mainly 5′ end transversions, and presence of inter– and intra– taxonomic complex sequence and microsatellites variations; a bias in transitions over transversions and indels between the different sequences compared is observed, which is however less marked between L. infantum and L. donovani. The study delivers a pool of well-documented polymorphic DNA markers, to develop molecular diagnostics assays to characterize and differentiate VL causing agents. PMID:25313833

Identification and Characterization of Microsatellite Markers Derived from the Whole Genome Analysis of Taenia solium.

PubMed

Pajuelo, Mónica J; Eguiluz, María; Dahlstrom, Eric; Requena, David; Guzmán, Frank; Ramirez, Manuel; Sheen, Patricia; Frace, Michael; Sammons, Scott; Cama, Vitaliano; Anzick, Sarah; Bruno, Dan; Mahanty, Siddhartha; Wilkins, Patricia; Nash, Theodore; Gonzalez, Armando; García, Héctor H; Gilman, Robert H; Porcella, Steve; Zimic, Mirko

2015-12-01

Infections with Taenia solium are the most common cause of adult acquired seizures worldwide, and are the leading cause of epilepsy in developing countries. A better understanding of the genetic diversity of T. solium will improve parasite diagnostics and transmission pathways in endemic areas thereby facilitating the design of future control measures and interventions. Microsatellite markers are useful genome features, which enable strain typing and identification in complex pathogen genomes. Here we describe microsatellite identification and characterization in T. solium, providing information that will assist in global efforts to control this important pathogen. For genome sequencing, T. solium cysts and proglottids were collected from Huancayo and Puno in Peru, respectively. Using next generation sequencing (NGS) and de novo assembly, we assembled two draft genomes and one hybrid genome. Microsatellite sequences were identified and 36 of them were selected for further analysis. Twenty T. solium isolates were collected from Tumbes in the northern region, and twenty from Puno in the southern region of Peru. The size-polymorphism of the selected microsatellites was determined with multi-capillary electrophoresis. We analyzed the association between microsatellite polymorphism and the geographic origin of the samples. The predicted size of the hybrid (proglottid genome combined with cyst genome) T. solium genome was 111 MB with a GC content of 42.54%. A total of 7,979 contigs (>1,000 nt) were obtained. We identified 9,129 microsatellites in the Puno-proglottid genome and 9,936 in the Huancayo-cyst genome, with 5 or more repeats, ranging from mono- to hexa-nucleotide. Seven microsatellites were polymorphic and 29 were monomorphic within the analyzed isolates. T. solium tapeworms were classified into two genetic groups that correlated with the North/South geographic origin of the parasites. The availability of draft genomes for T. solium represents a significant step towards the understanding the biology of the parasite. We report here a set of T. solium polymorphic microsatellite markers that appear promising for genetic epidemiology studies.

Genome Analysis of Listeria monocytogenes Sequence Type 8 Strains Persisting in Salmon and Poultry Processing Environments and Comparison with Related Strains

PubMed Central

Fagerlund, Annette; Langsrud, Solveig; Schirmer, Bjørn C. T.; Møretrø, Trond; Heir, Even

2016-01-01

Listeria monocytogenes is an important foodborne pathogen responsible for the disease listeriosis, and can be found throughout the environment, in many foods and in food processing facilities. The main cause of listeriosis is consumption of food contaminated from sources in food processing environments. Persistence in food processing facilities has previously been shown for the L. monocytogenes sequence type (ST) 8 subtype. In the current study, five ST8 strains were subjected to whole-genome sequencing and compared with five additionally available ST8 genomes, allowing comparison of strains from salmon, poultry and cheese industry, in addition to a human clinical isolate. Genome-wide analysis of single-nucleotide polymorphisms (SNPs) confirmed that almost identical strains were detected in a Danish salmon processing plant in 1996 and in a Norwegian salmon processing plant in 2001 and 2011. Furthermore, we show that L. monocytogenes ST8 was likely to have been transferred between two poultry processing plants as a result of relocation of processing equipment. The SNP data were used to infer the phylogeny of the ST8 strains, separating them into two main genetic groups. Within each group, the plasmid and prophage content was almost entirely conserved, but between groups, these sequences showed strong divergence. The accessory genome of the ST8 strains harbored genetic elements which could be involved in rendering the ST8 strains resilient to incoming mobile genetic elements. These included two restriction-modification loci, one of which was predicted to show phase variable recognition sequence specificity through site-specific domain shuffling. Analysis indicated that the ST8 strains harbor all important known L. monocytogenes virulence factors, and ST8 strains are commonly identified as the causative agents of invasive listeriosis. Therefore, the persistence of this L. monocytogenes subtype in food processing facilities poses a significant concern for food safety. PMID:26953695

Molecular epidemiology of Escherichia coli O157:H7 strains by bacteriophage lambda restriction fragment length polymorphism analysis: application to a multistate foodborne outbreak and a day-care center cluster.

PubMed

Samadpour, M; Grimm, L M; Desai, B; Alfi, D; Ongerth, J E; Tarr, P I

1993-12-01

Genomic DNAs prepared from 168 isolates of Escherichia coli O157:H7 were analyzed for restriction fragment length polymorphisms on Southern blots probed with bacteriophage lambda DNA. The isolates analyzed included strains from a recent large multistate outbreak of E. coli O157:H7 infection associated with consumption of poorly cooked beef in restaurants, a day-care center cluster, and temporally and geographically unrelated isolates. E. coli O157:H7 isolates recovered from the incriminated meat and from 61 (96.8%) of 63 patients from Washington and Nevada possessed identical lambda restriction fragment length patterns. The lambda restriction fragment length polymorphisms observed in 11 (91.7%) of 12 day-care center patients were identical, but they differed from that of the strain associated with the multistate outbreak. E. coli O157:H7 from 42 patients temporally or geographically unrelated to either cluster of infection possessed unique and different lambda restriction fragment length patterns, except for paired isolates from three separate clusters of infection. These data demonstrate that the hybridization of DNA digests of E. coli O157:H7 with radiolabelled bacteriophage lambda DNA can be a useful, stable, and discriminatory epidemiologic tool for analyzing the linkage between strains of E. coli O157:H7.

Introduction of a hydrolysis probe PCR assay for high-throughput screening of methicillin-resistant Staphylococcus aureus with the ability to include or exclude detection of Staphylococcus argenteus.

PubMed

Bogestam, Katja; Vondracek, Martin; Karlsson, Mattias; Fang, Hong; Giske, Christian G

2018-01-01

Many countries using sensitive screening methods for detection of carriage of methicillin-resistant Staphylococcus aureus (MRSA) have a sustained low incidence of MRSA infections. For diagnostic laboratories with high sample volumes, MRSA screening requires stability, low maintenance and high performance at a low cost. Herein we designed oligonucleotides for a new nuc targeted hydrolysis probe PCR to replace the standard in-house nuc SybrGreen PCR assay. This new, more time-efficient, PCR assay resulted in a 40% increase in daily sample capacity, with maintained high specificity and sensitivity. The assay was also able to detect Staphylococcus aureus clonal cluster 75 (CC75) lineage strains, recently re-classified as Staphylococcus argenteus, with a sensitivity considerably increased compared to our previous assay. While awaiting consensus if the CC75 lineage of S. aureus should be considered as S. argenteus, and whether methicillin-resistant S. argenteus should be included in the MRSA definition, many diagnostic laboratories need to update their MRSA assay sensitivity/specificity towards this lineage/species. The MRSA screening assay presented in this manuscript is comprised of nuc oligonucleotides separately targeting S. aureus and CC75 lineage strains/S. argenteus, thus providing high user flexibility for the detection of CC75 lineage strains/S. argenteus.

Effectiveness of the combined evaluation of KLK3 genetics and free-to-total prostate specific antigen ratio for prostate cancer diagnosis.

PubMed

Zambon, Carlo-Federico; Prayer-Galetti, Tommaso; Basso, Daniela; Padoan, Andrea; Rossi, Elisa; Secco, Silvia; Pelloso, Michela; Fogar, Paola; Navaglia, Filippo; Moz, Stefania; Zattoni, Filiberto; Plebani, Mario

2012-10-01

Of serum prostate specific antigen variability 40% depends on inherited factors. We ascertained whether the knowledge of KLK3 genetics would enhance prostate specific antigen diagnostic performance in patients with clinical suspicion of prostate cancer. We studied 1,058 men who consecutively underwent prostate biopsy for clinical suspicion of prostate cancer. At histology prostate cancer was present in 401 cases and absent in 657. Serum total prostate specific antigen and the free-to-total prostate specific antigen ratio were determined. Four polymorphisms of the KLK3 gene (rs2569733, rs2739448, rs925013 and rs2735839) and 1 polymorphism of the SRD5A2 gene (rs523349) were studied. The influence of genetics on prostate specific antigen variability was evaluated by multivariate linear regression analysis. The performance of total prostate specific antigen and the free-to-total prostate specific antigen ratio alone or combined with a genetically based patient classification were defined by ROC curve analyses. For prostate cancer diagnosis the free-to-total prostate specific antigen ratio index alone (cutoff 11%) was superior to total prostate specific antigen (cutoff 4 ng/ml) and to free-to-total prostate specific antigen ratio reflex testing (positive predictive value 61%, 43% and 54%, respectively). Prostate specific antigen correlated with KLK3 genetics (rs2735839 polymorphism p = 0.001, and rs2569733, rs2739448 and rs925013 haplotype combination p = 0.003). In patients with different KLK3 genetics 2 optimal free-to-total prostate specific antigen ratio cutoffs (11% and 14.5%) were found. For free-to-total prostate specific antigen ratio values between 11% and 14.5% the prostate cancer probability ranged from 30.0% to 47.4% according to patient genetics. The free-to-total prostate specific antigen ratio is superior to total prostate specific antigen for prostate cancer diagnosis, independent of total prostate specific antigen results. Free-to-total prostate specific antigen ratio findings below 11% are positively associated with prostate cancer and those above 14.5% are negatively associated with prostate cancer, while the interpretation of those between 11% and 14.5% is improved by patient KLK3 genetic analysis. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

A proposal to rationalize within-species plant virus nomenclature: benefits and implications of inaction.

PubMed

Jones, Roger A C; Kehoe, Monica A

2016-07-01

Current approaches used to name within-species, plant virus phylogenetic groups are often misleading and illogical. They involve names based on biological properties, sequence differences and geographical, country or place-association designations, or any combination of these. This type of nomenclature is becoming increasingly unsustainable as numbers of sequences of the same virus from new host species and different parts of the world increase. Moreover, this increase is accelerating as world trade and agriculture expand, and climate change progresses. Serious consequences for virus research and disease management might arise from incorrect assumptions made when current within-species phylogenetic group names incorrectly identify properties of group members. This could result in development of molecular tools that incorrectly target dangerous virus strains, potentially leading to unjustified impediments to international trade or failure to prevent such strains being introduced to countries, regions or continents formerly free of them. Dangerous strains might be missed or misdiagnosed by diagnostic laboratories and monitoring programs, and new cultivars with incorrect strain-specific resistances released. Incorrect deductions are possible during phylogenetic analysis of plant virus sequences and errors from strain misidentification during molecular and biological virus research activities. A nomenclature system for within-species plant virus phylogenetic group names is needed which avoids such problems. We suggest replacing all other naming approaches with Latinized numerals, restricting biologically based names only to biological strains and removing geographically based names altogether. Our recommendations have implications for biosecurity authorities, diagnostic laboratories, disease-management programs, plant breeders and researchers.

Complex Population Structure and Virulence Differences among Serotype 2 Streptococcus suis Strains Belonging to Sequence Type 28

PubMed Central

Athey, Taryn B. T.; Auger, Jean-Philippe; Teatero, Sarah; Dumesnil, Audrey; Takamatsu, Daisuke; Wasserscheid, Jessica; Dewar, Ken; Gottschalk, Marcelo; Fittipaldi, Nahuel

2015-01-01

Streptococcus suis is a major swine pathogen and a zoonotic agent. Serotype 2 strains are the most frequently associated with disease. However, not all serotype 2 lineages are considered virulent. Indeed, sequence type (ST) 28 serotype 2 S. suis strains have been described as a homogeneous group of low virulence. However, ST28 strains are often isolated from diseased swine in some countries, and at least four human ST28 cases have been reported. Here, we used whole-genome sequencing and animal infection models to test the hypothesis that the ST28 lineage comprises strains of different genetic backgrounds and different virulence. We used 50 S. suis ST28 strains isolated in Canada, the United States and Japan from diseased pigs, and one ST28 strain from a human case isolated in Thailand. We report a complex population structure among the 51 ST28 strains. Diversity resulted from variable gene content, recombination events and numerous genome-wide polymorphisms not attributable to recombination. Phylogenetic analysis using core genome single-nucleotide polymorphisms revealed four discrete clades with strong geographic structure, and a fifth clade formed by US, Thai and Japanese strains. When tested in experimental animal models, strains from this latter clade were significantly more virulent than a Canadian ST28 reference strain, and a closely related Canadian strain. Our results highlight the limitations of MLST for both phylogenetic analysis and virulence prediction and raise concerns about the possible emergence of ST28 strains in human clinical cases. PMID:26375680

Lactobacillus hammesii sp. nov., isolated from French sourdough.

PubMed

Valcheva, Rosica; Korakli, Maher; Onno, Bernard; Prévost, Hervé; Ivanova, Iskra; Ehrmann, Matthias A; Dousset, Xavier; Gänzle, Michael G; Vogel, Rudi F

2005-03-01

Twenty morphologically different strains were chosen from French wheat sourdough isolates. Cells were Gram-positive, non-spore-forming, non-motile rods. The isolates were identified using amplified-fragment length polymorphism, randomly amplified polymorphic DNA and 16S rRNA gene sequence analysis. All isolates were members of the genus Lactobacillus. They were identified as representing Lactobacillus plantarum, Lactobacillus paralimentarius, Lactobacillus sanfranciscensis, Lactobacillus spicheri and Lactobacillus sakei. However, two isolates (LP38(T) and LP39) could be clearly discriminated from recognized Lactobacillus species on the basis of genotyping methods. 16S rRNA gene sequence similarity and DNA-DNA relatedness data indicate that the two strains belong to a novel Lactobacillus species, for which the name Lactobacillus hammesii is proposed. The type strain is LP38(T) (=DSM 16381(T)=CIP 108387(T)=TMW 1.1236(T)).

New Mycobacterium tuberculosis Complex Sublineage, Brazzaville, Congo

PubMed Central

Malm, Sven; Linguissi, Laure S. Ghoma; Tekwu, Emmanuel M.; Vouvoungui, Jeannhey C.; Kohl, Thomas A.; Beckert, Patrick; Sidibe, Anissa; Rüsch-Gerdes, Sabine; Madzou-Laboum, Igor K.; Kwedi, Sylvie; Penlap Beng, Véronique; Frank, Matthias; Ntoumi, Francine

2017-01-01

Tuberculosis is a leading cause of illness and death in Congo. No data are available about the population structure and transmission dynamics of the Mycobacterium tuberculosis complex strains prevalent in this central Africa country. On the basis of single-nucleotide polymorphisms detected by whole-genome sequencing, we phylogenetically characterized 74 MTBC isolates from Brazzaville, the capital of Congo. The diversity of the study population was high; most strains belonged to the Euro-American lineage, which split into Latin American Mediterranean, Uganda I, Uganda II, Haarlem, X type, and a new dominant sublineage named Congo type (n = 26). Thirty strains were grouped in 5 clusters (each within 12 single-nucleotide polymorphisms), from which 23 belonged to the Congo type. High cluster rates and low genomic diversity indicate recent emergence and transmission of the Congo type, a new Euro-American sublineage of MTBC. PMID:28221129

New Mycobacterium tuberculosis Complex Sublineage, Brazzaville, Congo.

PubMed

Malm, Sven; Linguissi, Laure S Ghoma; Tekwu, Emmanuel M; Vouvoungui, Jeannhey C; Kohl, Thomas A; Beckert, Patrick; Sidibe, Anissa; Rüsch-Gerdes, Sabine; Madzou-Laboum, Igor K; Kwedi, Sylvie; Penlap Beng, Véronique; Frank, Matthias; Ntoumi, Francine; Niemann, Stefan

2017-03-01

Tuberculosis is a leading cause of illness and death in Congo. No data are available about the population structure and transmission dynamics of the Mycobacterium tuberculosis complex strains prevalent in this central Africa country. On the basis of single-nucleotide polymorphisms detected by whole-genome sequencing, we phylogenetically characterized 74 MTBC isolates from Brazzaville, the capital of Congo. The diversity of the study population was high; most strains belonged to the Euro-American lineage, which split into Latin American Mediterranean, Uganda I, Uganda II, Haarlem, X type, and a new dominant sublineage named Congo type (n = 26). Thirty strains were grouped in 5 clusters (each within 12 single-nucleotide polymorphisms), from which 23 belonged to the Congo type. High cluster rates and low genomic diversity indicate recent emergence and transmission of the Congo type, a new Euro-American sublineage of MTBC.

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

A Natural Polymorphism in rDNA Replication Origins Links Origin Activation with Calorie Restriction and Lifespan

PubMed Central

Kwan, Elizabeth X.; Foss, Eric J.; Tsuchiyama, Scott; Alvino, Gina M.; Kruglyak, Leonid; Kaeberlein, Matt; Raghuraman, M. K.; Brewer, Bonita J.; Kennedy, Brian K.; Bedalov, Antonio

2013-01-01

Aging and longevity are complex traits influenced by genetic and environmental factors. To identify quantitative trait loci (QTLs) that control replicative lifespan, we employed an outbred Saccharomyces cerevisiae model, generated by crossing a vineyard and a laboratory strain. The predominant QTL mapped to the rDNA, with the vineyard rDNA conferring a lifespan increase of 41%. The lifespan extension was independent of Sir2 and Fob1, but depended on a polymorphism in the rDNA origin of replication from the vineyard strain that reduced origin activation relative to the laboratory origin. Strains carrying vineyard rDNA origins have increased capacity for replication initiation at weak plasmid and genomic origins, suggesting that inability to complete genome replication presents a major impediment to replicative lifespan. Calorie restriction, a conserved mediator of lifespan extension that is also independent of Sir2 and Fob1, reduces rDNA origin firing in both laboratory and vineyard rDNA. Our results are consistent with the possibility that calorie restriction, similarly to the vineyard rDNA polymorphism, modulates replicative lifespan through control of rDNA origin activation, which in turn affects genome replication dynamics. PMID:23505383

A natural polymorphism in rDNA replication origins links origin activation with calorie restriction and lifespan.

PubMed

Kwan, Elizabeth X; Foss, Eric J; Tsuchiyama, Scott; Alvino, Gina M; Kruglyak, Leonid; Kaeberlein, Matt; Raghuraman, M K; Brewer, Bonita J; Kennedy, Brian K; Bedalov, Antonio

2013-01-01

Aging and longevity are complex traits influenced by genetic and environmental factors. To identify quantitative trait loci (QTLs) that control replicative lifespan, we employed an outbred Saccharomyces cerevisiae model, generated by crossing a vineyard and a laboratory strain. The predominant QTL mapped to the rDNA, with the vineyard rDNA conferring a lifespan increase of 41%. The lifespan extension was independent of Sir2 and Fob1, but depended on a polymorphism in the rDNA origin of replication from the vineyard strain that reduced origin activation relative to the laboratory origin. Strains carrying vineyard rDNA origins have increased capacity for replication initiation at weak plasmid and genomic origins, suggesting that inability to complete genome replication presents a major impediment to replicative lifespan. Calorie restriction, a conserved mediator of lifespan extension that is also independent of Sir2 and Fob1, reduces rDNA origin firing in both laboratory and vineyard rDNA. Our results are consistent with the possibility that calorie restriction, similarly to the vineyard rDNA polymorphism, modulates replicative lifespan through control of rDNA origin activation, which in turn affects genome replication dynamics.

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

Ribosomal DNA analysis of tsetse and non-tsetse transmitted Ethiopian Trypanosoma vivax strains in view of improved molecular diagnosis.

PubMed

Fikru, Regassa; Matetovici, Irina; Rogé, Stijn; Merga, Bekana; Goddeeris, Bruno Maria; Büscher, Philippe; Van Reet, Nick

2016-04-15

Animal trypanosomosis caused by Trypanosoma vivax (T. vivax) is a devastating disease causing serious economic losses. Most molecular diagnostics for T. vivax infection target the ribosomal DNA locus (rDNA) but are challenged by the heterogeneity among T. vivax strains. In this study, we investigated the rDNA heterogeneity of Ethiopian T. vivax strains in relation to their presence in tsetse-infested and tsetse-free areas and its effect on molecular diagnosis. We sequenced the rDNA loci of six Ethiopian (three from tsetse-infested and three from tsetse-free areas) and one Nigerian T. vivax strain. We analysed the obtained sequences in silico for primer-mismatches of some commonly used diagnostic PCR assays and for GC content. With these data, we selected some rDNA diagnostic PCR assays for evaluation of their diagnostic accuracy. Furthermore we constructed two phylogenetic networks based on sequences within the smaller subunit (SSU) of 18S and within the 5.8S and internal transcribed spacer 2 (ITS2) to assess the relatedness of Ethiopian T. vivax strains to strains from other African countries and from South America. In silico analysis of the rDNA sequence showed important mismatches of some published diagnostic PCR primers and high GC content of T. vivax rDNA. The evaluation of selected diagnostic PCR assays with specimens from cattle under natural T. vivax challenge showed that this high GC content interferes with the diagnostic accuracy of PCR, especially in cases of mixed infections with T. congolense. Adding betain to the PCR reaction mixture can enhance the amplification of T. vivax rDNA but decreases the sensitivity for T. congolense and Trypanozoon. The networks illustrated that Ethiopian T. vivax strains are considerably heterogeneous and two strains (one from tsetse-infested and one from tsetse-free area) are more related to the West African and South American strains than to the East African strains. The rDNA locus sequence of six Ethiopian T. vivax strains showed important differences and higher GC content compared to other animal trypanosomes but could not be related to their origin from tsetse-infested or tsetse-free area. The high GC content of T. vivax DNA renders accurate diagnosis of all pathogenic animal trypanosomes with one single PCR problematic. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Improved Hardware for Higher Spatial Resolution Strain-ENCoded (SENC) Breast MRI for Strain Measurements

PubMed Central

Harouni, Ahmed A.; Hossain, Jakir; Jacobs, Michael A.; Osman, Nael F.

2012-01-01

Introduction Early detection of breast lesions using mammography has resulted in lower mortality-rates. However, some breast lesions are mammography occult and magnetic resonance imaging (MRI) is recommended, but has lower specificity. It is possible to achieve higher specificity by using Strain-ENCoded (SENC) MRI and/or magnetic resonance elastography(MRE). SENC breast MRI can measure the strain properties of breast tissue. Similarly, MRE is used to measure elasticity (i.e., shear stiffness) of different tissue compositions interrogating the tissue mechanical properties. Reports have shown that malignant tumors are 3–13 times stiffer than normal tissue and benign tumors. Methods We have developed a Strain-ENCoded (SENC) breast hardware device capable of periodically compressing the breast, thus allowing for longer scanning time and measuring the strain characteristics of breast tissue. This hardware enabled us to use SENC MRI with high spatial resolution (1×1×5mm3) instead of Fast SENC(FSENC). Simple controls and multiple safety measures were added to ensure accurate, repeatable and safe in-vivo experiments. Results Phantom experiments showed that SENC breast MRI has higher SNR and CNR than FSENC under different scanning resolutions. Finally, the SENC breast device reproducibility measurements resulted in a difference of less than one mm with a 1% strain difference. Conclusion SENC breast MR images have higher SNR and CNR than FSENC images. Thus, combining SENC breast strain measurements with diagnostic breast MRI to differentiate benign from malignant lesions could potentially increase the specificity of diagnosis in the clinical setting. PMID:21440464

Comparison of strain and acoustic radiation force impulse elastography of breast lesions by qualitative evaluation.

PubMed

Zhao, Qing; Wang, Xiao-Lei; Sun, Jia-Wei; Jiang, Zhao-Peng; Tao, Lin; Zhou, Xian-Li

2018-04-13

To compare the diagnostic performance of conventional strain elastography (CSE) and acoustic radiation force impulse (ARFI) induced SE for qualitative assessment of breast lesions and evaluate the additional value of the two techniques combined with Breast Imaging Reporting and Data System (BI-RADS) respectively for the differentiation of benign and malignant breast lesions. In a cohort of 110 women, the conventional ultrasound (US) features and the elasticity scores of CSE and ARFI induced SE were recorded. The diagnostic performances of BI-RADS, elastography and BI-RADS plus elastography were evaluated, including the area under the receiver operating characteristic curve (AUROC), sensitivity, specificity and accuracy. Pathologically, there were forty-eight malignant and sixty-two benign breast lesions in the final analysis. The AUCs for CSE and ARFI induced SE are similar (CSE, 0.807; ARFI induced SE, 0.846; p > 0.05), however, the specificity of the latter method was significantly higher than that of CSE (83.9% vs. 58.1%, p = 0.004) in differentiating breast lesions. The accuracy and specificity of BI-RADS plus ARFI induced SE (84.5%, 80.6%, respectively) were significantly higher than BI-RADS alone (73.6%, 54.8%, respectively) and BI-RADS plus conventional SE (72.7%, 56.5%, respectively), respectively (p < 0.05) without loss of sensitivity. Our study showed that BI-RADS plus ARFI induced SE had a better diagnostic performance in the diagnosis of breast lesions in comparison with BI-RADS alone or BI-RADS plus CSE.

Prevalence of Streptococcus suis Genotypes in Wild Boars of Northwestern Germany▿

PubMed Central

Baums, Christoph G.; Verkühlen, Gerd Josef; Rehm, Thomas; Silva, Luciana M. G.; Beyerbach, Martin; Pohlmeyer, Klaus; Valentin-Weigand, Peter

2007-01-01

Invasive serotype 2 (cps2+) strains of Streptococcus suis cause meningitis in pigs and humans. Four case reports of S. suis meningitis in hunters suggest transmission of S. suis through the butchering of wild boars. Therefore, the objective of this study was to investigate the prevalence of potentially human-pathogenic S. suis strains in wild boars. S. suis was isolated from 92% of all tested tonsils (n = 200) from wild boars. A total of 244 S. suis isolates were genotyped using PCR assays for the detection of serotype-specific genes, the hemolysin gene sly, and the virulence-associated genes mrp and epf. The prevalence of the cps2+ genotype among strains from wild boars was comparable to that of control strains from domestic pig carriers. Ninety-five percent of the cps2+ wild boar strains were positive for mrp, sly, and epf*, the large variant of epf. Interestingly, epf* was significantly more frequently detected in cps2+ strains from wild boars than in those from domestic pigs; epf* is also typically found in European S. suis isolates from humans, including a meningitis isolate from a German hunter. These results suggest that at least 10% of wild boars in Northwestern Germany carry S. suis strains that are potentially virulent in humans. Additional amplified fragment length polymorphism analysis supported this hypothesis, since homogeneous clustering of the epf* mrp+ sly+ cps2+ strains from wild boars with invasive human and porcine strains was observed. PMID:17085699

Prevalence of Streptococcus suis genotypes in wild boars of Northwestern Germany.

PubMed

Baums, Christoph G; Verkühlen, Gerd Josef; Rehm, Thomas; Silva, Luciana M G; Beyerbach, Martin; Pohlmeyer, Klaus; Valentin-Weigand, Peter

2007-02-01

Invasive serotype 2 (cps2+) strains of Streptococcus suis cause meningitis in pigs and humans. Four case reports of S. suis meningitis in hunters suggest transmission of S. suis through the butchering of wild boars. Therefore, the objective of this study was to investigate the prevalence of potentially human-pathogenic S. suis strains in wild boars. S. suis was isolated from 92% of all tested tonsils (n=200) from wild boars. A total of 244 S. suis isolates were genotyped using PCR assays for the detection of serotype-specific genes, the hemolysin gene sly, and the virulence-associated genes mrp and epf. The prevalence of the cps2+ genotype among strains from wild boars was comparable to that of control strains from domestic pig carriers. Ninety-five percent of the cps2+ wild boar strains were positive for mrp, sly, and epf*, the large variant of epf. Interestingly, epf* was significantly more frequently detected in cps2+ strains from wild boars than in those from domestic pigs; epf* is also typically found in European S. suis isolates from humans, including a meningitis isolate from a German hunter. These results suggest that at least 10% of wild boars in Northwestern Germany carry S. suis strains that are potentially virulent in humans. Additional amplified fragment length polymorphism analysis supported this hypothesis, since homogeneous clustering of the epf* mrp+ sly+ cps2+ strains from wild boars with invasive human and porcine strains was observed.

First International External Quality Assessment Study on Molecular and Serological Methods for Yellow Fever Diagnosis

PubMed Central

Domingo, Cristina; Escadafal, Camille; Rumer, Leonid; Méndez, Jairo A.; García, Paquita; Sall, Amadou A.; Teichmann, Anette; Donoso-Mantke, Oliver; Niedrig, Matthias

2012-01-01

Objective We describe an external quality assurance (EQA) study designed to assess the efficiency and accurateness of molecular and serological methods used by expert laboratories performing YF diagnosis. Study Design For molecular diagnosis evaluation, a panel was prepared of 14 human plasma samples containing specific RNA of different YFV strains (YFV-17D, YFV South American strain [Brazil], YFV IvoryC1999 strain), and specificity samples containing other flaviviruses and negative controls. For the serological panel, 13 human plasma samples with anti-YFV-specific antibodies against different strains of YFV (YFV-17D strain, YFV IvoryC1999 strain, and YFV Brazilian strain), as well as specificity and negative controls, were included. Results Thirty-six laboratories from Europe, the Americas, Middle East, and Africa participated in these EQA activities. Only 16% of the analyses reported met all evaluation criteria with optimal performance. Serial dilutions of YFV-17D showed that in general the methodologies reported provided a suitable sensitivity. Failures were mainly due to the inability to detect wild-type strains or the presence of false positives. Performance in the serological diagnosis varied, mainly depending on the methodology used. Anti-YFV IgM detection was not performed in 16% of the reports using IIF or ELISA techniques, although it is preferable for the diagnosis of YFV acute infections. A good sensitivity profile was achieved in general; however, in the detection of IgM antibodies a lack of sensitivity of anti-YFV antibodies against the vaccine strain 17D was observed, and of the anti-YFV IgG antibodies against a West African strain. Neutralization assays showed a very good performance; however, the unexpected presence of false positives underlined the need of improving the running protocols. Conclusion This EQA provides information on each laboratory's efficacy of RT-PCR and serological YFV diagnosis techniques. The results indicate the need for improving serological and molecular diagnosis techniques and provide a follow-up of the diagnostic profiles. PMID:22570700

First international external quality assessment study on molecular and serological methods for yellow fever diagnosis.

PubMed

Domingo, Cristina; Escadafal, Camille; Rumer, Leonid; Méndez, Jairo A; García, Paquita; Sall, Amadou A; Teichmann, Anette; Donoso-Mantke, Oliver; Niedrig, Matthias

2012-01-01

We describe an external quality assurance (EQA) study designed to assess the efficiency and accurateness of molecular and serological methods used by expert laboratories performing YF diagnosis. For molecular diagnosis evaluation, a panel was prepared of 14 human plasma samples containing specific RNA of different YFV strains (YFV-17D, YFV South American strain [Brazil], YFV IvoryC1999 strain), and specificity samples containing other flaviviruses and negative controls. For the serological panel, 13 human plasma samples with anti-YFV-specific antibodies against different strains of YFV (YFV-17D strain, YFV IvoryC1999 strain, and YFV Brazilian strain), as well as specificity and negative controls, were included. Thirty-six laboratories from Europe, the Americas, Middle East, and Africa participated in these EQA activities. Only 16% of the analyses reported met all evaluation criteria with optimal performance. Serial dilutions of YFV-17D showed that in general the methodologies reported provided a suitable sensitivity. Failures were mainly due to the inability to detect wild-type strains or the presence of false positives. Performance in the serological diagnosis varied, mainly depending on the methodology used. Anti-YFV IgM detection was not performed in 16% of the reports using IIF or ELISA techniques, although it is preferable for the diagnosis of YFV acute infections. A good sensitivity profile was achieved in general; however, in the detection of IgM antibodies a lack of sensitivity of anti-YFV antibodies against the vaccine strain 17D was observed, and of the anti-YFV IgG antibodies against a West African strain. Neutralization assays showed a very good performance; however, the unexpected presence of false positives underlined the need of improving the running protocols. This EQA provides information on each laboratory's efficacy of RT-PCR and serological YFV diagnosis techniques. The results indicate the need for improving serological and molecular diagnosis techniques and provide a follow-up of the diagnostic profiles.

Secretome analysis of diarrhea-inducing strains of Escherichia coli

PubMed Central

Nirujogi, Raja Sekhar; Muthusamy, Babylakshmi; Kim, Min-Sik; Sathe, Gajanan J.; Lakshmi, P.T.V.; Kovbasnjuk, Olga N.; Prasad, T.S. Keshava; Wade, Mary; Jabbour, Rabih E.

2017-01-01

Secreted proteins constitute a major part of virulence factors that are responsible for pathogenesis caused by Gram-negative bacteria. Enterohemorrhagic Escherichia coli, O157:H7, is the major pathogen often causing outbreaks. However, studies have reported that the significant outbreaks caused by non-O157:H7 E. coli strains, also known as “Big-Six” serogroup strains, are increasing. There is no systematic study describing differential secreted proteins from these non-O157:H7 E. coli strains. In this study, we carried out MS-based differential secretome analysis using tandem mass tags labeling strategy of non-O157:H7 E. coli strains, O103, O111, O121, O145, O26, and O45. We identified 1241 proteins, of which 565 proteins were predicted to be secreted. We also found that 68 proteins were enriched in type III secretion system and several of them were differentially expressed across the strains. Additionally, we identified several strain-specific secreted proteins that could be used for developing potential markers for the identification and strain-level differentiation. To our knowledge, this study is the first comparative proteomic study on secretome of E. coli Big-Six serogroup and the several of these strain-specific secreted proteins can be further studied to develop potential markers for identification and strain-level differentiation. Moreover, the results of this study can be utilized in several applications, including food safety, diagnostics of E. coli outbreaks, and detection and identification of bio threats in biodefense. PMID:28070933

Potential of nanotechnology as a delivery platform against tuberculosis: current research review.

PubMed

Choudhary, S; Kusum Devi, V

2015-03-28

This review focusses on the current ongoing research in the field of tuberculosis comprising the resistant strains. It specifies a proper data analysis with results in concise form from areas gripping in: diagnostic nanotechnology, vaccine nanotechnology and the prime field of interest i.e., therapeutic nanotechnology. Primarily, therapeutic area recollects the research findings from advanced drug delivery (primary era) to the targeted drug delivery (modern era). The vaccine-based area derives the immune-specific targeting with enhanced emphasis on vaccine extraction and preparation of nanoparticles. Finally, the diagnostic area signifies the imaging techniques that may be employed in the diagnosis of TB. Not only that, there are some researches that emphasized on finding the comparable diagnostic differences between normal and resistant strains. With the advent of carbon nanotubes, metallic NPs, a newer hope has emerged out in diagnostic research, which may extend to therapeutic research applications too. Modifications of natural polymers, least or no use of organic solvents, size controlled NPs, optimized methodology, etc., are fields that need more effort to bypass toxicity. If above desired possibilities get the priority during research, it may lead to shift in the timeline towards much more oriented research. Copyright © 2015 Elsevier B.V. All rights reserved.

Genomic Correlates of Relationship QTL Involved in Fore- versus Hind Limb Divergence in Mice

PubMed Central

Pavlicev, Mihaela; Wagner, Günter P.; Noonan, James P.; Hallgrímsson, Benedikt; Cheverud, James M.

2013-01-01

Divergence of serially homologous elements of organisms is a common evolutionary pattern contributing to increased phenotypic complexity. Here, we study the genomic intervals affecting the variational independence of fore- and hind limb traits within an experimental mouse population. We use an advanced intercross of inbred mouse strains to map the loci associated with the degree of autonomy between fore- and hind limb long bone lengths (loci affecting the relationship between traits, relationship quantitative trait loci [rQTL]). These loci have been proposed to interact locally with the products of pleiotropic genes, thereby freeing the local trait from the variational constraint due to pleiotropic mutations. Using the known polymorphisms (single nucleotide polymorphisms [SNPs]) between the parental strains, we characterized and compared the genomic regions in which the rQTL, as well as their interaction partners (intQTL), reside. We find that these two classes of QTL intervals harbor different kinds of molecular variation. SNPs in rQTL intervals more frequently reside in limb-specific cis-regulatory regions than SNPs in intQTL intervals. The intQTL loci modified by the rQTL, in contrast, show the signature of protein-coding variation. This result is consistent with the widely accepted view that protein-coding mutations have broader pleiotropic effects than cis-regulatory polymorphisms. For both types of QTL intervals, the underlying candidate genes are enriched for genes involved in protein binding. This finding suggests that rQTL effects are caused by local interactions among the products of the causal genes harbored in rQTL and intQTL intervals. This is the first study to systematically document the population-level molecular variation underlying the evolution of character individuation. PMID:24065733

The role of the immunological background of mice in the genetic variability of Schistosoma mansoni as detected by random amplification of polymorphic DNA.

PubMed

Cossa-Moiane, I L; Mendes, T; Ferreira, T M; Mauricio, I; Calado, M; Afonso, A; Belo, S

2015-11-01

Schistosomiasis is a parasitic disease caused by flatworms of the genus Schistosoma. Among the Schistosoma species known to infect humans, S. mansoni is the most frequent cause of intestinal schistosomiasis in sub-Saharan Africa and South America: the World Health Organization estimates that about 200,000 deaths per year result from schistosomiasis in sub-Saharan Africa alone. The Schistosoma life cycle requires two different hosts: a snail as intermediate host and a mammal as definitive host. People become infected when they come into contact with water contaminated with free-living larvae (e.g. when swimming, fishing, washing). Although S. mansoni has mechanisms for escaping the host immune system, only a minority of infecting larvae develop into adults, suggesting that strain selection occurs at the host level. To test this hypothesis, we compared the Belo Horizonte (BH) strain of S. mansoni recovered from definitive hosts with different immunological backgrounds using random amplification of polymorphic DNA-polymerase chain reaction (RAPD-PCR). Schistosoma mansoni DNA profiles of worms obtained from wild-type (CD1 and C57BL/6J) and mutant (Jα18- / - and TGFβRIIdn) mice were analysed. Four primers produced polymorphic profiles, which can therefore potentially be used as reference biomarkers. All male worms were genetically distinct from females isolated from the same host, with female worms showing more specific fragments than males. Of the four host-derived schistosome populations, female and male adults recovered from TGFβRIIdn mice showed RAPD-PCR profiles that were most similar to each other. Altogether, these data indicate that host immunological backgrounds can influence the genetic diversity of parasite populations.

Inter- and Intra-subtype genotypic differences that differentiate Mycobacterium avium subspecies paratuberculosis strains

PubMed Central

2012-01-01

Background Mycobacterium avium subspecies paratuberculosis (Map) is the aetiological agent of Johne’s disease or paratuberculosis and is included within the Mycobacterium avium complex (MAC). Map strains are of two major types often referred to as ‘Sheep’ or ‘S-type’ and ‘Cattle’ or ‘C-type’. With the advent of more discriminatory typing techniques it has been possible to further classify the S-type strains into two groups referred to as Type I and Type III. This study was undertaken to genotype a large panel of S-type small ruminant isolates from different hosts and geographical origins and to compare them with a large panel of well documented C-type isolates to assess the genetic diversity of these strain types. Methods used included Mycobacterial Interspersed Repetitive Units - Variable-Number Tandem Repeat analysis (MIRU-VNTR), analysis of Large Sequence Polymorphisms by PCR (LSP analysis), Single Nucleotide Polymorphism (SNP) analysis of gyr genes, Pulsed-Field Gel Electrophoresis (PFGE) and Restriction Fragment Length Polymorphism analysis coupled with hybridization to IS900 (IS900-RFLP) analysis. Results The presence of LSPA4 and absence of LSPA20 was confirmed in all 24 Map S-type strains analysed. SNPs within the gyr genes divided the S-type strains into types I and III. Twenty four PFGE multiplex profiles and eleven different IS900-RFLP profiles were identified among the S-type isolates, some of them not previously published. Both PFGE and IS900-RFLP segregated the S-type strains into types I and III and the results concurred with those of the gyr SNP analysis. Nine MIRU-VNTR genotypes were identified in these isolates. MIRU-VNTR analysis differentiated Map strains from other members of Mycobacterium avium Complex, and Map S-type from C-type but not type I from III. Pigmented Map isolates were found of type I or III. Conclusion This is the largest panel of S-type strains investigated to date. The S-type strains could be further divided into two subtypes, I and III by some of the typing techniques (IS900-RFLP, PFGE and SNP analysis of the gyr genes). MIRU-VNTR did not divide the strains into the subtypes I and III but did detect genetic differences between isolates within each of the subtypes. Pigmentation is not exclusively associated with type I strains. PMID:23164429

Hemolysis, Toxicity, and Randomly Amplified Polymorphic DNA Analysis of Stachybotrys chartarum Strains

PubMed Central

Vesper, Stephen J.; Dearborn, Dorr G.; Yike, Iwona; Sorenson, W. G.; Haugland, Richard A.

1999-01-01

Stachybotrys chartarum is an indoor air, toxigenic fungus that has been associated with a number of human and veterinary health problems. Most notable among these has been a cluster of idiopathic pulmonary hemorrhage cases that were observed in the Cleveland, Ohio, area. In this study, 16 strains of S. chartarum isolated from case (n = 8) or control (n = 8) homes in Cleveland and 12 non-Cleveland strains from diverse geographic locations were analyzed for hemolytic activity, conidial toxicity, and randomly amplified polymorphic DNA banding patterns. In tests for hemolytic activity, strains were grown at 23°C on wet wallboard pieces for an 8-week test period. Conidia from these wallboard pieces were subcultured on sheep’s blood agar once a week over this period and examined for growth and clearing of the medium at 37 or 23°C. Five of the Cleveland strains (all from case homes) showed hemolytic activity at 37°C throughout the 8-week test compared to 3 of the non-Cleveland strains. Five of the Cleveland strains, compared to two of the non-Cleveland strains, produced highly toxic conidia (>90 μg of T2 toxin equivalents per g [wet weight] of conidia) after 10 and 30 days of growth on wet wallboard. Only 3 of the 28 strains examined both were consistently hemolytic and produced highly toxic conidia. Each of these strains was isolated from a house in Cleveland where an infant had idiopathic pulmonary hemorrhage. PMID:10388719

Hemolysis, toxicity, and randomly amplified polymorphic DNA analysis of Stachybotrys chartarum strains.

PubMed

Vesper, S J; Dearborn, D G; Yike, I; Sorenson, W G; Haugland, R A

1999-07-01

Stachybotrys chartarum is an indoor air, toxigenic fungus that has been associated with a number of human and veterinary health problems. Most notable among these has been a cluster of idiopathic pulmonary hemorrhage cases that were observed in the Cleveland, Ohio, area. In this study, 16 strains of S. chartarum isolated from case (n = 8) or control (n = 8) homes in Cleveland and 12 non-Cleveland strains from diverse geographic locations were analyzed for hemolytic activity, conidial toxicity, and randomly amplified polymorphic DNA banding patterns. In tests for hemolytic activity, strains were grown at 23 degrees C on wet wallboard pieces for an 8-week test period. Conidia from these wallboard pieces were subcultured on sheep's blood agar once a week over this period and examined for growth and clearing of the medium at 37 or 23 degrees C. Five of the Cleveland strains (all from case homes) showed hemolytic activity at 37 degrees C throughout the 8-week test compared to 3 of the non-Cleveland strains. Five of the Cleveland strains, compared to two of the non-Cleveland strains, produced highly toxic conidia (>90 microgram of T2 toxin equivalents per g [wet weight] of conidia) after 10 and 30 days of growth on wet wallboard. Only 3 of the 28 strains examined both were consistently hemolytic and produced highly toxic conidia. Each of these strains was isolated from a house in Cleveland where an infant had idiopathic pulmonary hemorrhage.

Next-generation sequencing: advances and applications in cancer diagnosis

PubMed Central

Serratì, Simona; De Summa, Simona; Pilato, Brunella; Petriella, Daniela; Lacalamita, Rosanna; Tommasi, Stefania; Pinto, Rosamaria

2016-01-01

Technological advances have led to the introduction of next-generation sequencing (NGS) platforms in cancer investigation. NGS allows massive parallel sequencing that affords maximal tumor genomic assessment. NGS approaches are different, and concern DNA and RNA analysis. DNA sequencing includes whole-genome, whole-exome, and targeted sequencing, which focuses on a selection of genes of interest for a specific disease. RNA sequencing facilitates the detection of alternative gene-spliced transcripts, posttranscriptional modifications, gene fusion, mutations/single-nucleotide polymorphisms, small and long noncoding RNAs, and changes in gene expression. Most applications are in the cancer research field, but lately NGS technology has been revolutionizing cancer molecular diagnostics, due to the many advantages it offers compared to traditional methods. There is greater knowledge on solid cancer diagnostics, and recent interest has been shown also in the field of hematologic cancer. In this review, we report the latest data on NGS diagnostic/predictive clinical applications in solid and hematologic cancers. Moreover, since the amount of NGS data produced is very large and their interpretation is very complex, we briefly discuss two bioinformatic aspects, variant-calling accuracy and copy-number variation detection, which are gaining a lot of importance in cancer-diagnostic assessment. PMID:27980425

Amplified Fragment Length Polymorphism Fingerprinting of Pseudomonas Strains from a Poultry Processing Plant

PubMed Central

Geornaras, Ifigenia; Kunene, Nokuthula F.; von Holy, Alexander; Hastings, John W.

1999-01-01

Molecular typing has been used previously to identify and trace dissemination of pathogenic and spoilage bacteria associated with food processing. Amplified fragment length polymorphism (AFLP) is a novel DNA fingerprinting technique which is considered highly reproducible and has high discriminatory power. This technique was used to fingerprint 88 Pseudomonas fluorescens and Pseudomonas putida strains that were previously isolated from plate counts of carcasses at six processing stages and various equipment surfaces and environmental sources of a poultry abattoir. Clustering of the AFLP patterns revealed a high level of diversity among the strains. Six clusters (clusters I through VI) were delineated at an arbitrary Dice coefficient level of 0.65; clusters III (31 strains) and IV (28 strains) were the largest clusters. More than one-half (52.3%) of the strains obtained from carcass samples, which may have represented the resident carcass population, grouped together in cluster III. By contrast, 43.2% of the strains from most of the equipment surfaces and environmental sources grouped together in cluster IV. In most cases, the clusters in which carcass strains from processing stages grouped corresponded to the clusters in which strains from the associated equipment surfaces and/or environmental sources were found. This provided evidence that there was cross-contamination between carcasses and the abattoir environment at the DNA level. The AFLP data also showed that strains were being disseminated from the beginning to the end of the poultry processing operation, since many strains associated with carcasses at the packaging stage were members of the same clusters as strains obtained from carcasses after the defeathering stage. PMID:10473382

Amplified fragment length polymorphism fingerprinting of Pseudomonas strains from a poultry processing plant.

PubMed

Geornaras, I; Kunene, N F; von Holy, A; Hastings, J W

1999-09-01

Molecular typing has been used previously to identify and trace dissemination of pathogenic and spoilage bacteria associated with food processing. Amplified fragment length polymorphism (AFLP) is a novel DNA fingerprinting technique which is considered highly reproducible and has high discriminatory power. This technique was used to fingerprint 88 Pseudomonas fluorescens and Pseudomonas putida strains that were previously isolated from plate counts of carcasses at six processing stages and various equipment surfaces and environmental sources of a poultry abattoir. Clustering of the AFLP patterns revealed a high level of diversity among the strains. Six clusters (clusters I through VI) were delineated at an arbitrary Dice coefficient level of 0.65; clusters III (31 strains) and IV (28 strains) were the largest clusters. More than one-half (52.3%) of the strains obtained from carcass samples, which may have represented the resident carcass population, grouped together in cluster III. By contrast, 43.2% of the strains from most of the equipment surfaces and environmental sources grouped together in cluster IV. In most cases, the clusters in which carcass strains from processing stages grouped corresponded to the clusters in which strains from the associated equipment surfaces and/or environmental sources were found. This provided evidence that there was cross-contamination between carcasses and the abattoir environment at the DNA level. The AFLP data also showed that strains were being disseminated from the beginning to the end of the poultry processing operation, since many strains associated with carcasses at the packaging stage were members of the same clusters as strains obtained from carcasses after the defeathering stage.

SnSAG5 is an alternative surface antigen of Sarcocystis neurona strains that is mutually exclusive to SnSAG1.

PubMed

Crowdus, Carolyn A; Marsh, Antoinette E; Saville, Willliam J; Lindsay, David S; Dubey, J P; Granstrom, David E; Howe, Daniel K

2008-11-25

Sarcocystis neurona is an obligate intracellular parasite that causes equine protozoal myeloencephalitis (EPM). Previous work has identified a gene family of paralogous surface antigens in S. neurona called SnSAGs. These surface proteins are immunogenic in their host animals, and are therefore candidate molecules for development of diagnostics and vaccines. However, SnSAG diversity exists in strains of S. neurona, including the absence of the major surface antigen gene SnSAG1. Instead, sequence for an alternative SnSAG has been revealed in two of the SnSAG1-deficient strains. Herein, we present data characterizing this new surface protein, which we have designated SnSAG5. The results indicated that the protein encoded by the SnSAG5 sequence is indeed a surface-associated molecule that has characteristics consistent with the other SAGs identified in S. neurona and related parasites. Importantly, Western blot analyses of a collection of S. neurona strains demonstrated that 6 of 13 parasite isolates express SnSAG5 as a dominant surface protein instead of SnSAG1. Conversely, SnSAG5 was not detected in SnSAG1-positive strains. One strain, which was isolated from the brain of a sea otter, did not express either SnSAG1 or SnSAG5. Genetic analysis with SnSAG5-specific primers confirmed the presence of the SnSAG5 gene in Western blot-positive strains, while also suggesting the presence of a novel SnSAG sequence in the SnSAG1-deficient, SnSAG5-deficient otter isolate. The findings provide further indication of S. neurona strain diversity, which has implications for diagnostic testing and development of vaccines against EPM as well as the population biology of Sarcocystis cycling in the opossum definitive host.

High Variation in Pathogenicity of Genetically Closely Related Strains of Xanthomonas albilineans, the Sugarcane Leaf Scald Pathogen, in Guadeloupe.

PubMed

Champoiseau, P; Daugrois, J-H; Pieretti, I; Cociancich, S; Royer, M; Rott, P

2006-10-01

ABSTRACT Pathogenicity of 75 strains of Xanthomonas albilineans from Guadeloupe was assessed by inoculation of sugarcane cv. B69566, which is susceptible to leaf scald, and 19 of the strains were selected as representative of the variation in pathogenicity observed based on stalk colonization. In vitro production of albicidin varied among these 19 strains, but the restriction fragment length polymorphism pattern of their albicidin biosynthesis genes was identical. Similarly, no genomic variation was found among strains by pulsed-field gel electrophoresis. Some variation among strains was found by amplified fragment length polymorphism, but no relationship between this genetic variation and variation in pathogenicity was found. Only 3 (pilB, rpfA, and xpsE) of 40 genes involved in pathogenicity of bacterial species closely related to X. albilineans could be amplified by polymerase chain reaction from total genomic DNA of all nine strains tested of X. albilineans differing in pathogenicity in Guadeloupe. Nucleotide sequences of these genes were 100% identical among strains, and a phylogenetic study with these genes and housekeeping genes efp and ihfA suggested that X. albilineans is on an evolutionary road between the X. campestris group and Xylella fastidiosa, another vascular plant pathogen. Sequencing of the complete genome of Xanthomonas albilineans could be the next step in deciphering molecular mechanisms involved in pathogenicity of X. albilineans.

Whole-genome sequencing of the efficient industrial fuel-ethanol fermentative Saccharomyces cerevisiae strain CAT-1.

PubMed

Babrzadeh, Farbod; Jalili, Roxana; Wang, Chunlin; Shokralla, Shadi; Pierce, Sarah; Robinson-Mosher, Avi; Nyren, Pål; Shafer, Robert W; Basso, Luiz C; de Amorim, Henrique V; de Oliveira, Antonio J; Davis, Ronald W; Ronaghi, Mostafa; Gharizadeh, Baback; Stambuk, Boris U

2012-06-01

The Saccharomyces cerevisiae strains widely used for industrial fuel-ethanol production have been developed by selection, but their underlying beneficial genetic polymorphisms remain unknown. Here, we report the draft whole-genome sequence of the S. cerevisiae strain CAT-1, which is a dominant fuel-ethanol fermentative strain from the sugarcane industry in Brazil. Our results indicate that strain CAT-1 is a highly heterozygous diploid yeast strain, and the ~12-Mb genome of CAT-1, when compared with the reference S228c genome, contains ~36,000 homozygous and ~30,000 heterozygous single nucleotide polymorphisms, exhibiting an uneven distribution among chromosomes due to large genomic regions of loss of heterozygosity (LOH). In total, 58 % of the 6,652 predicted protein-coding genes of the CAT-1 genome constitute different alleles when compared with the genes present in the reference S288c genome. The CAT-1 genome contains a reduced number of transposable elements, as well as several gene deletions and duplications, especially at telomeric regions, some correlated with several of the physiological characteristics of this industrial fuel-ethanol strain. Phylogenetic analyses revealed that some genes were likely associated with traits important for bioethanol production. Identifying and characterizing the allelic variations controlling traits relevant to industrial fermentation should provide the basis for a forward genetics approach for developing better fermenting yeast strains.

Comparison of Haemophilus parasuis reference strains and field isolates by using random amplified polymorphic DNA and protein profiles

PubMed Central

2012-01-01

Background Haemophilus parasuis is the causative agent of Glässer’s disease and is a pathogen of swine in high-health status herds. Reports on serotyping of field strains from outbreaks describe that approximately 30% of them are nontypeable and therefore cannot be traced. Molecular typing methods have been used as alternatives to serotyping. This study was done to compare random amplified polymorphic DNA (RAPD) profiles and whole cell protein (WCP) lysate profiles as methods for distinguishing H. parasuis reference strains and field isolates. Results The DNA and WCP lysate profiles of 15 reference strains and 31 field isolates of H. parasuis were analyzed using the Dice and neighbor joining algorithms. The results revealed unique and reproducible DNA and protein profiles among the reference strains and field isolates studied. Simpson’s index of diversity showed significant discrimination between isolates when three 10mer primers were combined for the RAPD method and also when both the RAPD and WCP lysate typing methods were combined. Conclusions The RAPD profiles seen among the reference strains and field isolates did not appear to change over time which may reflect a lack of DNA mutations in the genes of the samples. The recent field isolates had different WCP lysate profiles than the reference strains, possibly because the number of passages of the type strains may affect their protein expression. PMID:22703293

Development of two real-time polymerase chain reaction assays to detect Actinobacillus pleuropneumoniae serovars 1-9-11 and serovar 2.

PubMed

Marois-Créhan, Corinne; Lacouture, Sonia; Jacques, Mario; Fittipaldi, Nahuel; Kobisch, Marylène; Gottschalk, Marcelo

2014-01-01

Two real-time, or quantitative, polymerase chain reaction (qPCR) assays were developed to detect Actinobacillus pleuropneumoniae serovars 1-9-11 (highly related serovars with similar virulence potential) and serovar 2, respectively. The specificity of these assays was verified on a collection of 294 strains, which included all 16 reference A. pleuropneumoniae strains (including serovars 5a and 5b), 263 A. pleuropneumoniae field strains isolated between 1992 and 2009 in different countries, and 15 bacterial strains other than A. pleuropneumoniae. The detection levels of both qPCR tests were evaluated using 10-fold dilutions of chromosomal DNA from reference strains of A. pleuropneumoniae serovars 1 and 2, and the detection limit for both assays was 50 fg per assay. The analytical sensitivities of the qPCR tests were also estimated by using pure cultures and tonsils experimentally spiked with A. pleuropneumoniae. The detection threshold was 2.5 × 10(4) colony forming units (CFU)/ml and 2.9 × 10(5) CFU/0.1 g of tonsil, respectively, for both assays. These specific and sensitive tests can be used for the serotyping of A. pleuropneumoniae in diagnostic laboratories to control porcine pleuropneumonia.

Mitochondrial bioenergetics and drug-induced toxicity in a panel of mouse embryonic fibroblasts with mitochondrial DNA single nucleotide polymorphisms

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

Pereira, Claudia V.; Oliveira, Paulo J.; Will, Yvonne

2012-10-15

Mitochondrial DNA (mtDNA) variations including single nucleotide polymorphisms (SNPs) have been proposed to be involved in idiosyncratic drug reactions. However, current in vitro and in vivo models lack the genetic diversity seen in the human population. Our hypothesis is that different cell strains with distinct mtDNA SNPs may have different mitochondrial bioenergetic profiles and may therefore vary in their response to drug-induced toxicity. Therefore, we used an in vitro system composed of four strains of mouse embryonic fibroblasts (MEFs) with mtDNA polymorphisms. We sequenced mtDNA from embryonic fibroblasts isolated from four mouse strains, C57BL/6J, MOLF/EiJ, CZECHII/EiJ and PERA/EiJ, with themore » latter two being sequenced for the first time. The bioenergetic profile of the four strains of MEFs was investigated at both passages 3 and 10. Our results showed that there were clear differences among the four strains of MEFs at both passages, with CZECHII/EiJ having a lower mitochondrial robustness when compared to C57BL/6J, followed by MOLF/EiJ and PERA/EiJ. Seven drugs known to impair mitochondrial function were tested for their effect on the ATP content of the four strains of MEFs in both glucose- and galactose-containing media. Our results showed that there were strain-dependent differences in the response to some of the drugs. We propose that this model is a useful starting point to study compounds that may cause mitochondrial off-target toxicity in early stages of drug development, thus decreasing the number of experimental animals used. -- Highlights: ► mtDNA SNPs may be linked to individual predisposition to drug-induced toxicity. ► CZECHII/EiJ and PERA/EiJ mtDNA was sequenced for the first time in this study. ► Strain-dependent mitochondrial capacity differences were measured. ► Strain-dependent differences in response to mitochondrial toxicants were observed.« less

An Ecological Study of Lactococci Isolated from Raw Milk in the Camembert Cheese Registered Designation of Origin Area

PubMed Central

Corroler, D.; Mangin, I.; Desmasures, N.; Gueguen, M.

1998-01-01

The genetic diversity of lactococci isolated from raw milk in the Camembert cheese Registered Designation of Origin area was studied. Two seasonal samples (winter and summer) of raw milk were obtained from six farms in two areas (Bessin and Bocage Falaisien) of Normandy. All of the strains analyzed had a Lactococcus lactis subsp. lactis phenotype, whereas the randomly amplified polymorphic DNA (RAPD) technique genotypically identified the strains as members of L. lactis subsp. lactis or L. lactis subsp. cremoris. The genotypes were confirmed by performing standard PCR with primers corresponding to a region of the histidine biosynthesis operon. The geographic distribution of each subspecies of L. lactis was determined; 80% of the Bocage Falaisien strains were members of L. lactis subsp. lactis, and 30.5% of the Bessin strains were members of L. lactis subsp. lactis. A dendrogram was produced from a computer analysis of the RAPD profiles in order to evaluate the diversity of the lactococci below the subspecies level. The coefficient of similarity for 117 of the 139 strains identified as members of L. lactis subsp. cremoris was as high as 66%. The L. lactis subsp. lactis strains were more heterogeneous and formed 10 separate clusters (the level of similarity among the clusters was 18%). Reference strains of L. lactis subsp. lactis fell into 2 of these 10 clusters, demonstrating that lactococcal isolates are clearly different. As determined by the RAPD profiles, some L. lactis subsp. lactis strains were specific to the farms from which they originated and were recovered throughout the year (in both summer and winter). Therefore, the typicality of L. lactis subsp. lactis strains was linked to the farm of origin rather than the area. These findings emphasize the significance of designation of origin and the specificity of “Camembert de Normandie” cheese. PMID:9835555

An ecological study of lactococci isolated from raw milk in the camembert cheese registered designation of origin area.

PubMed

Corroler, D; Mangin, I; Desmasures, N; Gueguen, M

1998-12-01

The genetic diversity of lactococci isolated from raw milk in the Camembert cheese Registered Designation of Origin area was studied. Two seasonal samples (winter and summer) of raw milk were obtained from six farms in two areas (Bessin and Bocage Falaisien) of Normandy. All of the strains analyzed had a Lactococcus lactis subsp. lactis phenotype, whereas the randomly amplified polymorphic DNA (RAPD) technique genotypically identified the strains as members of L. lactis subsp. lactis or L. lactis subsp. cremoris. The genotypes were confirmed by performing standard PCR with primers corresponding to a region of the histidine biosynthesis operon. The geographic distribution of each subspecies of L. lactis was determined; 80% of the Bocage Falaisien strains were members of L. lactis subsp. lactis, and 30.5% of the Bessin strains were members of L. lactis subsp. lactis. A dendrogram was produced from a computer analysis of the RAPD profiles in order to evaluate the diversity of the lactococci below the subspecies level. The coefficient of similarity for 117 of the 139 strains identified as members of L. lactis subsp. cremoris was as high as 66%. The L. lactis subsp. lactis strains were more heterogeneous and formed 10 separate clusters (the level of similarity among the clusters was 18%). Reference strains of L. lactis subsp. lactis fell into 2 of these 10 clusters, demonstrating that lactococcal isolates are clearly different. As determined by the RAPD profiles, some L. lactis subsp. lactis strains were specific to the farms from which they originated and were recovered throughout the year (in both summer and winter). Therefore, the typicality of L. lactis subsp. lactis strains was linked to the farm of origin rather than the area. These findings emphasize the significance of designation of origin and the specificity of "Camembert de Normandie" cheese.

Development of a multiplex Q-PCR to detect Trichoderma harzianum Rifai strain T22 in plant roots.

PubMed

Horn, Ivo R; van Rijn, Menno; Zwetsloot, Tom J J; Basmagi, Said; Dirks-Mulder, Anita; van Leeuwen, Willem B; Ravensberg, Willem J; Gravendeel, Barbara

2016-02-01

The fungal species Trichoderma harzianum is widely used as a biological agent in crop protection. To verify the continued presence of this fungus on plant roots manually inoculated with T. harzianum strain T22, a Q-PCR was designed using specific probes for this particular strain. To develop these molecular diagnostic tools, genome mining was first carried out to retrieve putative new regions by which different strains of T. harzianum could be distinguished. Subsequently, Sanger sequencing of the L-aminoacid oxidase gene (aox1) in T. harzianum was applied to determine the mutations differing between various strains isolated from the Trichoderma collection of Koppert Biological Systems. Based on the sequence information obtained, a set of hydrolysis probes was subsequently developed which discriminated T. harzianum T22 strains varying in only a single nucleotide. Probes designed for two strains uniquely recognized the respective strains in Q-PCR with a detection limit of 12,5ng DNA. Titration assays in which T. harzianum DNA from distinct strains was varied further underscored the specificity of the probes. Lastly, fungal DNA extracted from roots of greenhouse cultured tomato plants was analyzed using the probe-based assay. DNA from T. harzianum strain T22 could readily be identified on roots of greenhouse reared tomato plants inoculated with varying concentrations up to one week after treatment with a detection limit of 3e6 colony forming units of T. harzianum T22. We conclude that the Q-PCR method is a reliable and robust method for assessing the presence and quantity of T. harzianum strain T22 in manually inoculated plant material. Our method provides scope for the development of DNA based strain specific identification of additional strains of Trichoderma and other fungal biological control agents. Copyright © 2015 Elsevier B.V. All rights reserved.

Amino acid and structural variability of Yersinia pestis LcrV protein

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

Anisimov, A P; Dentovskaya, S V; Panfertsev, E A

2009-11-09

The LcrV protein is a multifunctional virulence factor and protective antigen of the plague bacterium which is generally conserved between the epidemic strains of Yersinia pestis. They investigated the diversity in the LcrV sequences among non-epidemic Y. pestis strains which have a limited virulence in selected animal models and for humans. Sequencing of lcrV genes from ten Y. pestis strains belonging to different phylogenetic groups (subspecies) showed that the LcrV proteins possess four major variable hotspots at positions 18, 72, 273, and 324-326. These major variations, together with other minor substitutions in amino acid sequences, allowed them to classify themore » LcrV alleles into five sequence types (A-E). They observed that the strains of different Y. pestis subspecies can have the same typ of LcrV, and different types of LcrV can exist within the same natural plague focus. The LcrV polymorphisms were structurally analyzed by comparing the modeled structures of LcrV from all available strains. All changes except one occurred either in flexible regions or on the surface of the protein, but local chemical properties (i.e. those of a hydrophobic, hydrophilic, amphipathic, or charged nature) were conserved across all of the strains. Polymorphisms in flexible and surface regions are likely subject to less selective pressure, and have a limited impact on the structure. In contrast, the substitution of tryptophan at position 113 with either glutamic acid or glycine likely has a serious influence on the regional structure of the protein, and these mutations might have an effect on the function of LcrV. The polymorphisms at positions 18, 72 and 273 were accountable for differences in oligomerization of LcrV. The importance of the latter property in emergence of epidemic strains of Y. pestis during evolution of this pathogen will need to be further investigated.« less

Molecular typing of Brucella melitensis endemic strains and differentiation from the vaccine strain Rev-1.

PubMed

Noutsios, Georgios T; Papi, Rigini M; Ekateriniadou, Loukia V; Minas, Anastasios; Kyriakidis, Dimitrios A

2012-03-01

In the present study forty-four Greek endemic strains of Br. melitensis and three reference strains were genotyped by Multi locus Variable Number Tandem Repeat (ML-VNTR) analysis based on an eight-base pair tandem repeat sequence that was revealed in eight loci of Br. melitensis genome. The forty-four strains were discriminated from the vaccine strain Rev-1 by Restriction Fragment Length Polymorphism (RFLP) and Denaturant Gradient Gel Electrophoresis (DGGE). The ML-VNTR analysis revealed that endemic, reference and vaccine strains are genetically closely related, while most of the loci tested (1, 2, 4, 5 and 7) are highly polymorphic with Hunter-Gaston Genetic Diversity Index (HGDI) values in the range of 0.939 to 0.775. Analysis of ML-VNTRs loci stability through in vitro passages proved that loci 1 and 5 are non stable. Therefore, vaccine strain can be discriminated from endemic strains by allele's clusters of loci 2, 4, 6 and 7. RFLP and DGGE were also employed to analyse omp2 gene and reveled different patterns among Rev-1 and endemic strains. In RFLP, Rev-1 revealed three fragments (282, 238 and 44 bp), while endemic strains two fragments (238 and 44 bp). As for DGGE, the electrophoretic mobility of Rev-1 is different from the endemic strains due to heterologous binding of DNA chains of omp2a and omp2b gene. Overall, our data show clearly that it is feasible to genotype endemic strains of Br. melitensis and differentiate them from vaccine strain Rev-1 with ML-VNTR, RFLP and DGGE techniques. These tools can be used for conventional investigations in brucellosis outbreaks.

Potential Application of Digitally Linked Tuberculosis Diagnostics for Real-Time Surveillance of Drug-Resistant Tuberculosis Transmission: Validation and Analysis of Test Results.

PubMed

Ng, Kamela Charmaine; Meehan, Conor Joseph; Torrea, Gabriela; Goeminne, Léonie; Diels, Maren; Rigouts, Leen; de Jong, Bouke Catherine; André, Emmanuel

2018-02-27

Tuberculosis (TB) is the highest-mortality infectious disease in the world and the main cause of death related to antimicrobial resistance, yet its surveillance is still paper-based. Rifampicin-resistant TB (RR-TB) is an urgent public health crisis. The World Health Organization has, since 2010, endorsed a series of rapid diagnostic tests (RDTs) that enable rapid detection of drug-resistant strains and produce large volumes of data. In parallel, most high-burden countries have adopted connectivity solutions that allow linking of diagnostics, real-time capture, and shared repository of these test results. However, these connected diagnostics and readily available test results are not used to their full capacity, as we have yet to capitalize on fully understanding the relationship between test results and specific rpoB mutations to elucidate its potential application to real-time surveillance. We aimed to validate and analyze RDT data in detail, and propose the potential use of connected diagnostics and associated test results for real-time evaluation of RR-TB transmission. We selected 107 RR-TB strains harboring 34 unique rpoB mutations, including 30 within the rifampicin resistance-determining region (RRDR), from the Belgian Coordinated Collections of Microorganisms, Antwerp, Belgium. We subjected these strains to Xpert MTB/RIF, GenoType MTBDRplus v2.0, and Genoscholar NTM + MDRTB II, the results of which were validated against the strains' available rpoB gene sequences. We determined the reproducibility of the results, analyzed and visualized the probe reactions, and proposed these for potential use in evaluating transmission. The RDT probe reactions detected most RRDR mutations tested, although we found a few critical discrepancies between observed results and manufacturers' claims. Based on published frequencies of probe reactions and RRDR mutations, we found specific probe reactions with high potential use in transmission studies: Xpert MTB/RIF probes A, Bdelayed, C, and Edelayed; Genotype MTBDRplus v2.0 WT2, WT5, and WT6; and Genoscholar NTM + MDRTB II S1 and S3. Inspection of probe reactions of disputed mutations may potentially resolve discordance between genotypic and phenotypic test results. We propose a novel approach for potential real-time detection of RR-TB transmission through fully using digitally linked TB diagnostics and shared repository of test results. To our knowledge, this is the first pragmatic and scalable work in response to the consensus of world-renowned TB experts in 2016 on the potential of diagnostic connectivity to accelerate efforts to eliminate TB. This is evidenced by the ability of our proposed approach to facilitate comparison of probe reactions between different RDTs used in the same setting. Integrating this proposed approach as a plug-in module to a connectivity platform will increase usefulness of connected TB diagnostics for RR-TB outbreak detection through real-time investigation of suspected RR-TB transmission cases based on epidemiologic linking. ©Kamela Charmaine Ng, Conor Joseph Meehan, Gabriela Torrea, Léonie Goeminne, Maren Diels, Leen Rigouts, Bouke Catherine de Jong, Emmanuel André. Originally published in JMIR Medical Informatics (http://medinform.jmir.org), 27.02.2018.

Development of a species-diagnostic marker for identification of the stingless bee Trigona pagdeni in Thailand.

PubMed

Thummajitsakul, Sirikul; Klinbunga, Sirawut; Sittipraneed, Siriporn

2010-04-01

A species-diagnostic SCAR marker for identification of the stingless bee (Trigona pagdeni Schwarz) was successfully developed. Initially, amplified fragment length polymorphism analysis was carried out across representatives of 12 stingless bee species using 64 primer combinations. A 284 bp band restrictively found in T. pagdeni was cloned and sequenced. A primer pair (CUTP1-F/R) was designed and tested for species-specificity in 15 stingless bees. The expected 163 bp fragment was successfully amplified in all examined individuals of T. pagdeni (129/129). Nevertheless, cross-species amplification was also observed in T. fimbriata (1/3), T. collina (11/112), T. laeviceps (1/12), and T. fuscobalteata (15/15), but not in other species. SSCP analysis of CUTP1 further differentiated T. fuscobalteata and T. collina from T. pagdeni. Although T. laeviceps, T. fimbriata, and T. pagdeni shared an identical SSCP genotype, they are not taxonomically problematic species.

Polymorphisms and resistance mutations in the protease and reverse transcriptase genes of HIV-1 F subtype Romanian strains.

PubMed

Paraschiv, Simona; Otelea, Dan; Dinu, Magdalena; Maxim, Daniela; Tinischi, Mihaela

2007-03-01

To evaluate the prevalence of resistance mutations in the genome of HIV-1 F subtype strains isolated from Romanian antiretroviral (ARV) treatment-naïve patients and to assess the phylogenetic relatedness of these strains with other HIV-1 strains. Twenty-nine HIV-1 strains isolated from treatment-naïve adolescents (n=15) and adults (n=14) were included in this study. Resistance genotyping was performed by using Big Dye Terminator chemistry provided by the ViroSeq Genotyping System. The sequences of the protease and reverse transcriptase genes were aligned (ClustalW) and a phylogenetic tree was built (MEGA 3 software). For subtyping purposes, all the nucleotide sequences were submitted to the Stanford database. All the studied strains were found to harbor accessory mutations in the protease gene. The most frequent mutation was M36I (29 of 29 strains), followed by L63T, K20R, and L10V. The number of polymorphisms associated with protease inhibitor resistance was different for the two age groups. Intraphylogenetic divergence was greater for adults than for adolescents infected in childhood. All the strains were found to belong to the F1 subtype. The phylogenetic analysis revealed that Romanian strains clustered together, but distinctly from F1 HIV-1 strains isolated in other parts of the world (Brazil, Finland, and Belgium). Protease secondary mutations are present with high frequency in the HIV-1 F subtype strains isolated from Romanian ARV treatment-naïve patients, but no major resistance mutations were found.

Quantification of Siderophore and Hemolysin from Stachybotrys chartarum Strains, Including a Strain Isolated from the Lung of a Child with Pulmonary Hemorrhage and Hemosiderosis

PubMed Central

Vesper, Stephen J.; Dearborn, Dorr G.; Elidemir, Okan; Haugland, Richard A.

2000-01-01

A strain of Stachybotrys chartarum was recently isolated from the lung of a pulmonary hemorrhage and hemosiderosis (PH) patient in Texas (designated the Houston strain). This is the first time that S. chartarum has been isolated from the lung of a PH patient. In this study, the Houston strain and 10 strains of S. chartarum isolated from case (n = 5) or control (n = 5) homes in Cleveland were analyzed for hemolytic activity, siderophore production, and relatedness as measured by random amplified polymorphic DNA analysis. PMID:10831457

Identification of promoter polymorphisms in the cytochrome P450 CYP6AY1 linked with insecticide resistance in the brown planthopper, Nilaparvata lugens.

PubMed

Pang, R; Li, Y; Dong, Y; Liang, Z; Zhang, Y; Zhang, W

2014-12-01

Imidacloprid resistance in the brown planthopper, Nilaparvata lugens, is primarily the result of the over-expression of cytochrome P450 monooxygenases. Here, a field-collected strain of N. lugens was shown to be highly resistant to both imidacloprid and buprofezin. Insecticide exposure and quantitative real-time PCR revealed that its resistance was mainly associated with a cytochrome P450 gene, CYP6AY1. CYP6AY1 is known to metabolize imidacloprid but its effect on buprofezin is unclear. In the 5'-untranslated region of CYP6AY1, a novel alternative splicing was detected. After a 1990-bp promoter region was cloned, its basal luciferase activity was assessed. Furthermore, genotyping studies identified 12 variations in the promoter region that discriminated between the field-collected and control strain. Finally, survival bioassays revealed a single nucleotide polymorphism and an insertion-deletion polymorphism linked to buprofezin and imidacloprid resistance. Mutagenesis of these sites enhanced the promoter activity of CYP6AY1. These results suggest that promoter polymorphisms may affect P450-mediated multiple insecticide resistance of pests. © 2014 The Royal Entomological Society.

Loop-mediated amplification of the Clavibacter michiganensis subsp. michiganensis micA gene is highly specific.

PubMed

Yasuhara-Bell, Jarred; Kubota, Ryo; Jenkins, Daniel M; Alvarez, Anne M

2013-12-01

Loop-mediated amplification (LAMP) was used to specifically identify Clavibacter michiganensis subsp. michiganensis, causal agent of bacterial canker of tomato. LAMP primers were developed to detect micA, a chromosomally stable gene that encodes a type II lantibiotic, michiganin A, which inhibits growth of other C. michiganensis subspecies. In all, 409 bacterial strains (351 C. michiganensis subsp. michiganensis and 58 non-C. michiganensis subsp. michiganensis) from a worldwide collection were tested with LAMP to determine its specificity. LAMP results were compared with genetic profiles established using polymerase chain reaction (PCR) amplification of seven genes (dnaA, ppaJ, pat-1, chpC, tomA, ppaA, and ppaC). C. michiganensis subsp. michiganensis strains produced eight distinct profiles. The LAMP reaction identified all C. michiganensis subsp. michiganensis strains and discriminated them from other C. michiganensis subspecies and non-Clavibacter bacteria. LAMP has advantages over immunodiagnostic and other molecular detection methods because of its specificity and isothermal nature, which allows for easy field application. The LAMP reaction is also not affected by as many inhibitors as PCR. This diagnostic tool has potential to provide an easy, one-step test for rapid identification of C. michiganensis subsp. michiganensis.

Lactobacillus nantensis sp. nov., isolated from French wheat sourdough.

PubMed

Valcheva, Rosica; Ferchichi, Mounir F; Korakli, Maher; Ivanova, Iskra; Gänzle, Michael G; Vogel, Rudi F; Prévost, Hervé; Onno, Bernard; Dousset, Xavier

2006-03-01

A polyphasic taxonomic study of the bacterial flora isolated from traditional French wheat sourdough, using phenotypic characterization and phylogenetic as well as genetic methods, revealed a consistent group of isolates that could not be assigned to any recognized species. These results were confirmed by randomly amplified polymorphic DNA and amplified fragment length polymorphism fingerprinting analyses. Cells were Gram-positive, homofermentative rods. Comparative 16S rRNA gene sequence analysis of the representative strain LP33T indicated that these strains belong to the genus Lactobacillus and that they formed a branch distinct from their closest relatives Lactobacillus farciminis, Lactobacillus alimentarius, Lactobacillus paralimentarius and Lactobacillus mindensis. DNA-DNA reassociation experiments with the three phylogenetically closest Lactobacillus species confirmed that LP33T (= DSM 16982T = CIP 108546T = TMW 1.1265T) represents the type strain of a novel species, for which the name Lactobacillus nantensis sp. nov. is proposed.

The Evolution of Vp1 Gene in Enterovirus C Species Sub-Group That Contains Types CVA-21, CVA-24, EV-C95, EV-C96 and EV-C99

PubMed Central

Smura, Teemu; Blomqvist, Soile; Vuorinen, Tytti; Ivanova, Olga; Samoilovich, Elena; Al-Hello, Haider; Savolainen-Kopra, Carita; Hovi, Tapani; Roivainen, Merja

2014-01-01

Genus Enterovirus (Family Picornaviridae,) consists of twelve species divided into genetically diverse types by their capsid protein VP1 coding sequences. Each enterovirus type can further be divided into intra-typic sub-clusters (genotypes). The aim of this study was to elucidate what leads to the emergence of novel enterovirus clades (types and genotypes). An evolutionary analysis was conducted for a sub-group of Enterovirus C species that contains types Coxsackievirus A21 (CVA-21), CVA-24, Enterovirus C95 (EV-C95), EV-C96 and EV-C99. VP1 gene datasets were collected and analysed to infer the phylogeny, rate of evolution, nucleotide and amino acid substitution patterns and signs of selection. In VP1 coding gene, high intra-typic sequence diversities and robust grouping into distinct genotypes within each type were detected. Within each type the majority of nucleotide substitutions were synonymous and the non-synonymous substitutions tended to cluster in distinct highly polymorphic sites. Signs of positive selection were detected in some of these highly polymorphic sites, while strong negative selection was indicated in most of the codons. Despite robust clustering to intra-typic genotypes, only few genotype-specific ‘signature’ amino acids were detected. In contrast, when different enterovirus types were compared, there was a clear tendency towards fixation of type-specific ‘signature’ amino acids. The results suggest that permanent fixation of type-specific amino acids is a hallmark associated with evolution of different enterovirus types, whereas neutral evolution and/or (frequency-dependent) positive selection in few highly polymorphic amino acid sites are the dominant forms of evolution when strains within an enterovirus type are compared. PMID:24695547

The evolution of Vp1 gene in enterovirus C species sub-group that contains types CVA-21, CVA-24, EV-C95, EV-C96 and EV-C99.

PubMed

Smura, Teemu; Blomqvist, Soile; Vuorinen, Tytti; Ivanova, Olga; Samoilovich, Elena; Al-Hello, Haider; Savolainen-Kopra, Carita; Hovi, Tapani; Roivainen, Merja

2014-01-01

Genus Enterovirus (Family Picornaviridae,) consists of twelve species divided into genetically diverse types by their capsid protein VP1 coding sequences. Each enterovirus type can further be divided into intra-typic sub-clusters (genotypes). The aim of this study was to elucidate what leads to the emergence of novel enterovirus clades (types and genotypes). An evolutionary analysis was conducted for a sub-group of Enterovirus C species that contains types Coxsackievirus A21 (CVA-21), CVA-24, Enterovirus C95 (EV-C95), EV-C96 and EV-C99. VP1 gene datasets were collected and analysed to infer the phylogeny, rate of evolution, nucleotide and amino acid substitution patterns and signs of selection. In VP1 coding gene, high intra-typic sequence diversities and robust grouping into distinct genotypes within each type were detected. Within each type the majority of nucleotide substitutions were synonymous and the non-synonymous substitutions tended to cluster in distinct highly polymorphic sites. Signs of positive selection were detected in some of these highly polymorphic sites, while strong negative selection was indicated in most of the codons. Despite robust clustering to intra-typic genotypes, only few genotype-specific 'signature' amino acids were detected. In contrast, when different enterovirus types were compared, there was a clear tendency towards fixation of type-specific 'signature' amino acids. The results suggest that permanent fixation of type-specific amino acids is a hallmark associated with evolution of different enterovirus types, whereas neutral evolution and/or (frequency-dependent) positive selection in few highly polymorphic amino acid sites are the dominant forms of evolution when strains within an enterovirus type are compared.

DNA Probes Show Genetic Variation in Cyanobacterial Symbionts of the Azolla Fern and a Closer Relationship to Free-Living Nostoc Strains than to Free-Living Anabaena Strains

PubMed Central

Plazinski, Jacek; Zheng, Qi; Taylor, Rona; Croft, Lynn; Rolfe, Barry G.; Gunning, Brian E. S.

1990-01-01

Twenty-two isolates of Anabaena azollae derived from seven Azolla species from various geographic and ecological sources were characterized by DNA-DNA hybridization. Cloned DNA fragments derived from the genomic sequences of three different A. azollae isolates were used to detect restriction fragment length polymorphism among all symbiotic anabaenas. DNA clones were radiolabeled and hybridized against southern blot transfers of genomic DNAs of different isolates of A. azollae digested with restriction endonucleases. Eight DNA probes were selected to identify the Anabaena strains tested. Two were strain specific and hybridized only to A. azollae strains isolated from Azolla microphylla or Azolla caroliniana. One DNA probe was section specific (hybridized only to anabaenas isolated from Azolla ferns representing the section Euazolla), and five other probes gave finer discrimination among anabaenas representing various ecotypes of Azolla species. These cloned genomic DNA probes identified 11 different genotypes of A. azollae isolates. These included three endosymbiotic genotypes within Azolla filiculoides species and two genotypes within both A. caroliniana and Azolla pinnata endosymbionts. Although we were not able to discriminate among anabaenas extracted from different ecotypes of Azolla nilotica, Azolla mexicina, Azolla rubra and Azolla microphylla species, each of the endosymbionts was easily identified as a unique genotype. When total DNA isolated from free-living Anabaena sp. strain PCC7120 was screened, none of the genomic DNA probes gave detectable positive hybridization. Total DNA of Nostoc cycas PCC7422 hybridized with six of eight genomic DNA fragments. These data imply that the dominant symbiotic organism in association with Azolla spp. is more closely related to Nostoc spp. than to free-living Anabaena spp. Images PMID:16348182

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.

Parent-Child Diagnostic Agreement on Anxiety Symptoms with a Structured Diagnostic Interview for Mental Disorders in Children.

PubMed

Popp, Lukka; Neuschwander, Murielle; Mannstadt, Sandra; In-Albon, Tina; Schneider, Silvia

2017-01-01

Objective: In clinical structured diagnostic interviews, diagnoses based on parent and child reports have low to moderate agreement. The aims of the present study are (1) to examine diagnostic agreement on anxiety disorders between parents and children on the levels of current and lifetime diagnostic category and diagnoses focusing in particular on diagnostic criteria and (2) to identify parent- and child-related predictors for diagnostic agreement. Method: The sample consisted of 166 parent-child dyads interviewed with the Structured Diagnostic Interview for Mental Disorders in Children (Kinder-DIPS, Schneider et al., 2009). The children (51.8% girls) were between the ages of 7 and 18 years ( M = 10.94; SD = 2.22). Results: Overall, parent-child agreement on the diagnostic category of anxiety disorder ( k = 0.21; k = 0.22) and the specific anxiety diagnoses (base rate > 10%) of social phobia, specific phobia and separation anxiety disorder ( k = 0.24-0.52; k = 0.19-0.43) and corresponding diagnostic criteria ( k = 0.22-0.67; k = 0.24-0.41) were low to moderate with the highest agreement on separation anxiety disorder ( k > 0.43). Lower maternal depression, and higher social support reported by mother and father were associated with higher parent-child agreement. Maternal depression was indicated as the strongest predictor. Parental sense of competence, parental anxiety, the amount of parent-child interaction and the child's age and gender had no predictive value. Conclusions: Parent-child agreement can be expected to be higher on the level of anxiety criteria compared to specific anxiety diagnoses and diagnostic anxiety category. Psychological strains in the family-especially maternal depression and low social support-lower the parent-child agreement on anxiety symptoms. Child- and relation-related variables (age, gender, amount of time parent(s) and children interact) play no role in the prediction of low parent-child agreement.

Parent-Child Diagnostic Agreement on Anxiety Symptoms with a Structured Diagnostic Interview for Mental Disorders in Children

PubMed Central

Popp, Lukka; Neuschwander, Murielle; Mannstadt, Sandra; In-Albon, Tina; Schneider, Silvia

2017-01-01

Objective: In clinical structured diagnostic interviews, diagnoses based on parent and child reports have low to moderate agreement. The aims of the present study are (1) to examine diagnostic agreement on anxiety disorders between parents and children on the levels of current and lifetime diagnostic category and diagnoses focusing in particular on diagnostic criteria and (2) to identify parent- and child-related predictors for diagnostic agreement. Method: The sample consisted of 166 parent-child dyads interviewed with the Structured Diagnostic Interview for Mental Disorders in Children (Kinder-DIPS, Schneider et al., 2009). The children (51.8% girls) were between the ages of 7 and 18 years (M = 10.94; SD = 2.22). Results: Overall, parent-child agreement on the diagnostic category of anxiety disorder (k = 0.21; k = 0.22) and the specific anxiety diagnoses (base rate > 10%) of social phobia, specific phobia and separation anxiety disorder (k = 0.24–0.52; k = 0.19–0.43) and corresponding diagnostic criteria (k = 0.22–0.67; k = 0.24–0.41) were low to moderate with the highest agreement on separation anxiety disorder (k > 0.43). Lower maternal depression, and higher social support reported by mother and father were associated with higher parent-child agreement. Maternal depression was indicated as the strongest predictor. Parental sense of competence, parental anxiety, the amount of parent-child interaction and the child's age and gender had no predictive value. Conclusions: Parent-child agreement can be expected to be higher on the level of anxiety criteria compared to specific anxiety diagnoses and diagnostic anxiety category. Psychological strains in the family—especially maternal depression and low social support—lower the parent-child agreement on anxiety symptoms. Child- and relation-related variables (age, gender, amount of time parent(s) and children interact) play no role in the prediction of low parent-child agreement. PMID:28396644

Advances toward fully automated in vivo assessment of oral epithelial dysplasia by nuclear endomicroscopy-A pilot study.

PubMed

Liese, Jan; Winter, Karsten; Glass, Änne; Bertolini, Julia; Kämmerer, Peer Wolfgang; Frerich, Bernhard; Schiefke, Ingolf; Remmerbach, Torsten W

2017-11-01

Uncertainties in detection of oral epithelial dysplasia (OED) frequently result from sampling error especially in inflammatory oral lesions. Endomicroscopy allows non-invasive, "en face" imaging of upper oral epithelium, but parameters of OED are unknown. Mucosal nuclei were imaged in 34 toluidine blue-stained oral lesions with a commercial endomicroscopy. Histopathological diagnosis showed four biopsies in "dys-/neoplastic," 23 in "inflammatory," and seven in "others" disease groups. Strength of different assessment strategies of nuclear scoring, nuclear count, and automated nuclear analysis were measured by area under ROC curve (AUC) to identify histopathological "dys-/neoplastic" group. Nuclear objects from automated image analysis were visually corrected. Best-performing parameters of nuclear-to-image ratios were the count of large nuclei (AUC=0.986) and 6-nearest neighborhood relation (AUC=0.896), and best parameters of nuclear polymorphism were the count of atypical nuclei (AUC=0.996) and compactness of nuclei (AUC=0.922). Excluding low-grade OED, nuclear scoring and count reached 100% sensitivity and 98% specificity for detection of dys-/neoplastic lesions. In automated analysis, combination of parameters enhanced diagnostic strength. Sensitivity of 100% and specificity of 87% were seen for distances of 6-nearest neighbors and aspect ratios even in uncorrected objects. Correction improved measures of nuclear polymorphism only. The hue of background color was stronger than nuclear density (AUC=0.779 vs 0.687) to detect dys-/neoplastic group indicating that macroscopic aspect is biased. Nuclear-to-image ratios are applicable for automated optical in vivo diagnostics for oral potentially malignant disorders. Nuclear endomicroscopy may promote non-invasive, early detection of dys-/neoplastic lesions by reducing sampling error. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Diagnostic accuracy of rectal mucosa biopsy testing for chronic wasting disease within white-tailed deer (Odocoileus virginianus) herds in North America:Effects of age,sex,polymorphism at PRNP codon 96,and disease progression

USDA-ARS?s Scientific Manuscript database

An effective live animal diagnostic test is needed to assist in the control of chronic wasting disease (CWD), which has spread through captive and wild herds of white-tailed deer (Odocoileus virginianus) in Canada and the United States. In the present study, the diagnostic accuracy of rectal mucosa ...

Comparative genomics of citric-acid producing Aspergillus niger ATCC 1015 versus enzyme-producing CBS 513.88

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

Andersen, Mikael R.; Salazar, Margarita; Schaap, Peter

2011-06-01

The filamentous fungus Aspergillus niger exhibits great diversity in its phenotype. It is found globally, both as marine and terrestrial strains, produces both organic acids and hydrolytic enzymes in high amounts, and some isolates exhibit pathogenicity. Although the genome of an industrial enzyme-producing A. niger strain (CBS 513.88) has already been sequenced, the versatility and diversity of this species compels additional exploration. We therefore undertook whole genome sequencing of the acidogenic A. niger wild type strain (ATCC 1015), and produced a genome sequence of very high quality. Only 15 gaps are present in the sequence and half the telomeric regionsmore » have been elucidated. Moreover, sequence information from ATCC 1015 was utilized to improve the genome sequence of CBS 513.88. Chromosome-level comparisons uncovered several genome rearrangements, deletions, a clear case of strain-specific horizontal gene transfer, and identification of 0.8 megabase of novel sequence. Single nucleotide polymorphisms per kilobase (SNPs/kb) between the two strains were found to be exceptionally high (average: 7.8, maximum: 160 SNPs/kb). High variation within the species was confirmed with exo-metabolite profiling and phylogenetics. Detailed lists of alleles were generated, and genotypic differences were observed to accumulate in metabolic pathways essential to acid production and protein synthesis. A transcriptome analysis revealed up-regulation of the electron transport chain, specifically the alternative oxidative pathway in ATCC 1015, while CBS 513.88 showed significant up regulation of genes associated with biosynthesis of amino acids that are abundant in glucoamylase A, tRNA-synthases and protein transporters.« less

Defining species-specific immunodominant B cell epitopes for molecular serology of Chlamydia species.

PubMed

Rahman, K Shamsur; Chowdhury, Erfan U; Poudel, Anil; Ruettger, Anke; Sachse, Konrad; Kaltenboeck, Bernhard

2015-05-01

Urgently needed species-specific enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against Chlamydia spp. have been elusive due to high cross-reactivity of chlamydial antigens. To identify Chlamydia species-specific B cell epitopes for such assays, we ranked the potential epitopes of immunodominant chlamydial proteins that are polymorphic among all Chlamydia species. High-scoring peptides were synthesized with N-terminal biotin, followed by a serine-glycine-serine-glycine spacer, immobilized onto streptavidin-coated microtiter plates, and tested with mono-specific mouse hyperimmune sera against each Chlamydia species in chemiluminescent ELISAs. For each of nine Chlamydia species, three to nine dominant polymorphic B cell epitope regions were identified on OmpA, CT618, PmpD, IncA, CT529, CT442, IncG, Omp2, TarP, and IncE proteins. Peptides corresponding to 16- to 40-amino-acid species-specific sequences of these epitopes reacted highly and with absolute specificity with homologous, but not heterologous, Chlamydia monospecies-specific sera. Host-independent reactivity of such epitopes was confirmed by testing of six C. pecorum-specific peptides from five proteins with C. pecorum-reactive sera from cattle, the natural host of C. pecorum. The probability of cross-reactivity of peptide antigens from closely related chlamydial species or strains correlated with percent sequence identity and declined to zero at <50% sequence identity. Thus, phylograms of B cell epitope regions predict the specificity of peptide antigens for rational use in the genus-, species-, or serovar-specific molecular serology of Chlamydia spp. We anticipate that these peptide antigens will improve chlamydial serology by providing easily accessible assays to nonspecialist laboratories. Our approach also lends itself to the identification of relevant epitopes of other microbial pathogens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Defining Species-Specific Immunodominant B Cell Epitopes for Molecular Serology of Chlamydia Species

PubMed Central

Rahman, K. Shamsur; Chowdhury, Erfan U.; Poudel, Anil; Ruettger, Anke; Sachse, Konrad

2015-01-01

Urgently needed species-specific enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against Chlamydia spp. have been elusive due to high cross-reactivity of chlamydial antigens. To identify Chlamydia species-specific B cell epitopes for such assays, we ranked the potential epitopes of immunodominant chlamydial proteins that are polymorphic among all Chlamydia species. High-scoring peptides were synthesized with N-terminal biotin, followed by a serine-glycine-serine-glycine spacer, immobilized onto streptavidin-coated microtiter plates, and tested with mono-specific mouse hyperimmune sera against each Chlamydia species in chemiluminescent ELISAs. For each of nine Chlamydia species, three to nine dominant polymorphic B cell epitope regions were identified on OmpA, CT618, PmpD, IncA, CT529, CT442, IncG, Omp2, TarP, and IncE proteins. Peptides corresponding to 16- to 40-amino-acid species-specific sequences of these epitopes reacted highly and with absolute specificity with homologous, but not heterologous, Chlamydia monospecies-specific sera. Host-independent reactivity of such epitopes was confirmed by testing of six C. pecorum-specific peptides from five proteins with C. pecorum-reactive sera from cattle, the natural host of C. pecorum. The probability of cross-reactivity of peptide antigens from closely related chlamydial species or strains correlated with percent sequence identity and declined to zero at <50% sequence identity. Thus, phylograms of B cell epitope regions predict the specificity of peptide antigens for rational use in the genus-, species-, or serovar-specific molecular serology of Chlamydia spp. We anticipate that these peptide antigens will improve chlamydial serology by providing easily accessible assays to nonspecialist laboratories. Our approach also lends itself to the identification of relevant epitopes of other microbial pathogens. PMID:25761461

Rapid and specific detection of Asian- and African-lineage Zika viruses

PubMed Central

Chotiwan, Nunya; Brewster, Connie D.; Magalhaes, Tereza; Weger-Lucarelli, James; Duggal, Nisha K.; Rückert, Claudia; Nguyen, Chilinh; Garcia Luna, Selene M.; Fauver, Joseph R.; Andre, Barb; Gray, Meg; Black, William C.; Kading, Rebekah C.; Ebel, Gregory D.; Kuan, Guillermina; Balmaseda, Angel; Jaenisch, Thomas; Marques, Ernesto T. A.; Brault, Aaron C.; Harris, Eva; Foy, Brian D.; Quackenbush, Sandra L.; Perera, Rushika; Rovnak, Joel

2017-01-01

Understanding the dynamics of Zika virus transmission and formulating rational strategies for its control require precise diagnostic tools that are also appropriate for resource-poor environments. We have developed a rapid and sensitive loop-mediated isothermal amplification (LAMP) assay that distinguishes Zika viruses of Asian and African lineages. The assay does not detect chikungunya virus or flaviviruses such as dengue, yellow fever, or West Nile viruses. The assay conditions allowed direct detection of Zika virus RNA in cultured infected cells; in mosquitoes; in virus-spiked samples of human blood, plasma, saliva, urine, and semen; and in infected patient serum, plasma, and semen samples without the need for RNA isolation or reverse transcription. The assay offers rapid, specific, sensitive, and inexpensive detection of the Asian-lineage Zika virus strain that is currently circulating in the Western hemisphere, and can also detect the African-lineage Zika virus strain using separate, specific primers. PMID:28469032

Genotypic Analysis of Escherichia coli Strains from Poultry Carcasses and Their Susceptibilities to Antimicrobial Agents

PubMed Central

Geornaras, Ifigenia; Hastings, John W.; von Holy, Alexander

2001-01-01

Plasmid profiling and amplified fragment length polymorphism (AFLP) analysis were used to genotype 50 Escherichia coli strains from poultry carcasses. Thirty different plasmid profiles were evident, and clustering of the AFLP data showed that they were a distinctly heterogeneous group of strains. Susceptibility testing against five antimicrobial agents used in the South African poultry industry showed all strains to be susceptible to danofloxacin and colistin, while the majority (96%) were resistant to two tetracyclines. PMID:11282652

Population genomic analysis uncovers environmental stress-driven selection and adaptation of Lentinula edodes population in China.

PubMed

Xiao, Yang; Cheng, Xuanjin; Liu, Jun; Li, Chuang; Nong, Wenyan; Bian, Yinbing; Cheung, Man Kit; Kwan, Hoi Shan

2016-11-10

The elucidation of genome-wide variations could help reveal aspects of divergence, domestication, and adaptation of edible mushrooms. Here, we resequenced the whole genomes of 39 wild and 21 cultivated strains of Chinese Lentinula edodes, the shiitake mushroom. We identified three distinct genetic groups in the Chinese L. edodes population with robust differentiation. Results of phylogenetic and population structure analyses suggest that the cultivated strains and most of the wild trains of L. edodes in China possess different gene pools and two outlier strains show signatures of hybridization between groups. Eighty-four candidate genes contributing to population divergence were detected in outlier analysis, 18 of which are involved in response to environmental stresses. Gene enrichment analysis of group-specific single nucleotide polymorphisms showed that the cultivated strains were genetically diversified in biological processes related to stress response. As the formation of fruiting bodies is a stress-response process, we postulate that environment factors, such as temperature, drove the population divergence of L. edodes in China by natural or artificial selection. We also found phenotypic variations between groups and identified some wild strains that have potential to diversify the genetic pool for improving agricultural traits of L. edodes cultivars in China.

Population genomic analysis uncovers environmental stress-driven selection and adaptation of Lentinula edodes population in China

PubMed Central

Xiao, Yang; Cheng, Xuanjin; Liu, Jun; Li, Chuang; Nong, Wenyan; Bian, Yinbing; Cheung, Man Kit; Kwan, Hoi Shan

2016-01-01

The elucidation of genome-wide variations could help reveal aspects of divergence, domestication, and adaptation of edible mushrooms. Here, we resequenced the whole genomes of 39 wild and 21 cultivated strains of Chinese Lentinula edodes, the shiitake mushroom. We identified three distinct genetic groups in the Chinese L. edodes population with robust differentiation. Results of phylogenetic and population structure analyses suggest that the cultivated strains and most of the wild trains of L. edodes in China possess different gene pools and two outlier strains show signatures of hybridization between groups. Eighty-four candidate genes contributing to population divergence were detected in outlier analysis, 18 of which are involved in response to environmental stresses. Gene enrichment analysis of group-specific single nucleotide polymorphisms showed that the cultivated strains were genetically diversified in biological processes related to stress response. As the formation of fruiting bodies is a stress-response process, we postulate that environment factors, such as temperature, drove the population divergence of L. edodes in China by natural or artificial selection. We also found phenotypic variations between groups and identified some wild strains that have potential to diversify the genetic pool for improving agricultural traits of L. edodes cultivars in China. PMID:27830835

An integrated metagenomics pipeline for strain profiling reveals novel patterns of bacterial transmission and biogeography

PubMed Central

Nayfach, Stephen; Rodriguez-Mueller, Beltran; Garud, Nandita

2016-01-01

We present the Metagenomic Intra-species Diversity Analysis System (MIDAS), which is an integrated computational pipeline for quantifying bacterial species abundance and strain-level genomic variation, including gene content and single-nucleotide polymorphisms (SNPs), from shotgun metagenomes. Our method leverages a database of more than 30,000 bacterial reference genomes that we clustered into species groups. These cover the majority of abundant species in the human microbiome but only a small proportion of microbes in other environments, including soil and seawater. We applied MIDAS to stool metagenomes from 98 Swedish mothers and their infants over one year and used rare SNPs to track strains between hosts. Using this approach, we found that although species compositions of mothers and infants converged over time, strain-level similarity diverged. Specifically, early colonizing bacteria were often transmitted from an infant’s mother, while late colonizing bacteria were often transmitted from other sources in the environment and were enriched for spore-formation genes. We also applied MIDAS to 198 globally distributed marine metagenomes and used gene content to show that many prevalent bacterial species have population structure that correlates with geographic location. Strain-level genetic variants present in metagenomes clearly reveal extensive structure and dynamics that are obscured when data are analyzed at a coarser taxonomic resolution. PMID:27803195

Evaluation of a Modified Single-Enzyme Amplified-Fragment Length Polymorphism Technique for Fingerprinting and Differentiating of Mycobacterium kansasii Type I Isolates

PubMed Central

Gaafar, Ayman; Josebe Unzaga, M.; Cisterna, Ramón; Clavo, Felicitas Elena; Urra, Elena; Ayarza, Rafael; Martín, Gloria

2003-01-01

The usefulness of single-enzyme amplified-fragment length polymorphism (AFLP) analysis for the subtyping of Mycobacterium kansasii type I isolates was evaluated. This simplified technique classified 253 type I strains into 12 distinct clusters. The discriminating power of this technique was high, and the technique easily distinguished between the epidemiologically unrelated control strains and our clinical isolates. Overall, the technique was relatively rapid and technically simple, yet it gave reproducible and discriminatory results. This technique provides a powerful typing tool which may be helpful in solving many questions concerning the reservoirs, pathogenicities, and modes of transmission of these isolates. PMID:12904399

Linkage of genes for laminin B1 and B2 subunits on chromosome 1 in mouse.

PubMed

Elliott, R W; Barlow, D; Hogan, B L

1985-08-01

We have used cDNA clones for the B1 and B2 subunits of laminin to find restriction fragment length DNA polymorphisms for the genes encoding these polypeptides in the mouse. Three alleles were found for LamB2 and two for LamB1 among the inbred mouse strains. The segregation of these polymorphisms among recombinant inbred strains showed that these genes are tightly linked in the central region of mouse Chromosome 1 between Sas-1 and Ly-m22, 7.4 +/- 3.2 cM distal to the Pep-3 locus. There is no evidence in the mouse for pseudogenes for these proteins.

Characterization of ROP18 alleles in human toxoplasmosis.

PubMed

Sánchez, Víctor; de-la-Torre, Alejandra; Gómez-Marín, Jorge Enrique

2014-04-01

The role of the virulent gene ROP18 polymorphisms is not known in human toxoplasmosis. A total of 320 clinical samples were analyzed. In samples positive for ROP18 gene, we determined by an allele specific PCR, if patients got the upstream insertion positive ROP18 sequence Toxoplasma strain (mouse avirulent strain) or the upstream insertion negative ROP18 sequence Toxoplasma strain (mouse virulent strain). We designed an ELISA assay for antibodies against ROP18 derived peptides from the three major clonal lineages of Toxoplasma. 20 clinical samples were of quality for ROP18 allele analysis. In patients with ocular toxoplasmosis, a higher inflammatory reaction on eye was associated to a PCR negative result for the upstream region of ROP18. 23.3%, 33% and 16.6% of serums from individuals with ocular toxoplasmosis were positive for type I, type II and type III ROP18 derived peptides, respectively but this assay was affected by cross reaction. The absence of Toxoplasma ROP18 promoter insertion sequence in ocular toxoplasmosis was correlated with severe ocular inflammatory response. Determination of antibodies against ROP18 protein was not useful for serotyping in human toxoplasmosis. © 2013.

Mycelial Propagation and Molecular Phylogenetic Relationships of Commercially Cultivated Agrocybe cylindracea based on ITS Sequences and RAPD

PubMed Central

Alam, Nuhu; Kim, Jeong Hwa; Shim, Mi Ja; Lee, U Youn

2010-01-01

This study evaluated the optimal vegetative growth conditions and molecular phylogenetic relationships of eleven strains of Agrocybe cylindracea collected from different ecological regions of Korea, China and Taiwan. The optimal temperature and pH for mycelial growth were observed at 25℃ and 6. Potato dextrose agar and Hennerberg were the favorable media for vegetative growth, whereas glucose tryptone was unfavorable. Dextrin, maltose, and fructose were the most effective carbon sources. The most suitable nitrogen sources were arginine and glycine, whereas methionine, alanine, histidine, and urea were least effective for the mycelial propagation of A. cylindracea. The internal transcribed spacer (ITS) regions of rDNA were amplified using PCR. The sequence of ITS2 was more variable than that of ITS1, while the 5.8S sequences were identical. The reciprocal homologies of the ITS sequences ranged from 98 to 100%. The strains were also analyzed by random amplification of polymorphic DNA (RAPD) using 20 arbitrary primers. Fifteen primers efficiently amplified the genomic DNA. The average number of polymorphic bands observed per primer was 3.8. The numbers of amplified bands varied based on the primers and strains, with polymorphic fragments ranging from 0.1 to 2.9 kb. The results of RAPD analysis were similar to the ITS region sequences. The results revealed that RAPD and ITS techniques were well suited for detecting the genetic diversity of all A. cylindracea strains tested. PMID:23956633

Variation along ITS markers across strains of Fibrocapsa japonica (Raphidophyceae) suggests hybridisation events and recent range expansion

NASA Astrophysics Data System (ADS)

Kooistra, Wiebe H. C. F.; de Boer, M. Karin; Vrieling, Engel G.; Connell, Laurie B.; Gieskes, Winfried W. C.

2001-12-01

The flagellate micro-alga Fibrocapsa japonica can form harmful algal blooms along all temperate coastal regions of the world. The species was first observed in coastal waters of Japan and the western US in the 1970s; it has been reported regularly worldwide since. To unravel whether this apparent range expansion can be tracked, we assessed genetic variation among nuclear ribosomal DNA ITS sequences, obtained from sixteen global strains collected over the course of three decades. Ten sequence positions showed polymorphism across the strains. Nine out of these revealed ambiguities in several or most sequences sampled. The oldest strain collected (LB-2161) was the only one without such intra-individual polymorphism. In the others, the proportion of ambiguities at variable sites increased with more recent collection date. The pattern does not result from loss of variation due to sexual reproduction and random drift in culture because sister cultures CS-332 and NIES-136 showed virtually the same ITS-pattern after seven years of separation. Neither are the patterns explained by recent range expansion of a single genotype, because in that case one would expect lowest genetic diversity in the recently invaded North Sea; instead, polymorphism is highest there. Recent ballast-water-mediated mixing of formerly isolated populations and subsequent ongoing sexual reproduction among them can explain the increase in ambiguities. The species' capacity to form harmful blooms may well have been enhanced through increased genetic diversity of regional populations.

[Algorithm of toxigenic genetically altered Vibrio cholerae El Tor biovar strain identification].

PubMed

Smirnova, N I; Agafonov, D A; Zadnova, S P; Cherkasov, A V; Kutyrev, V V

2014-01-01

Development of an algorithm of genetically altered Vibrio cholerae biovar El Tor strai identification that ensures determination of serogroup, serovar and biovar of the studied isolate based on pheno- and genotypic properties, detection of genetically altered cholera El Tor causative agents, their differentiation by epidemic potential as well as evaluation of variability of key pathogenicity genes. Complex analysis of 28 natural V. cholerae strains was carried out by using traditional microbiological methods, PCR and fragmentary sequencing. An algorithm of toxigenic genetically altered V. cholerae biovar El Tor strain identification was developed that includes 4 stages: determination of serogroup, serovar and biovar based on phenotypic properties, confirmation of serogroup and biovar based on molecular-genetic properties determination of strains as genetically altered, differentiation of genetically altered strains by their epidemic potential and detection of ctxB and tcpA key pathogenicity gene polymorphism. The algorithm is based on the use of traditional microbiological methods, PCR and sequencing of gene fragments. The use of the developed algorithm will increase the effectiveness of detection of genetically altered variants of the cholera El Tor causative agent, their differentiation by epidemic potential and will ensure establishment of polymorphism of genes that code key pathogenicity factors for determination of origins of the strains and possible routes of introduction of the infection.

DNA amplification polymorphisms of the cultivated mushroom Agaricus bisporus.

PubMed Central

Khush, R S; Becker, E; Wach, M

1992-01-01

Single 10-bp primers were used to generate random amplified polymorphic DNA (RAPD) markers from commercial and wild strains of the cultivated mushroom Agaricus bisporus via the polymerase chain reaction. Of 20 primers tested, 19 amplified A. bisporus DNA, each producing 5 to 15 scorable markers ranging from 0.5 to 3.0 kbp. RAPD markers identified seven distinct genotypes among eight heterokaryotic strains; two of the commercial strains were shown to be related to each other through single-spore descent. Homokaryons recovered from protoplast regenerants of heterokaryotic strains carried a subset of the RAPD markers found in the heterokaryon, and both of the haploid nuclei from two heterokaryons were distinguishable. RAPD markers also served to verify the creation of a hybrid heterokaryon and to analyze meiotic progeny from this new strain: most of the basidiospores displayed RAPD fingerprints identical to that of the parental heterokaryon, although a few selected slow growers were homoallelic at a number of loci that were heteroallelic in the parent, suggesting that they represented rare homokaryotic basidiospores; crossover events between a RAPD marker locus and its respective centromere appeared to be infrequent. These results demonstrate that RAPD markers provide an efficient alternative for strain fingerprinting and a versatile tool for genetic studies and manipulations of A. bisporus. Images PMID:1444410

Infectious polymorphic toxins delivered by outer membrane exchange discriminate kin in myxobacteria.

PubMed

Vassallo, Christopher N; Cao, Pengbo; Conklin, Austin; Finkelstein, Hayley; Hayes, Christopher S; Wall, Daniel

2017-08-18

Myxobacteria are known for complex social behaviors including outer membrane exchange (OME), in which cells exchange large amounts of outer membrane lipids and proteins upon contact. The TraA cell surface receptor selects OME partners based on a variable domain. However, traA polymorphism alone is not sufficient to precisely discriminate kin. Here, we report a novel family of OME-delivered toxins that promote kin discrimination of OME partners. These SitA lipoprotein toxins are polymorphic and widespread in myxobacteria. Each sitA is associated with a cognate sitI immunity gene, and in some cases a sitB accessory gene. Remarkably, we show that SitA is transferred serially between target cells, allowing the toxins to move cell-to-cell like an infectious agent. Consequently, SitA toxins define strong identity barriers between strains and likely contribute to population structure, maintenance of cooperation, and strain diversification. Moreover, these results highlight the diversity of systems evolved to deliver toxins between bacteria.

Epidemiological investigation of nosocomial outbreak of staphylococcal skin diseases in neonatal ward.

PubMed

Kurlenda, J; Grinholc, M; Krzysztoń-Russjan, J; Wiśniewska, K

2009-05-01

During a 1-month period, eight neonates developed staphylococcal skin disease diagnosed as a bullous impetigo in the maternity unit of the Provincial Hospital in Gdansk. An epidemiological investigation based on phenotyping and genotyping methods was performed. All neonates involved in the outbreak, their mothers and 15 staff members were screened for carriage of Staphylococcus aureus by nasal swabs. Isolated strains were compared with strains cultured from affected skin and purulent conjunctiva of infected newborns. Isolates were analyzed for the presence of the etA and etB genes using polymerase chain reaction and genotyped by pulsed-field gel electrophoresis (PFGE) and coa gene polymorphism. The analyzed S. aureus strains were methicillin-sensitive and could be divided into two groups according to antibiotyping, phage typing, coa polymorphism and PFGE pattern. The first group consisted of etA and etB negative strains, and the second one involved only the etB positive ones. Our results have shown that there were two different clusters of infection caused by two populations of S. aureus strains. Among the 15 medical staff members screened we have found seven carriers. However, phage typing revealed that distinct strains unrelated to the outbreak isolates were carried. Although we have not been able to establish the source of bacteria involved in the outbreak, our results suggest that for both groups, mothers could be the source of the infecting strains.

Persistence of Lactobacillus fermentum RC-14 and Lactobacillus rhamnosus GR-1 but Not L. rhamnosus GG in the Human Vagina as Demonstrated by Randomly Amplified Polymorphic DNA

PubMed Central

Gardiner, Gillian E.; Heinemann, Christine; Bruce, Andrew W.; Beuerman, Dee; Reid, Gregor

2002-01-01

Lactobacillus rhamnosus GR-1 and L. fermentum RC-14 are well-characterized probiotic strains with efficacy in the prevention and treatment of urogenital infections in women. The aim of the present study was to apply a molecular biology-based methodology for the detection of these strains and L. rhamnosus GG (a commercially available intestinal probiotic) in the human vagina in order to assess probiotic persistence at this site. Ten healthy women inserted vaginally a capsule containing either a combination of strains GR-1 and RC-14 or the GG strain for 3 consecutive nights. Vaginal swabs taken before and at various time points after probiotic insertion were analyzed, and the Lactobacillus flora was assessed by randomly amplified polymorphic DNA (RAPD) analysis. This method generated discrete DNA fingerprints for GR-1, RC-14, and GG and enabled successful detection of these strains in the vagina. Strain GR-1 and/or strain RC-14 was found to persist in the vaginal tract for up to 19 days after vaginal instillation, while L. rhamnosus GG was detectable for up to 5 days postadministration. In conclusion, the fates of probiotic L. rhamnosus and L. fermentum strains were successfully monitored in the human vagina by RAPD analysis. This technique provides molecular biology-based evidence that RC-14 and GR-1, strains selected as urogenital probiotics, persist in the human vagina and may be more suited to vaginal colonization than L. rhamnosus GG. This highlights the importance of proper selection of strains for urogenital probiotic applications. PMID:11777835

[Comparison of efficacy of tests for differentiation of typical and atypical strains of Yersinia pestis and Yersinia pseudotuberculosis].

PubMed

Arsen'eva, T E; Lebedeva, S A; Trukhachev, A L; Vasil'eva, E A; Ivanova, V S; Bozhko, N V

2010-01-01

To characterize species specificity of officially recommended tests for differentiation of Yersiniapestis and Yersinia pseudotuberculosis and propose additional tests allowing for more accurate identification. Natural, laboratory and typical strains oftwo Yersinia species were studied using microbiological, molecular and biochemical methods. For PCR species-specific primers complementary to certain fragments of chromosomal DNA of each species as well as to several plasmid genes of Y. pestis were used. It was shown that such attributes of Y. pestis as form of colonies, fermentation ofrhamnose, melibiose and urea, susceptibility to diagnostic phages, nutritional requirements could be lost in pestis bacterial species or detected in pseudotuberculosis species. Such attribute as mobility as well as positive result of CoA-reaction on fraction V antigen are more reliable. Guaranteed differentiation of typical and changed according to differential tests strains is provided only by PCR-analysis with primers vlml2for/ISrev216 and JS respectively, which are homologous to certain chromosome fragments of one of two Yersinia species.

Designing malaria vaccines to circumvent antigen variability✩

PubMed Central

Ouattara, Amed; Barry, Alyssa E.; Dutta, Sheetij; Remarque, Edmond J.; Beeson, James G.; Plowe, Christopher V.

2016-01-01

Prospects for malaria eradication will be greatly enhanced by an effective vaccine, but parasite genetic diversity poses a major impediment to malaria vaccine efficacy. In recent pre-clinical and field trials, vaccines based on polymorphic Plasmodium falciparum antigens have shown efficacy only against homologous strains, raising the specter of allele-specific immunity such as that which plagues vaccines against influenza and HIV. The most advanced malaria vaccine, RTS,S, targets relatively conserved epitopes on the P. falciparum circumsporozoite protein. After more than 40 years of development and testing, RTS,S, has shown significant but modest efficacy against clinical malaria in phase 2 and 3 trials. Ongoing phase 2 studies of an irradiated sporozoite vaccine will ascertain whether the full protection against homologous experimental malaria challenge conferred by high doses of a whole organism vaccine can provide protection against diverse strains in the field. Here we review and evaluate approaches being taken to design broadly cross-protective malaria vaccines. PMID:26475447

An Evaluation of the Genetic Diversity of Xylella fastidiosa Isolated from Diseased Citrus and Coffee in São Paulo, Brazil.

PubMed

Qin, X; Miranda, V S; Machado, M A; Lemos, E G; Hartung, J S

2001-06-01

ABSTRACT Strains of Xylella fastidiosa, isolated from sweet orange trees (Citrus sinensis) and coffee trees (Coffea arabica) with symptoms of citrus variegated chlorosis and Requeima do Café, respectively, were indistinguishable based on repetitive extragenic palindromic polymerase chain reaction (PCR) and enterobacterial repetitive intergenic consensus PCR assays. These strains were also indistinguishable with a previously described PCR assay that distinguished the citrus strains from all other strains of Xylella fastidiosa. Because we were not able to document any genomic diversity in our collection of Xylella fastidiosa strains isolated from diseased citrus, the observed gradient of increasing disease severity from southern to northern regions of São Paulo State is unlikely due to the presence of significantly different strains of the pathogen in the different regions. When comparisons were made to reference strains of Xylella fastidiosa isolated from other hosts using these methods, four groups were consistently identified consistent with the hosts and regions from which the strains originated: citrus and coffee, grapevine and almond, mulberry, and elm, plum, and oak. Independent results from random amplified polymorphic DNA (RAPD) PCR assays were also consistent with these results; however, two of the primers tested in RAPD-PCR were able to distinguish the coffee and citrus strains. Sequence comparisons of a PCR product amplified from all strains of Xylella fastidiosa confirmed the presence of a CfoI polymorphism that can be used to distinguish the citrus strains from all others. The ability to distinguish Xylella fastidiosa strains from citrus and coffee with a PCR-based assay will be useful in epidemiological and etiological studies of this pathogen.

[RAPD analysis of Aspergilli and its application in brewing industry].

PubMed

Pan, Li; Wang, Bin; Guo, Yong

2007-06-01

Phylogenetic analysis of sixteen Aspergilli was done by RAPD technology, using Aspergillus oryzae AS3.951, Aspergillus flavus GIM3.18 and Aspergillus sojae AS3.495 as controls. First, genome DNA of the sixteen test strains were prepared by improved extraction method, and their quality was verified by electrophoresis and spectrophotometry. They displayed an identical band (approximately 20 kb) in agarose gel electrophoresis, which conformed to the fact that these strains all belong to Aspergillus. OD260/OD280 of the prepared DNA ranged from 1.80 to 1.90, illustrating that they were good enough to be used as templates in the following RAPD-PCR experiment. Then, three appropriate primers (Primerl, Primer2, Primer5) for RAPD-PCR were screened from nine random primers, and repetitive experiments demonstrated that the RAPD-PCR polymorphic patterns of the sixteen test strains based on these three primers were stable. There were usually 8-14 bands in their RADP-PCR patterns, where the number of the main bands was 4-9 and the secondary bands were abundant. There were totally 181 bands in their RAPD-PCR patterns, where the percentage of polymorphic bands reached to 40.9% (74 bands). The similarity coefficient between the strains was calculated based on their RAPD-PCR patterns, ranging from 8.0% to 96.6%. All these data suggests that the genetic polymorphism of the strains is abundant and they have evident genetic differentiation. The phylogenetic tree of the sixteen test strains was reconstructed according to their RAPD-PCR patterns with Primer1, Primer2 and Primer5. It basically corresponded to traditional morphological taxonomy, demonstrating that the application of RAPD molecular marker in the phylogenetic analysis of these Aspergilli is feasible. Besides, the aflatoxin-producing strains (GIM3.17, CICC2219, CICC2357, CICC2390, CICC2402, CICC2404) could be easily discriminated by RAPD molecular marker, whereas it is difficult to distinguish them by conventional morphological taxonomy. Consequently, RAPD molecular marker provides a novel clue to discriminating aflatoxin-producing strains in brewing industry.

Analysis of infant isolates of Bifidobacterium breve by comparative genome hybridization indicates the existence of new subspecies with marked infant specificity.

PubMed

Boesten, Rolf; Schuren, Frank; Wind, Richèle D; Knol, Jan; de Vos, Willem M

2011-09-01

A total of 20 Bifidobacterium strains were isolated from fecal samples of 4 breast- and bottle-fed infants and all were characterized as Bifidobacterium breve based on 16S rRNA gene sequence and metabolic analysis. These isolates were further characterized and compared to the type strains of B. breve and 7 other Bifidobacterium spp. by comparative genome hybridization. For this purpose, we constructed and used a DNA-based microarray containing over 2000 randomly cloned DNA fragments from B. breve type strain LMG13208. This molecular analysis revealed a high degree of genomic variation between the isolated strains and allowed the vast majority to be grouped into 4 clusters. One cluster contained a single isolate that was virtually indistinguishable from the B. breve type strain. The 3 other clusters included 19 B. breve strains that differed considerably from all type strains. Remarkably, each of the 4 clusters included strains that were isolated from a single infant, indicating that a niche adaptation may contribute to variation within the B. breve species. Based on genomic hybridization data, the new B. breve isolates were estimated to contain approximately 60-90% of the genes of the B. breve type strain, attesting to the existence of various subspecies within the species B. breve. Further bioinformatic analysis identified several hundred diagnostic clones specific to the genomic clustering of the B. breve isolates. Molecular analysis of representatives of these revealed that annotated genes from the conserved B. breve core encoded mainly housekeeping functions, while the strain-specific genes were predicted to code for functions related to life style, such as carbohydrate metabolism and transport. This is compatible with genetic adaptation of the strains to their niche, a combination of infants and diet. Copyright © 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Allele-dependent differences in quorum-sensing dynamics result in variant expression of virulence genes in Staphylococcus aureus.

PubMed

Geisinger, Edward; Chen, John; Novick, Richard P

2012-06-01

Agr is an autoinducing, quorum-sensing system that functions in many Gram-positive species and is best characterized in the pathogen Staphylococcus aureus, in which it is a global regulator of virulence gene expression. Allelic variations in the agr genes have resulted in the emergence of four quorum-sensing specificity groups in S. aureus, which correlate with different strain pathotypes. The basis for these predilections is unclear but is hypothesized to involve the phenomenon of quorum-sensing interference between strains of different agr groups, which may drive S. aureus strain isolation and divergence. Whether properties intrinsic to each agr allele directly influence virulence phenotypes within S. aureus is unknown. In this study, we examined group-specific differences in agr autoinduction and virulence gene regulation by utilizing congenic strains, each harboring a unique S. aureus agr allele, enabling a dissection of agr locus-dependent versus genotype-dependent effects on quorum-sensing dynamics and virulence factor production. Employing a reporter fusion to the principal agr promoter, P3, we observed allele-dependent differences in the timing and magnitude of agr activation. These differences were mediated by polymorphisms within the agrBDCA genes and translated to significant variations in the expression of a key transcriptional regulator, Rot, and of several important exoproteins and surface factors involved in pathogenesis. This work uncovers the contribution of divergent quorum-sensing alleles to variant expression of virulence determinants within a bacterial species.

Salmonella enterica Prophage Sequence Profiles Reflect Genome Diversity and Can Be Used for High Discrimination Subtyping.

PubMed

Mottawea, Walid; Duceppe, Marc-Olivier; Dupras, Andrée A; Usongo, Valentine; Jeukens, Julie; Freschi, Luca; Emond-Rheault, Jean-Guillaume; Hamel, Jeremie; Kukavica-Ibrulj, Irena; Boyle, Brian; Gill, Alexander; Burnett, Elton; Franz, Eelco; Arya, Gitanjali; Weadge, Joel T; Gruenheid, Samantha; Wiedmann, Martin; Huang, Hongsheng; Daigle, France; Moineau, Sylvain; Bekal, Sadjia; Levesque, Roger C; Goodridge, Lawrence D; Ogunremi, Dele

2018-01-01

Non-typhoidal Salmonella is a leading cause of foodborne illness worldwide. Prompt and accurate identification of the sources of Salmonella responsible for disease outbreaks is crucial to minimize infections and eliminate ongoing sources of contamination. Current subtyping tools including single nucleotide polymorphism (SNP) typing may be inadequate, in some instances, to provide the required discrimination among epidemiologically unrelated Salmonella strains. Prophage genes represent the majority of the accessory genes in bacteria genomes and have potential to be used as high discrimination markers in Salmonella . In this study, the prophage sequence diversity in different Salmonella serovars and genetically related strains was investigated. Using whole genome sequences of 1,760 isolates of S. enterica representing 151 Salmonella serovars and 66 closely related bacteria, prophage sequences were identified from assembled contigs using PHASTER. We detected 154 different prophages in S. enterica genomes. Prophage sequences were highly variable among S. enterica serovars with a median ± interquartile range (IQR) of 5 ± 3 prophage regions per genome. While some prophage sequences were highly conserved among the strains of specific serovars, few regions were lineage specific. Therefore, strains belonging to each serovar could be clustered separately based on their prophage content. Analysis of S . Enteritidis isolates from seven outbreaks generated distinct prophage profiles for each outbreak. Taken altogether, the diversity of the prophage sequences correlates with genome diversity. Prophage repertoires provide an additional marker for differentiating S. enterica subtypes during foodborne outbreaks.

Comparative Genomics of Completely Sequenced Lactobacillus helveticus Genomes Provides Insights into Strain-Specific Genes and Resolves Metagenomics Data Down to the Strain Level.

PubMed

Schmid, Michael; Muri, Jonathan; Melidis, Damianos; Varadarajan, Adithi R; Somerville, Vincent; Wicki, Adrian; Moser, Aline; Bourqui, Marc; Wenzel, Claudia; Eugster-Meier, Elisabeth; Frey, Juerg E; Irmler, Stefan; Ahrens, Christian H

2018-01-01

Although complete genome sequences hold particular value for an accurate description of core genomes, the identification of strain-specific genes, and as the optimal basis for functional genomics studies, they are still largely underrepresented in public repositories. Based on an assessment of the genome assembly complexity for all lactobacilli, we used Pacific Biosciences' long read technology to sequence and de novo assemble the genomes of three Lactobacillus helveticus starter strains, raising the number of completely sequenced strains to 12. The first comparative genomics study for L. helveticus -to our knowledge-identified a core genome of 988 genes and sets of unique, strain-specific genes ranging from about 30 to more than 200 genes. Importantly, the comparison of MiSeq- and PacBio-based assemblies uncovered that not only accessory but also core genes can be missed in incomplete genome assemblies based on short reads. Analysis of the three genomes revealed that a large number of pseudogenes were enriched for functional Gene Ontology categories such as amino acid transmembrane transport and carbohydrate metabolism, which is in line with a reductive genome evolution in the rich natural habitat of L. helveticus . Notably, the functional Clusters of Orthologous Groups of proteins categories "cell wall/membrane biogenesis" and "defense mechanisms" were found to be enriched among the strain-specific genes. A genome mining effort uncovered examples where an experimentally observed phenotype could be linked to the underlying genotype, such as for cell envelope proteinase PrtH3 of strain FAM8627. Another possible link identified for peptidoglycan hydrolases will require further experiments. Of note, strain FAM22155 did not harbor a CRISPR/Cas system; its loss was also observed in other L. helveticus strains and lactobacillus species, thus questioning the value of the CRISPR/Cas system for diagnostic purposes. Importantly, the complete genome sequences proved to be very useful for the analysis of natural whey starter cultures with metagenomics, as a larger percentage of the sequenced reads of these complex mixtures could be unambiguously assigned down to the strain level.

Comparative Genomics of Completely Sequenced Lactobacillus helveticus Genomes Provides Insights into Strain-Specific Genes and Resolves Metagenomics Data Down to the Strain Level

PubMed Central

Schmid, Michael; Muri, Jonathan; Melidis, Damianos; Varadarajan, Adithi R.; Somerville, Vincent; Wicki, Adrian; Moser, Aline; Bourqui, Marc; Wenzel, Claudia; Eugster-Meier, Elisabeth; Frey, Juerg E.; Irmler, Stefan; Ahrens, Christian H.

2018-01-01

Although complete genome sequences hold particular value for an accurate description of core genomes, the identification of strain-specific genes, and as the optimal basis for functional genomics studies, they are still largely underrepresented in public repositories. Based on an assessment of the genome assembly complexity for all lactobacilli, we used Pacific Biosciences' long read technology to sequence and de novo assemble the genomes of three Lactobacillus helveticus starter strains, raising the number of completely sequenced strains to 12. The first comparative genomics study for L. helveticus—to our knowledge—identified a core genome of 988 genes and sets of unique, strain-specific genes ranging from about 30 to more than 200 genes. Importantly, the comparison of MiSeq- and PacBio-based assemblies uncovered that not only accessory but also core genes can be missed in incomplete genome assemblies based on short reads. Analysis of the three genomes revealed that a large number of pseudogenes were enriched for functional Gene Ontology categories such as amino acid transmembrane transport and carbohydrate metabolism, which is in line with a reductive genome evolution in the rich natural habitat of L. helveticus. Notably, the functional Clusters of Orthologous Groups of proteins categories “cell wall/membrane biogenesis” and “defense mechanisms” were found to be enriched among the strain-specific genes. A genome mining effort uncovered examples where an experimentally observed phenotype could be linked to the underlying genotype, such as for cell envelope proteinase PrtH3 of strain FAM8627. Another possible link identified for peptidoglycan hydrolases will require further experiments. Of note, strain FAM22155 did not harbor a CRISPR/Cas system; its loss was also observed in other L. helveticus strains and lactobacillus species, thus questioning the value of the CRISPR/Cas system for diagnostic purposes. Importantly, the complete genome sequences proved to be very useful for the analysis of natural whey starter cultures with metagenomics, as a larger percentage of the sequenced reads of these complex mixtures could be unambiguously assigned down to the strain level. PMID:29441050

Transcriptomic Analyses Elucidate Adaptive Differences of Closely Related Strains of Pseudomonas aeruginosa in Fuel

PubMed Central

Gunasekera, Thusitha S.; Bowen, Loryn L.; Zhou, Carol E.; Howard-Byerly, Susan C.; Foley, William S.; Striebich, Richard C.; Dugan, Larry C.

2017-01-01

ABSTRACT Pseudomonas aeruginosa can utilize hydrocarbons, but different strains have various degrees of adaptation despite their highly conserved genome. P. aeruginosa ATCC 33988 is highly adapted to hydrocarbons, while P. aeruginosa strain PAO1, a human pathogen, is less adapted and degrades jet fuel at a lower rate than does ATCC 33988. We investigated fuel-specific transcriptomic differences between these strains in order to ascertain the underlying mechanisms utilized by the adapted strain to proliferate in fuel. During growth in fuel, the genes related to alkane degradation, heat shock response, membrane proteins, efflux pumps, and several novel genes were upregulated in ATCC 33988. Overexpression of alk genes in PAO1 provided some improvement in growth, but it was not as robust as that of ATCC 33988, suggesting the role of other genes in adaptation. Expression of the function unknown gene PA5359 from ATCC 33988 in PAO1 increased the growth in fuel. Bioinformatic analysis revealed that PA5359 is a predicted lipoprotein with a conserved Yx(FWY)xxD motif, which is shared among bacterial adhesins. Overexpression of the putative resistance-nodulation-division (RND) efflux pump PA3521 to PA3523 increased the growth of the ATCC 33988 strain, suggesting a possible role in fuel tolerance. Interestingly, the PAO1 strain cannot utilize n-C8 and n-C10. The expression of green fluorescent protein (GFP) under the control of alkB promoters confirmed that alk gene promoter polymorphism affects the expression of alk genes. Promoter fusion assays further confirmed that the regulation of alk genes was different in the two strains. Protein sequence analysis showed low amino acid differences for many of the upregulated genes, further supporting transcriptional control as the main mechanism for enhanced adaptation. IMPORTANCE These results support that specific signal transduction, gene regulation, and coordination of multiple biological responses are required to improve the survival, growth, and metabolism of fuel in adapted strains. This study provides new insight into the mechanistic differences between strains and helpful information that may be applied in the improvement of bacterial strains for resistance to biotic and abiotic factors encountered during bioremediation and industrial biotechnological processes. PMID:28314727

Restriction-Site-Specific PCR as a Rapid Test To Detect Enterohemorrhagic Escherichia coli O157:H7 Strains in Environmental Samples

PubMed Central

Kimura, Richard; Mandrell, Robert E.; Galland, John C.; Hyatt, Doreene; Riley, Lee W.

2000-01-01

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important food-borne pathogen in industrialized countries. We developed a rapid and simple test for detecting E. coli O157:H7 using a method based on restriction site polymorphisms. Restriction-site-specific PCR (RSS-PCR) involves the amplification of DNA fragments using primers based on specific restriction enzyme recognition sequences, without the use of endonucleases, to generate a set of amplicons that yield “fingerprint” patterns when resolved electrophoretically on an agarose gel. The method was evaluated in a blinded study of E. coli isolates obtained from environmental samples collected at beef cattle feedyards. The 54 isolates were all initially identified by a commonly used polyclonal antibody test as belonging to O157:H7 serotype. They were retested by anti-O157 and anti-H7 monoclonal antibody enzyme-linked immunosorbent assay (ELISA). The RSS-PCR method identified all 28 isolates that were shown to be E. coli O157:H7 by the monoclonal antibody ELISA as belonging to the O157:H7 serotype. Of the remaining 26 ELISA-confirmed non-O157:H7 strains, the method classified 25 strains as non-O157:H7. The specificity of the RSS-PCR results correlated better with the monoclonal antibody ELISA than with the polyclonal antibody latex agglutination tests. The RSS-PCR method may be a useful test to distinguish E. coli O157:H7 from a large number of E. coli isolates from environmental samples. PMID:10831431

Correlation between genetic variability and virulence factors in clinical strains of Malassezia pachydermatis of animal origin.

PubMed

Buommino, Elisabetta; Nocera, Francesca Paola; Parisi, Annamaria; Rizzo, Antonietta; Donnarumma, Giovanna; Mallardo, Karina; Fiorito, Filomena; Baroni, Adone; De Martino, Luisa

2016-07-01

Malassezia pachydermatis is a yeast belonging to the microbiota of the skin and mucous membranes of dog and cat, but it can also act as pathogen, causing dermatitis. The aim of this work was to evaluate the genetic variability of M. pachydermatis strains isolated from symptomatic dogs and cats and determine a correlation between genotype and phenotype. For this purpose eleven strains of M. pachydermatis were molecularly classified by nested-polymerase chain reaction (nested-PCR) based on ITS-1 and ITS-2 regions, specific for fungal rRNA genes. Furthermore, random amplification of polymorphic DNA (RAPD) was applied for genetic typing of M. pachydermatis isolates identifying four different genotypes. Strains belonging to genotype 1 produced the highest amount of biofilm and phospholipase activity. The inflammatory response induced by M. pachydermatis strains in immortalized human keratinocytes (HaCat cells) was significantly different when we compared the results obtained from each strain. In particular, HaCat cells infected with the strains belonging to genotypes 1 and 2 triggered the highest levels of increase in TLR-2, IL-1β, IL-6, IL-8, COX-2 and MMP-9 expression. By contrast, cells infected with the strains of genotype 3 and those of genotype 4 did not significantly induce TLR-2 and cytokines. The results obtained might suggest a possible association between genotype and virulence factors expressed by M. pachydermatis strains. This highlights the need for a more accurate identification of the yeast to improve the therapeutic approach and to monitor the onset of human infections caused by this emergent zoonotic pathogen.

Initiation of a pan-genomic research project for Xylella fastidiosa

USDA-ARS?s Scientific Manuscript database

Differences in genomic structure and nucleotide polymorphism among strains form the genetic basis for adaptability of a bacterial species. This can be described by a bacterial pan-genome, which is defined as the full complement of genes in all strains of a species. The pan-genome is composed of a "c...

Rapid and Reliable Single Nucleotide Polymorphism-Based Differentiation of Brucella Live Vaccine Strains from Field Strains

USDA-ARS?s Scientific Manuscript database

Brucellosis is a major zoonotic disease responsible for substantial social and economic problems, particularly in the developing world. One element that can implemented as part of control programs tackling animal disease is the use of one of the OIE recommended vaccines to protect against either Bru...

Sulfadiazine resistance in Toxoplasma gondii: no involvement of overexpression or polymorphisms in genes of therapeutic targets and ABC transporters

PubMed Central

Doliwa, Christelle; Escotte-Binet, Sandie; Aubert, Dominique; Sauvage, Virginie; Velard, Frédéric; Schmid, Aline; Villena, Isabelle

2013-01-01

Several treatment failures have been reported for the treatment of toxoplasmic encephalitis, chorioretinitis, and congenital toxoplasmosis. Recently we found three Toxoplasma gondii strains naturally resistant to sulfadiazine and we developed in vitro two sulfadiazine resistant strains, RH-RSDZ and ME-49-RSDZ, by gradual pressure. In Plasmodium, common mechanisms of drug resistance involve, among others, mutations and/or amplification within genes encoding the therapeutic targets dhps and dhfr and/or the ABC transporter genes family. To identify genotypic and/or phenotypic markers of resistance in T. gondii, we sequenced and analyzed the expression levels of therapeutic targets dhps and dhfr, three ABC genes, two Pgp, TgABC.B1 and TgABC.B2, and one MRP, TgABC.C1, on sensitive strains compared to sulfadiazine resistant strains. Neither polymorphism nor overexpression was identified. Contrary to Plasmodium, in which mutations and/or overexpression within gene targets and ABC transporters are involved in antimalarial resistance, T. gondii sulfadiazine resistance is not related to these toxoplasmic genes studied. PMID:23707894

Genetic diversity among silkworm (Bombyx mori L., Lep., Bombycidae) germplasms revealed by microsatellites.

PubMed

Li, Muwang; Shen, Li; Xu, Anying; Miao, Xuexia; Hou, Chengxiang; Sun, Pingjiang; Zhang, Yuehua; Huang, Yongping

2005-10-01

To determine genetic relationships among strains of silkworm, Bombyx mori L., 31 strains with different origins, number of generations per year, number of molts per generation, and morphological characters were studied using simple sequence repeat (SSR) markers. Twenty-six primer pairs flanking microsatellite sequences in the silkworm genome were assayed. All were polymorphic and unambiguously separated silkworm strains from each other. A total of 188 alleles were detected with a mean value of 7.2 alleles/locus (range 2-17). The average heterozygosity value for each SSR locus ranged from 0 to 0.60, and the highest one was 0.96 (Fl0516 in 4013). The mean polymorphism index content (PIC) was 0.66 (range 0.12-0.89). Unweighted pair group method with arithmetic means (UPGMA) cluster analysis of Nei's genetic distance grouped silkworm strains based on their origin. Seven major ecotypic silkworm groups were analyzed. Principal components analysis (PCA) for SSR data support their UPGMA clustering. The results indicated that SSR markers are an efficient tool for fingerprinting cultivars and conducting genetic-diversity studies in the silkworm.

Molecular typing of Sarcocystis neurona: current status and future trends.

PubMed

Elsheikha, Hany M; Mansfield, Linda S

2007-10-21

Sarcocystis neurona is an important protozoal pathogen because it causes the serious neurological disease equine protozoal myeloencephalitis (EPM). The capacity of this organism to cause a wide spectrum of neurological signs in horses and the broad geographic distribution of observed cases in the Americas drive the need for sensitive, reliable and rapid typing methods to characterize strains. Various molecular methods have been developed and used to diagnose EPM due to S. neurona, to identify S. neurona isolates and to determine the heterogeneity and evolutionary relatedness within this species and related Sarcocystis spp. These methods included sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immuno-fluorescent assay (IFA), slide agglutination test (SAT), SnSAG-specific ELISA, random amplified polymorphic DNA (RAPD), PCR-based restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP) fingerprinting, and sequence analysis of surface protein genes, ribosomal genes, microsatellite alleles and other molecular markers. Here, the utility of these molecular methods is reviewed and evaluated with respect to the need for molecular approaches that utilize well-characterized polymorphic, simple, independent, and stable genetic markers. These tools have the potential to add to knowledge of the genetic population structure of S. neurona and to provide new insights into the pathogenesis of EPM and S. neurona epidemiology. In particular, these methods provide new tools to address the hypothesis that particular genetic variants are associated with adverse clinical outcomes (severe pathotypes). The ultimate goal is to utilize them in future studies to improve treatment and prevention strategies.

Orangutan Alu quiescence reveals possible source element: support for ancient backseat drivers

PubMed Central

2012-01-01

Background Sequence analysis of the orangutan genome revealed that recent proliferative activity of Alu elements has been uncharacteristically quiescent in the Pongo (orangutan) lineage, compared with all previously studied primate genomes. With relatively few young polymorphic insertions, the genomic landscape of the orangutan seemed like the ideal place to search for a driver, or source element, of Alu retrotransposition. Results Here we report the identification of a nearly pristine insertion possessing all the known putative hallmarks of a retrotranspositionally competent Alu element. It is located in an intronic sequence of the DGKB gene on chromosome 7 and is highly conserved in Hominidae (the great apes), but absent from Hylobatidae (gibbon and siamang). We provide evidence for the evolution of a lineage-specific subfamily of this shared Alu insertion in orangutans and possibly the lineage leading to humans. In the orangutan genome, this insertion contains three orangutan-specific diagnostic mutations which are characteristic of the youngest polymorphic Alu subfamily, AluYe5b5_Pongo. In the Homininae lineage (human, chimpanzee and gorilla), this insertion has acquired three different mutations which are also found in a single human-specific Alu insertion. Conclusions This seemingly stealth-like amplification, ongoing at a very low rate over millions of years of evolution, suggests that this shared insertion may represent an ancient backseat driver of Alu element expansion. PMID:22541534

Orangutan Alu quiescence reveals possible source element: support for ancient backseat drivers.

PubMed

Walker, Jerilyn A; Konkel, Miriam K; Ullmer, Brygg; Monceaux, Christopher P; Ryder, Oliver A; Hubley, Robert; Smit, Arian Fa; Batzer, Mark A

2012-04-30

Sequence analysis of the orangutan genome revealed that recent proliferative activity of Alu elements has been uncharacteristically quiescent in the Pongo (orangutan) lineage, compared with all previously studied primate genomes. With relatively few young polymorphic insertions, the genomic landscape of the orangutan seemed like the ideal place to search for a driver, or source element, of Alu retrotransposition. Here we report the identification of a nearly pristine insertion possessing all the known putative hallmarks of a retrotranspositionally competent Alu element. It is located in an intronic sequence of the DGKB gene on chromosome 7 and is highly conserved in Hominidae (the great apes), but absent from Hylobatidae (gibbon and siamang). We provide evidence for the evolution of a lineage-specific subfamily of this shared Alu insertion in orangutans and possibly the lineage leading to humans. In the orangutan genome, this insertion contains three orangutan-specific diagnostic mutations which are characteristic of the youngest polymorphic Alu subfamily, AluYe5b5_Pongo. In the Homininae lineage (human, chimpanzee and gorilla), this insertion has acquired three different mutations which are also found in a single human-specific Alu insertion. This seemingly stealth-like amplification, ongoing at a very low rate over millions of years of evolution, suggests that this shared insertion may represent an ancient backseat driver of Alu element expansion.

[Intraspecific chromosomal variability in human pathogenic fungi, especially in Histoplasma capsulatum].

PubMed

Romero-Martínez, Rafael; Canteros, Cristina; Taylor, Maria Lucia

2004-12-01

The ploidy, karyotype, and chromosome length polymorphism (CLP) of human pathogenic fungi were revised with emphasis on Histoplasma capsulatum, the causative agent of the systemic mycosis, histoplasmosis. Currently, different systems of gel electrophoresis are being used to determine fungal electrokaryotypes (EK). By renaturation kinetic and genomic reconstruction in H. capsulatum strains (G-186AS and Downs), estimated genome sizes of 23 and 32 Mb were determined for both strains, respectively. The haploid state was proposed for both strains, although aneuploidy was suggested for the Downs strain. Contour-clamped homogeneous electric field (CHEF), field inversion gel electrophoresis (FIGE), and Southern blot using different probes showed the presence of six to seven chromosomes in the Downs strain (low virulence), whereas four chromosomes were identified in the G-186B strain (high virulence). The use of these methods in the three major H. capsulatum reference strains (G-217B and Downs from the United States of America, G-186B from Panama) revealed distinct chromosome sizes, from 0.5 to 5.7 Mb, with CLP associated with chromosomes size and mobility. Recently, by CHEF, using 19 H. capsulatum isolates from Latin-America and the G-186B strain, five to seven chromosomes with 1.1 to 11.2 Mb molecular sizes were revealed, which again suggested CLP in H. capsulatum. However, to elucidate the EKs polymorphism in H. capsulatum and its relationship with the isolates phenotype more studies are needed to understand the mechanisms controlling ploidy variability.

Comparative evaluation of the Ridascreen Verotoxin enzyme immunoassay for detection of Shiga-toxin producing strains of Escherichia coli (STEC) from food and other sources.

PubMed

Beutin, L; Steinrück, H; Krause, G; Steege, K; Haby, S; Hultsch, G; Appel, B

2007-03-01

To evaluate the suitability of the commercially distributed Ridascreen Verotoxin enzyme immunoassay (EIA) for detection of known genetic types of the Vero (Shiga) toxins 1 (Stx1) and 2 (Stx2) families and to determine its relative sensitivity and specificity. The Ridascreen-EIA was compared with the Vero cell assay, a P(1)-glycoprotein receptor EIA and with stx gene-specific PCs for detection of Stx with 43 Shiga toxin-producing strains of Escherichia coli (STEC) reference strains and with 241 test strains. The Ridascreen-EIA detects strains producing Stx1 and variants Stx1c and Stx1d, as well as Stx2 and variants Stx2d1, Stx2d2, Stx2e, Stx2d, Stx2-O118 (Stx2d-ount), Stx2-NV206, Stx2f and Stx2g. The assay showed a relative sensitivity of 95.7% and a relative specificity of 98.7%. Some of the Stx2-O118-, Stx2e- and Stx2g-producing STEC were not detected with the Ridascreen-EIA probably because of low amount of toxin produced by these strains. The Ridascreen-EIA is able to detect all known types of Stx and is applicable for routine screening of bacterial isolates owing to its high specificity. It is less applicable for testing samples where low amounts of Stx are expected, such as mixed cultures and certain Stx2 variants. This study presents a first comprehensive evaluation of the Ridascreen-EIA, a rapid standardized STEC screening test for routine diagnostic laboratories. Data are presented on the type of the spectrum of Stx that are detected with this immunoassay and its advantages and limits for practical use.

Development and systematic validation of qPCR assays for rapid and reliable differentiation of Xylella fastidiosa strains causing citrus variegated chlorosis.

PubMed

Li, Wenbin; Teixeira, Diva C; Hartung, John S; Huang, Qi; Duan, Yongping; Zhou, Lijuan; Chen, Jianchi; Lin, Hong; Lopes, Silvio; Ayres, A Juliano; Levy, Laurene

2013-01-01

The xylem-limited, Gram-negative, fastidious plant bacterium Xylella fastidiosa is the causal agent of citrus variegated chlorosis (CVC), a destructive disease affecting approximately half of the citrus plantations in the State of São Paulo, Brazil. The disease was recently found in Central America and is threatening the multi-billion U.S. citrus industry. Many strains of X. fastidiosa are pathogens or endophytes in various plants growing in the U.S., and some strains cross infect several host plants. In this study, a TaqMan-based assay targeting the 16S rDNA signature region was developed for the identification of X. fastidiosa at the species level. Another TaqMan-based assay was developed for the specific identification of the CVC strains. Both new assays have been systematically validated in comparison with the primer/probe sets from four previously published assays on one platform and under similar PCR conditions, and shown to be superior. The species specific assay detected all X. fastidiosa strains and did not amplify any other citrus pathogen or endophyte tested. The CVC-specific assay detected all CVC strains but did not amplify any non-CVC X. fastidiosa nor any other citrus pathogen or endophyte evaluated. Both sets were multiplexed with a reliable internal control assay targeting host plant DNA, and their diagnostic specificity and sensitivity remained unchanged. This internal control provides quality assurance for DNA extraction, performance of PCR reagents, platforms and operators. The limit of detection for both assays was equivalent to 2 to 10 cells of X. fastidiosa per reaction for field citrus samples. Petioles and midribs of symptomatic leaves of sweet orange harbored the highest populations of X. fastidiosa, providing the best materials for detection of the pathogen. These new species specific assay will be invaluable for molecular identification of X. fastidiosa at the species level, and the CVC specific assay will be very powerful for the specific identification of X. fastidiosa strains that cause citrus variegated chlorosis. Published by Elsevier B.V.

Diagnostic power of longitudinal strain at rest for the detection of obstructive coronary artery disease in patients with type 2 diabetes mellitus.

PubMed

Zuo, Houjuan; Yan, Jiangtao; Zeng, Hesong; Li, Wenyu; Li, Pengcheng; Liu, Zhengxiang; Cui, Guanglin; Lv, Jiagao; Wang, Daowen; Wang, Hong

2015-01-01

Global longitudinal strain (GLS) measured by 2-D speckle-tracking echocardiography (2-D STE) at rest has been recognized as a sensitive parameter in the detection of significant coronary artery disease (CAD). However, the diagnostic power of 2-D STE in the detection of significant CAD in patients with diabetes mellitus is unknown. Two-dimensional STE features were studied in total of 143 consecutive patients who underwent echocardiography and coronary angiography. Left ventricular global and segmental peak systolic longitudinal strains (PSLSs) were quantified by speckle-tracking imaging. In the presence of obstructive CAD (defined as stenosis ≥75%), global PSLS was significantly lower in patients with diabetes mellitus than in patients without (16.65 ± 2.29% vs. 17.32 ± 2.27%, p < 0.05). Receiver operating characteristic analysis revealed that global PSLS could effectively detect obstructive CAD in patients without diabetes mellitus (cutoff value: -18.35%, sensitivity: 78.8%, specificity: 77.5%). However, global PSLS could detect obstructive CAD in diabetic patients at a lower cutoff value with inadequate sensitivity and specificity (cutoff value: -17.15%; sensitivity: 61.1%, specificity: 52.9%). In addition, the results for segmental PSLS were similar to those for global PSLS. In conclusion, global and segmental PSLSs at rest were significantly lower in patients with both obstructive CAD and diabetes mellitus than in patients with obstructive CAD only; thus, PSLSs at rest might not be a useful parameter in the detection of obstructive CAD in patients with diabetes mellitus. Copyright © 2015 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

Detection, Characterization, and Typing of Shiga Toxin-Producing Escherichia coli.

PubMed

Parsons, Brendon D; Zelyas, Nathan; Berenger, Byron M; Chui, Linda

2016-01-01

Shiga toxin-producing Escherichia coli (STEC) are responsible for gastrointestinal diseases reported in numerous outbreaks around the world. Given the public health importance of STEC, effective detection, characterization and typing is critical to any medical laboratory system. While non-O157 serotypes account for the majority of STEC infections, frontline microbiology laboratories may only screen for STEC using O157-specific agar-based methods. As a result, non-O157 STEC infections are significantly under-reported. This review discusses recent advances on the detection, characterization and typing of STEC with emphasis on work performed at the Alberta Provincial Laboratory for Public Health (ProvLab). Candidates for the detection of all STEC serotypes include chromogenic agars, enzyme immunoassays (EIA) and quantitative real time polymerase chain reaction (qPCR). Culture methods allow further characterization of isolates, whereas qPCR provides the greatest sensitivity and specificity, followed by EIA. The virulence gene profiles using PCR arrays and stx gene subtypes can subsequently be determined. Different non-O157 serotypes exhibit markedly different virulence gene profiles and a greater prevalence of stx1 than stx2 subtypes compared to O157:H7 isolates. Finally, recent innovations in whole genome sequencing (WGS) have allowed it to emerge as a candidate for the characterization and typing of STEC in diagnostic surveillance isolates. Methods of whole genome analysis such as single nucleotide polymorphisms and k-mer analysis are concordant with epidemiological data and standard typing methods, such as pulsed-field gel electrophoresis and multiple-locus variable number tandem repeat analysis while offering additional strain differentiation. Together these findings highlight improved strategies for STEC detection using currently available systems and the development of novel approaches for future surveillance.

Detection, Characterization, and Typing of Shiga Toxin-Producing Escherichia coli

PubMed Central

Parsons, Brendon D.; Zelyas, Nathan; Berenger, Byron M.; Chui, Linda

2016-01-01

Shiga toxin-producing Escherichia coli (STEC) are responsible for gastrointestinal diseases reported in numerous outbreaks around the world. Given the public health importance of STEC, effective detection, characterization and typing is critical to any medical laboratory system. While non-O157 serotypes account for the majority of STEC infections, frontline microbiology laboratories may only screen for STEC using O157-specific agar-based methods. As a result, non-O157 STEC infections are significantly under-reported. This review discusses recent advances on the detection, characterization and typing of STEC with emphasis on work performed at the Alberta Provincial Laboratory for Public Health (ProvLab). Candidates for the detection of all STEC serotypes include chromogenic agars, enzyme immunoassays (EIA) and quantitative real time polymerase chain reaction (qPCR). Culture methods allow further characterization of isolates, whereas qPCR provides the greatest sensitivity and specificity, followed by EIA. The virulence gene profiles using PCR arrays and stx gene subtypes can subsequently be determined. Different non-O157 serotypes exhibit markedly different virulence gene profiles and a greater prevalence of stx1 than stx2 subtypes compared to O157:H7 isolates. Finally, recent innovations in whole genome sequencing (WGS) have allowed it to emerge as a candidate for the characterization and typing of STEC in diagnostic surveillance isolates. Methods of whole genome analysis such as single nucleotide polymorphisms and k-mer analysis are concordant with epidemiological data and standard typing methods, such as pulsed-field gel electrophoresis and multiple-locus variable number tandem repeat analysis while offering additional strain differentiation. Together these findings highlight improved strategies for STEC detection using currently available systems and the development of novel approaches for future surveillance. PMID:27148176

Leveraging sequence-based faecal microbial community survey data to identify a composite biomarker for colorectal cancer.

PubMed

Shah, Manasi S; DeSantis, Todd Z; Weinmaier, Thomas; McMurdie, Paul J; Cope, Julia L; Altrichter, Adam; Yamal, Jose-Miguel; Hollister, Emily B

2018-05-01

Colorectal cancer (CRC) is the second leading cause of cancer-associated mortality in the USA. The faecal microbiome may provide non-invasive biomarkers of CRC and indicate transition in the adenoma-carcinoma sequence. Re-analysing raw sequence and metadata from several studies uniformly, we sought to identify a composite and generalisable microbial marker for CRC. Raw 16S rRNA gene sequence data sets from nine studies were processed with two pipelines, (1) QIIME closed reference (QIIME-CR) or (2) a strain-specific method herein termed SS-UP (Strain Select, UPARSE bioinformatics pipeline). A total of 509 samples (79 colorectal adenoma, 195 CRC and 235 controls) were analysed. Differential abundance, meta-analysis random effects regression and machine learning analyses were carried out to determine the consistency and diagnostic capabilities of potential microbial biomarkers. Definitive taxa, including Parvimonas micra ATCC 33270, Streptococcus anginosus and yet-to-be-cultured members of Proteobacteria, were frequently and significantly increased in stools from patients with CRC compared with controls across studies and had high discriminatory capacity in diagnostic classification. Microbiome-based CRC versus control classification produced an area under receiver operator characteristic (AUROC) curve of 76.6% in QIIME-CR and 80.3% in SS-UP. Combining clinical and microbiome markers gave a diagnostic AUROC of 83.3% for QIIME-CR and 91.3% for SS-UP. Despite technological differences across studies and methods, key microbial markers emerged as important in classifying CRC cases and such could be used in a universal diagnostic for the disease. The choice of bioinformatics pipeline influenced accuracy of classification. Strain-resolved microbial markers might prove crucial in providing a microbial diagnostic for CRC. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.