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Sample records for subsp tularensis clades

  1. Multiple Francisella tularensis Subspecies and Clades, Tularemia Outbreak, Utah

    PubMed Central

    Petersen, Jeannine M.; Carlson, Jennifer K.; Dietrich, Gabrielle; Eisen, Rebecca J.; Coombs, Jana; Janusz, Aimee M.; Summers, JoDee; Ben Beard, C.

    2008-01-01

    In July 2007, a deer fly–associated outbreak of tularemia occurred in Utah. Human infections were caused by 2 clades (A1 and A2) of Francisella tularensis subsp. tularensis. Lagomorph carcasses from the area yielded evidence of infection with A1 and A2, as well as F. tularensis subsp. holarctica. These findings indicate that multiple subspecies and clades can cause disease in a localized outbreak of tularemia. PMID:19046524

  2. Genome-Wide DNA Microarray Analysis of Francisella tularensis Strains Demonstrates Extensive Genetic Conservation within the Species but Identifies Regions That Are Unique to the Highly Virulent F. tularensis subsp. tularensis

    PubMed Central

    Broekhuijsen, Martien; Larsson, Pär; Johansson, Anders; Byström, Mona; Eriksson, Ulla; Larsson, Eva; Prior, Richard G.; Sjöstedt, Anders; Titball, Richard W.; Forsman, Mats

    2003-01-01

    Francisella tularensis is a potent pathogen and a possible bioterrorism agent. Little is known, however, to explain the molecular basis for its virulence and the distinct differences in virulence found between the four recognized subspecies, F. tularensis subsp. tularensis, F. tularensis subsp. mediasiatica, F. tularensis subsp. holarctica, and F. tularensis subsp. novicida. We developed a DNA microarray based on 1,832 clones from a shotgun library used for sequencing of the highly virulent strain F. tularensis subsp. tularensis Schu S4. This allowed a genome-wide analysis of 27 strains representing all four subspecies. Overall, the microarray analysis confirmed a limited genetic variation within the species F. tularensis, and when the strains were compared, at most 3.7% of the probes showed differential hybridization. Cluster analysis of the hybridization data revealed that the causative agents of type A and type B tularemia, i.e., F. tularensis subsp. tularensis and F. tularensis subsp. holarctica, respectively, formed distinct clusters. Despite marked differences in their virulence and geographical origin, a high degree of genomic similarity between strains of F. tularensis subsp. tularensis and F. tularensis subsp. mediasiatica was apparent. Strains from Japan clustered separately, as did strains of F. tularensis subsp. novicida. Eight regions of difference (RD) 0.6 to 11.5 kb in size, altogether comprising 21 open reading frames, were identified that distinguished strains of the moderately virulent subspecies F. tularensis subsp. holarctica and the highly virulent subspecies F. tularensis subsp. tularensis. One of these regions, RD1, allowed for the first time the development of an F. tularensis-specific PCR assay that discriminates each of the four subspecies. PMID:12843022

  3. Hare-to-human transmission of Francisella tularensis subsp. holarctica, Germany.

    PubMed

    Otto, Peter; Kohlmann, Rebekka; Müller, Wolfgang; Julich, Sandra; Geis, Gabriele; Gatermann, Sören G; Peters, Martin; Wolf, Peter Johannes; Karlsson, Edvin; Forsman, Mats; Myrtennäs, Kerstin; Tomaso, Herbert

    2015-01-01

    In November 2012, a group of 7 persons who participated in a hare hunt in North Rhine-Westphalia, Germany, acquired tularemia. Two F. tularensis subsp. holarctica isolates were cultivated from human and hare biopsy material. Both isolates belonged to the FTN002-00 genetic subclade (derived for single nucleotide polymorphisms B.10 and B.18), thus indicating likely hare-to-human transmission.

  4. Genomic Deletion Marking an Emerging Subclone of Francisella tularensis subsp. holarctica in France and the Iberian Peninsula▿ †

    PubMed Central

    Dempsey, M. P.; Dobson, M.; Zhang, C.; Zhang, M.; Lion, C.; Gutiérrez-Martín, C. B.; Iwen, P. C.; Fey, P. D.; Olson, M. E.; Niemeyer, D.; Francesconi, S.; Crawford, R.; Stanley, M.; Rhodes, J.; Wagner, D. M.; Vogler, A. J.; Birdsell, D.; Keim, P.; Johansson, A.; Hinrichs, S. H.; Benson, A. K.

    2007-01-01

    Francisella tularensis subsp. holarctica is widely disseminated in North America and the boreal and temperate regions of the Eurasian continent. Comparative genomic analyses identified a 1.59-kb genomic deletion specific to F. tularensis subsp. holarctica isolates from Spain and France. Phylogenetic analysis of strains carrying this deletion by multiple-locus variable-number tandem repeat analysis showed that the strains comprise a highly related set of genotypes, implying that these strains were recently introduced or recently emerged by clonal expansion in France and the Iberian Peninsula. PMID:17890329

  5. Comparison of bacterial culture and polymerase chain reaction (PCR) for the detection of F. tularensis subsp. holarctica in wild animals.

    PubMed

    Sting, Reinhard; Runge, Martin; Eisenberg, Tobias; Braune, Silke; Müller, Wolfgang; Otto, Peter

    2013-01-01

    Detection of the zoonotic pathogen Francisella tularensis subsp. holarctica (EF tularensis) in wild animals with culture techniques as well as polymerase chain reaction were compared and discussed on the basis of the investigation of 60 animals. The samples originated from 55 European brown hares (Lepus europaeus), two red foxes (Vulpes vulpes) and one each from a wild rabbit (Oryctolagus cuniculus), a European beaver (Castor fiber), and a lemur (Lemur catta). When comparing the growth of 28 F. tularensis isolates on the cysteine blood agar and the modified Martin-Lewis-agar used in this study, cultivation was successful for 26 isolates on both media, but for two isolates only on the cysteine blood agar. Out of 43 carcasses 19 tested positive in bacteriological culture and PCR. Two culture positive samples of tonsils originating from foxes could not be confirmed by PCR, although PCR was positive in 22 samples that missed growth of F. tularensis. Comparative studies on cultural detection of E. tularensis were performed on samples of 16 hares from lung, spleen, liver and gut and in one case with a peritoneal swab. In at least one of these localizations cultivation of the pathogen was successful. Detection rate was reduced to 94% (15 of 16 hares) considering only the results of the cultures of the lungs and spleens. For a sensitive and rapid detection of F. tularensis subsp. holarctica, the PCR is a suitable method thereby avoiding hazardous multiplying of the pathogen. However, cultivation of F. tularensis is often a prerequisite for further studies on antibiotic resistance patterns of the pathogen, molecular epidemiological and pathological analyses of tularaemia.

  6. High-Quality Draft Genome Sequence of Francisella tularensis subsp. holarctica Strain 08T0073 Isolated from a Wild European Hare

    PubMed Central

    Thomas, Prasad; Myrtennäs, Kerstin; Forsman, Mats; Braune, Silke; Runge, Martin; Tomaso, Herbert

    2017-01-01

    ABSTRACT Here, we report a high-quality draft genome sequence of Francisella tularensis subsp. holarctica strain 08T0073, isolated from the cadaver of a wild European hare (Lepus europaeus) found near Helmstedt, Lower Saxony, Germany, in 2007. In Germany, infected hares are a major source of tularemia in humans. PMID:28336603

  7. Phylogeographical pattern of Francisella tularensis in a nationwide outbreak of tularaemia in Norway, 2011.

    PubMed

    Afset, J E; Larssen, K W; Bergh, K; Larkeryd, A; Sjodin, A; Johansson, A; Forsman, M

    2015-05-14

    In 2011, a nationwide outbreak of tularaemia occurred in Norway with 180 recorded cases. It was associated with the largest peak in lemming density seen in 40 years. Francisella tularensis was isolated from 18 patients. To study the geographical distribution of F.tularensis genotypes in Norway and correlate genotype with epidemiology and clinical presentation,we performed whole genome sequencing of patient isolates. All 18 genomes from the outbreak carried genetic signatures of F. tularensis subsp. holarctica and were assigned to genetic clades using canonical single nucleotide polymorphisms. Ten isolates were assigned to major genetic clade B.6 (subclade B.7),seven to clade B.12, and one to clade B.4. The B.6 subclade B.7 was most common in southern and central Norway, while clade B.12 was evenly distributed between the southern, central and northern parts of the country. There was no association between genotype and clinical presentation of tularaemia, time of year or specimen type. We found extensive sequence similarity with F. tularensis subsp. holarctica genomes from high-endemic tularaemia areas in Sweden.Finding nearly identical genomes across large geographical distances in Norway and Sweden imply a life cycle of the bacterium without replication between the outbreaks and raise new questions about long-range migration mechanisms.

  8. Genomic analyses of Francisella tularensis strains confirm disease transmission from drinking water sources, Turkey, 2008, 2009 and 2012.

    PubMed

    Karadenizli, A; Forsman, M; Şimşek, H; Taner, M; Öhrman, C; Myrtennäs, K; Lärkeryd, A; Johansson, A; Özdemir, L; Sjödin, A

    2015-05-28

    Waterborne epidemics of tularaemia caused by Francisella tularensis are increasingly reported in Turkey. We have used whole genome sequencing to investigate if F. tularensis isolated from patients could be traced back to drinking water sources. Tonsil swabs from 33 patients diagnosed with oropharyngeal tularaemia in three outbreaks and 140 water specimens were analysed. F. tularensis subsp. holarctica was confirmed by microagglutination and PCR in 12 patients and five water specimens. Genomic analysis of three pairs of patient and water isolates from outbreaks in Sivas, Çorum, and Kocaeli showed the isolates to belong to two new clusters of the F. tularensis B.12 genetic clade. The clusters were defined by 19 and 15 single nucleotide polymorphisms (SNPs) in a multiple alignment based on 507 F. tularensis genomes. One synonymous SNP was chosen as a new canonical SNP (canSNP) for each cluster for future use in diagnostic assays. No SNP was identified between the genomes from the patient–water pair of isolates from Kocaeli, one SNP between the pair of isolates from Sivas, whereas the pair from Çorum differed at seven SNPs. These results illustrate the power of whole genome sequencing for tracing F. tularensis patient isolates back to their environmental source.

  9. Respiratory Tularemia: Francisella Tularensis and Microarray Probe Designing

    PubMed Central

    Ranjbar, Reza; Behzadi, Payam; Mammina, Caterina

    2016-01-01

    Background: Francisella tularensis (F. tularensis) is the etiological microorganism for tularemia. There are different forms of tularemia such as respiratory tularemia. Respiratory tularemia is the most severe form of tularemia with a high rate of mortality; if not treated. Therefore, traditional microbiological tools and Polymerase Chain Reaction (PCR) are not useful for a rapid, reliable, accurate, sensitive and specific diagnosis. But, DNA microarray technology does. DNA microarray technology needs to appropriate microarray probe designing. Objective: The main goal of this original article was to design suitable long oligo microarray probes for detection and identification of F. tularensis. Method: For performing this research, the complete genomes of F. tularensis subsp. tularensis FSC198, F. tularensis subsp. holarctica LVS, F. tularensis subsp. mediasiatica, F. tularensis subsp. novicida (F. novicida U112), and F. philomiragia subsp. philomiragia ATCC 25017 were studied via NCBI BLAST tool, GView and PanSeq Servers and finally the microarray probes were produced and processed via AlleleID 7.7 software and Oligoanalyzer tool, respectively. Results: In this in silico investigation, a number of long oligo microarray probes were designed for detecting and identifying F. tularensis. Among these probes, 15 probes were recognized as the best candidates for microarray chip designing. Conclusion: Calibrated microarray probes reduce the biasis of DNA microarray technology as an advanced, rapid, accurate and cost-effective molecular diagnostic tool with high specificity and sensitivity. Professional microarray probe designing provides us with much more facility and flexibility regarding preparation of a microarray diagnostic chip. PMID:28077973

  10. Targeted inactivation of francisella tularensis genes by group II introns.

    PubMed

    Rodriguez, Stephen A; Yu, Jieh-Juen; Davis, Greg; Arulanandam, Bernard P; Klose, Karl E

    2008-05-01

    Studies of the molecular mechanisms of pathogenesis of Francisella tularensis, the causative agent of tularemia, have been hampered by a lack of genetic techniques for rapid targeted gene disruption in the most virulent subspecies. Here we describe efficient targeted gene disruption in F. tularensis utilizing mobile group II introns (targetrons) specifically optimized for F. tularensis. Utilizing a targetron targeted to blaB, which encodes ampicillin resistance, we showed that the system works at high efficiency in three different subspecies: F. tularensis subsp. tularensis, F. tularensis subsp. holarctica, and "F. tularensis subsp. novicida." A targetron was also utilized to inactivate F. tularensis subsp. holarctica iglC, a gene required for virulence. The iglC gene is located within the Francisella pathogenicity island (FPI), which has been duplicated in the most virulent subspecies. Importantly, the iglC targetron targeted both copies simultaneously, resulting in a strain mutated in both iglC genes in a single step. This system will help illuminate the contributions of specific genes, and especially those within the FPI, to the pathogenesis of this poorly studied organism.

  11. Relationship of Bacillus amyloliquefaciens clades associated with strains DSM 7T and FZB42T: a proposal for Bacillus amyloliquefaciens subsp. amyloliquefaciens subsp. nov. and Bacillus amyloliquefaciens subsp. plantarum subsp. nov. based on complete genome sequence comparisons.

    PubMed

    Borriss, Rainer; Chen, Xiao-Hua; Rueckert, Christian; Blom, Jochen; Becker, Anke; Baumgarth, Birgit; Fan, Ben; Pukall, Rüdiger; Schumann, Peter; Spröer, Cathrin; Junge, Helmut; Vater, Joachim; Pühler, Alfred; Klenk, Hans-Peter

    2011-08-01

    The whole-genome-sequenced rhizobacterium Bacillus amyloliquefaciens FZB42(T) (Chen et al., 2007) and other plant-associated strains of the genus Bacillus described as belonging to the species Bacillus amyloliquefaciens or Bacillus subtilis are used commercially to promote the growth and improve the health of crop plants. Previous investigations revealed that a group of strains represented a distinct ecotype related to B. amyloliquefaciens; however, the exact taxonomic position of this group remains elusive (Reva et al., 2004). In the present study, we demonstrated the ability of a group of Bacillus strains closely related to strain FZB42(T) to colonize Arabidopsis roots. On the basis of their phenotypic traits, the strains were similar to Bacillus amyloliquefaciens DSM 7(T) but differed considerably from this type strain in the DNA sequences of genes encoding 16S rRNA, gyrase subunit A (gyrA) and histidine kinase (cheA). Phylogenetic analysis performed with partial 16S rRNA, gyrA and cheA gene sequences revealed that the plant-associated strains of the genus Bacillus, including strain FZB42(T), formed a lineage, which could be distinguished from the cluster of strains closely related to B. amyloliquefaciens DSM 7(T). DNA-DNA hybridizations (DDH) performed with genomic DNA from strains DSM 7(T) and FZB42(T) yielded relatedness values of 63.7-71.2 %. Several methods of genomic analysis, such as direct whole-genome comparison, digital DDH and microarray-based comparative genomichybridization (M-CGH) were used as complementary tests. The group of plant-associated strains could be distinguished from strain DSM 7(T) and the type strain of B. subtilis by differences in the potential to synthesize non-ribosomal lipopeptides and polyketides. Based on the differences found in the marker gene sequences and the whole genomes of these strains, we propose two novel subspecies, designated B. amyloliquefaciens subsp. plantarum subsp. nov., with the type strain FZB42(T) ( = DSM

  12. Detection of Francisella tularensis in voles in Finland.

    PubMed

    Rossow, Heidi; Sissonen, Susanna; Koskela, Katja A; Kinnunen, Paula M; Hemmilä, Heidi; Niemimaa, Jukka; Huitu, Otso; Kuusi, Markku; Vapalahti, Olli; Henttonen, Heikki; Nikkari, Simo

    2014-03-01

    Francisella tularensis is a highly virulent intracellular bacterium causing the zoonotic disease tularemia. It recurrently causes human and animal outbreaks in northern Europe, including Finland. Although F. tularensis infects several mammal species, only rodents and lagomorphs seem to have importance in its ecology. Peak densities of rodent populations may trigger tularemia outbreaks in humans; however, it is still unclear to which extent rodents or other small mammals maintain F. tularensis in nature. The main objective of this study was to obtain information about the occurrence of F. tularensis in small mammals in Finland. We snap-trapped 547 wild small mammals representing 11 species at 14 locations around Finland during 6 years and screened them for the presence of F. tularensis DNA using PCR analysis. High copy number of F. tularensis-specific DNA was detected in tissue samples of five field voles (Microtus agrestis) originating from one location and 2 years. According to DNA sequences of the bacterial 23S ribosomal RNA gene amplified from F. tularensis-infected voles, the infecting agent belongs to the subspecies holarctica. To find out the optimal tissue for tularemia screening in voles, we compared the amounts of F. tularensis DNA in lungs, liver, spleen, and kidney of the infected animals. F. tularensis DNA was detectable in high levels in all four organs except for one animal, whose kidney was F. tularensis DNA-negative. Thus, at least liver, lung, and spleen seem suitable for F. tularensis screening in voles. Thus, liver, lung, and spleen all seem suitable for F. tularensis screening in voles. In conclusion, field voles can be heavily infected with F. tularensis subsp. holarctica and thus potentially serve as the source of infection in humans and other mammals.

  13. Detection of Francisella tularensis in Voles in Finland

    PubMed Central

    Sissonen, Susanna; Koskela, Katja A.; Kinnunen, Paula M.; Hemmilä, Heidi; Niemimaa, Jukka; Huitu, Otso; Kuusi, Markku; Vapalahti, Olli; Henttonen, Heikki; Nikkari, Simo

    2014-01-01

    Abstract Francisella tularensis is a highly virulent intracellular bacterium causing the zoonotic disease tularemia. It recurrently causes human and animal outbreaks in northern Europe, including Finland. Although F. tularensis infects several mammal species, only rodents and lagomorphs seem to have importance in its ecology. Peak densities of rodent populations may trigger tularemia outbreaks in humans; however, it is still unclear to which extent rodents or other small mammals maintain F. tularensis in nature. The main objective of this study was to obtain information about the occurrence of F. tularensis in small mammals in Finland. We snap-trapped 547 wild small mammals representing 11 species at 14 locations around Finland during 6 years and screened them for the presence of F. tularensis DNA using PCR analysis. High copy number of F. tularensis-specific DNA was detected in tissue samples of five field voles (Microtus agrestis) originating from one location and 2 years. According to DNA sequences of the bacterial 23S ribosomal RNA gene amplified from F. tularensis–infected voles, the infecting agent belongs to the subspecies holarctica. To find out the optimal tissue for tularemia screening in voles, we compared the amounts of F. tularensis DNA in lungs, liver, spleen, and kidney of the infected animals. F. tularensis DNA was detectable in high levels in all four organs except for one animal, whose kidney was F. tularensis DNA-negative. Thus, at least liver, lung, and spleen seem suitable for F. tularensis screening in voles. Thus, liver, lung, and spleen all seem suitable for F. tularensis screening in voles. In conclusion, field voles can be heavily infected with F. tularensis subsp. holarctica and thus potentially serve as the source of infection in humans and other mammals. PMID:24575824

  14. German Francisella tularensis isolates from European brown hares (Lepus europaeus) reveal genetic and phenotypic diversity

    PubMed Central

    2013-01-01

    Background Tularemia is a zoonotic disease caused by Francisella tularensis that has been found in many different vertebrates. In Germany most human infections are caused by contact with infected European brown hares (Lepus europaeus). The aim of this study was to elucidate the epidemiology of tularemia in hares using phenotypic and genotypic characteristics of F. tularensis. Results Cultivation of F. tularensis subsp. holarctica bacteria from organ material was successful in 31 of 52 hares that had a positive PCR result targeting the Ft-M19 locus. 17 isolates were sensitive to erythromycin and 14 were resistant. Analysis of VNTR loci (Ft-M3, Ft-M6 and Ft-M24), INDELs (Ftind33, Ftind38, Ftind49, RD23) and SNPs (B.17, B.18, B.19, and B.20) was shown to be useful to investigate the genetic relatedness of Francisella strains in this set of strains. The 14 erythromycin resistant isolates were assigned to clade B.I, and 16 erythromycin sensitive isolates to clade B.IV and one isolate was found to belong to clade B.II. MALDI-TOF mass spectrometry (MS) was useful to discriminate strains to the subspecies level. Conclusions F. tularensis seems to be a re-emerging pathogen in Germany. The pathogen can easily be identified using PCR assays. Isolates can also be identified within one hour using MALDI-TOF MS in laboratories where specific PCR assays are not established. Further analysis of strains requires genotyping tools. The results from this study indicate a geographical segregation of the phylogenetic clade B.I and B.IV, where B.I strains localize primarily within eastern Germany and B.IV strains within western Germany. This phylogeographical pattern coincides with the distribution of biovar I (erythromycin sensitive) and biovar II (erythromycin resistance) strains. When time and costs are limiting parameters small numbers of isolates can be analysed using PCR assays combined with DNA sequencing with a focus on genetic loci that are most likely discriminatory among

  15. A 14.7 kDa protein from Francisella tularensis subsp. novicida (named FTN_1133), involved in the response to oxidative stress induced by organic peroxides, is not endowed with thiol-dependent peroxidase activity.

    PubMed

    Meireles, Diogo de Abreu; Alegria, Thiago Geronimo Pires; Alves, Simone Vidigal; Arantes, Carla Rani Rocha; Netto, Luis Eduardo Soares

    2014-01-01

    Francisella genus comprises Gram-negative facultative intracellular bacteria that are among the most infectious human pathogens. A protein of 14.7 KDa named as FTN_1133 was previously described as a novel hydroperoxide resistance protein in F. tularensis subsp. novicida, implicated in organic peroxide detoxification and virulence. Here, we describe a structural and biochemical characterization of FTN_1133. Contrary to previous assumptions, multiple amino acid sequence alignment analyses revealed that FTN_1133 does not share significant similarity with proteins of the Ohr/OsmC family or any other Cys-based, thiol dependent peroxidase, including conserved motifs around reactive cysteine residues. Circular dichroism analyses were consistent with the in silico prediction of an all-α-helix secondary structure. The pKa of its single cysteine residue, determined by a monobromobimane alkylation method, was shown to be 8.0±0.1, value that is elevated when compared with other Cys-based peroxidases, such as peroxiredoxins and Ohr/OsmC proteins. Attempts to determine a thiol peroxidase activity for FTN_1133 failed, using both dithiols (DTT, thioredoxin and lipoamide) and monothiols (glutathione or 2-mercaptoethanol) as reducing agents. Heterologous expression of FTN_1133 gene in ahpC and oxyR mutants of E. coli showed no complementation. Furthermore, analysis of FTN_1133 protein by non-reducing SDS-PAGE showed that an inter-molecular disulfide bond (not detected in Ohr proteins) can be generated under hydroperoxide treatment, but the observed rates were not comparable to those observed for other thiol-dependent peroxidases. All the biochemical and structural data taken together indicated that FTN_1133 displayed distinct characteristics from other thiol dependent peroxidases and, therefore, suggested that FTN_1133 is not directly involved in hydroperoxide detoxification.

  16. First indication for a functional CRISPR/Cas system in Francisella tularensis.

    PubMed

    Schunder, Eva; Rydzewski, Kerstin; Grunow, Roland; Heuner, Klaus

    2013-03-01

    Francisella tularensis is a zoonotic agent and the subspecies novicida is proposed to be a water-associated bacterium. The intracellular pathogen F. tularensis causes tularemia in humans and is known for its potential to be used as a biological threat. We analyzed the genome sequence of F. tularensis subsp. novicida U112 in silico for the presence of a putative functional CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system. CRISPR/Cas systems are known to encode an RNA-guided adaptive immunity-like system to protect bacteria against invading genetic elements like bacteriophages and plasmids. In this work, we present a first indication that F. tularensis subsp. novicida encodes a functional CRISPR/Cas defence system. Additionally, we identified various spacer DNAs homologous to a putative phage present within the genome of F. tularensis subsp. novicida-like strain 3523. CRISPR/Cas is also present in F. tularensis subsp. tularensis, holarctica, and mediasiatica, but these systems seem to be non-functional.

  17. Molecular method for discrimination between Francisella tularensis and Francisella-like endosymbionts.

    PubMed

    Escudero, Raquel; Toledo, A; Gil, Horacio; Kovácsová, Katarina; Rodríguez-Vargas, Manuela; Jado, Isabel; García-Amil, Cristina; Lobo, Bruno; Bhide, Mangesh; Anda, Pedro

    2008-09-01

    Environmental studies on the distribution of Francisella spp. are hampered by the frequency of Francisella-like endosymbionts that can produce a misleading positive result. A new, efficient molecular method for detection of Francisella tularensis and its discrimination from Francisella-like endosymbionts, as well as two variants associated with human disease (unusual F. tularensis strain FnSp1 and F. tularensis subsp. novicida-like strain 3523), is described. The method is highly specific and sensitive, detecting up to one plasmid copy or 10 genome equivalents.

  18. Comparative Transcriptional Analyses of Francisella tularensis and Francisella novicida

    PubMed Central

    Waldo, Robert H.; Belland, Robert J.; Klose, Karl E.

    2016-01-01

    Francisella tularensis is composed of a number of subspecies with varied geographic distribution, host ranges, and virulence. In view of these marked differences, comparative functional genomics may elucidate some of the molecular mechanism(s) behind these differences. In this study a shared probe microarray was designed that could be used to compare the transcriptomes of Francisella tularensis subsp. tularensis Schu S4 (Ftt), Francisella tularensis subsp. holarctica OR960246 (Fth), Francisella tularensis subsp. holarctica LVS (LVS), and Francisella novicida U112 (Fn). To gain insight into expression differences that may be related to the differences in virulence of these subspecies, transcriptomes were measured from each strain grown in vitro under identical conditions, utilizing a shared probe microarray. The human avirulent Fn strain exhibited high levels of transcription of genes involved in general metabolism, which are pseudogenes in the human virulent Ftt and Fth strains, consistent with the process of genome decay in the virulent strains. Genes encoding an efflux system (emrA2 cluster of genes), siderophore (fsl operon), acid phosphatase, LPS synthesis, polyamine synthesis, and citrulline ureidase were all highly expressed in Ftt when compared to Fn, suggesting that some of these may contribute to the relative high virulence of Ftt. Genes expressed at a higher level in Ftt when compared to the relatively less virulent Fth included genes encoding isochorismatases, cholylglycine hydrolase, polyamine synthesis, citrulline ureidase, Type IV pilus subunit, and the Francisella Pathogenicity Island protein PdpD. Fth and LVS had very few expression differences, consistent with the derivation of LVS from Fth. This study demonstrated that a shared probe microarray designed to detect transcripts in multiple species/subspecies of Francisella enabled comparative transcriptional analyses that may highlight critical differences that underlie the relative pathogenesis of

  19. A multiplex real-time PCR assay for the detection and differentiation of Francisella tularensis subspecies.

    PubMed

    Gunnell, Mark K; Lovelace, Charity D; Satterfield, Benjamin A; Moore, Emily A; O'Neill, Kim L; Robison, Richard A

    2012-11-01

    Francisella tularensis is the aetiological agent of tularaemia, a zoonotic disease with worldwide prevalence. F. tularensis is a highly pathogenic organism and has been designated a category A biothreat agent by the Centers for Disease Control and Prevention. Tularaemia is endemic in much of the USA, Europe and parts of Asia. It is transmitted by numerous vectors and vehicles such as deer flies, ticks and rabbits. Currently, there are four recognized subspecies of F. tularensis: tularensis (type A), holarctica (type B), mediasiatica and novicida. Within the type A classification there are two subclassifications, type A.I and A.II, each with a specific geographical distribution across the USA. F. tularensis subsp. holartica (type B) is found in both the USA and Europe. Because of virulence differences among subtypes, it is important that health departments, hospitals and other government agencies are able to quickly identify each subtype. The purpose of this study was to develop a multiplex real-time PCR assay for the identification and discrimination of type A.I, type A.II, type B and novicida subspecies of F. tularensis. The assay was validated using 119 isolates of F. tularensis, three of its nearest neighbours and 14 other bacterial pathogens. This assay proved to be ~98 % successful at identifying the known subspecies of F. tularensis and could prove to be a useful tool in the characterization of this important pathogen.

  20. Identification of two substrates of FTS_1067 protein - An essential virulence factor of Francisella tularensis.

    PubMed

    Spidlova, Petra; Senitkova, Iva; Link, Marek; Stulik, Jiri

    2016-11-15

    Francisella tularensis is a highly virulent intracellular pathogen with the capacity to infect a variety of hosts including humans. One of the most important proteins involved in F. tularensis virulence and pathogenesis is the protein DsbA. This protein is annotated as a lipoprotein with disulfide oxidoreductase/isomerase activity. Therefore, its interactions with different substrates, including probable virulence factors, to assist in their proper folding are anticipated. We aimed to use the immunopurification approach to find DsbA (gene locus FTS_1067) interacting partners in F. tularensis subsp. holarctica strain FSC200 and compare the identified substrates with proteins which were found in our previous comparative proteome analysis. As a result of our work two FTS_1067 substrates, D-alanyl-D-alanine carboxypeptidase family protein and HlyD family secretion protein, were identified. Bacterial two-hybrid systems were further used to test their relevance in confirming FTS_1067 protein interactions.

  1. Towards Development of Improved Serodiagnostics for Tularemia by Use of Francisella tularensis Proteome Microarrays.

    PubMed

    Nakajima, Rie; Escudero, Raquel; Molina, Douglas M; Rodríguez-Vargas, Manuela; Randall, Arlo; Jasinskas, Algis; Pablo, Jozelyn; Felgner, Philip L; AuCoin, David P; Anda, Pedro; Davies, D Huw

    2016-07-01

    Tularemia in humans is caused mainly by two subspecies of the Gram-negative facultative anaerobe Francisella tularensis: F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B). The current serological test for tularemia is based on agglutination of whole organisms, and the reactive antigens are not well understood. Previously, we profiled the antibody responses in type A and B tularemia cases in the United States using a proteome microarray of 1,741 different proteins derived from the type A strain Schu S4. Fifteen dominant antigens able to detect antibodies to both types of infection were identified, although these were not validated in a different immunoassay format. Since type A and B subspecies are closely related, we hypothesized that Schu S4 antigens would also have utility for diagnosing type B tularemia caused by strains from other geographic locations. To test this, we probed the Schu S4 array with sera from 241 type B tularemia cases in Spain. Despite there being no type A strains in Spain, we confirmed the responses against some of the same potential serodiagnostic antigens reported previously, as well as determined the responses against additional potential serodiagnostic antigens. Five potential serodiagnostic antigens were evaluated on immunostrips, and two of these (FTT1696/GroEL and FTT0975/conserved hypothetical protein) discriminated between the Spanish tularemia cases and healthy controls. We conclude that antigens from the type A strain Schu S4 are suitable for detection of antibodies from patients with type B F. tularensis infections and that these can be used for the diagnosis of tularemia in a deployable format, such as the immunostrip.

  2. Towards Development of Improved Serodiagnostics for Tularemia by Use of Francisella tularensis Proteome Microarrays

    PubMed Central

    Nakajima, Rie; Escudero, Raquel; Molina, Douglas M.; Rodríguez-Vargas, Manuela; Randall, Arlo; Jasinskas, Algis; Pablo, Jozelyn; Felgner, Philip L.; AuCoin, David P.; Anda, Pedro

    2016-01-01

    Tularemia in humans is caused mainly by two subspecies of the Gram-negative facultative anaerobe Francisella tularensis: F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B). The current serological test for tularemia is based on agglutination of whole organisms, and the reactive antigens are not well understood. Previously, we profiled the antibody responses in type A and B tularemia cases in the United States using a proteome microarray of 1,741 different proteins derived from the type A strain Schu S4. Fifteen dominant antigens able to detect antibodies to both types of infection were identified, although these were not validated in a different immunoassay format. Since type A and B subspecies are closely related, we hypothesized that Schu S4 antigens would also have utility for diagnosing type B tularemia caused by strains from other geographic locations. To test this, we probed the Schu S4 array with sera from 241 type B tularemia cases in Spain. Despite there being no type A strains in Spain, we confirmed the responses against some of the same potential serodiagnostic antigens reported previously, as well as determined the responses against additional potential serodiagnostic antigens. Five potential serodiagnostic antigens were evaluated on immunostrips, and two of these (FTT1696/GroEL and FTT0975/conserved hypothetical protein) discriminated between the Spanish tularemia cases and healthy controls. We conclude that antigens from the type A strain Schu S4 are suitable for detection of antibodies from patients with type B F. tularensis infections and that these can be used for the diagnosis of tularemia in a deployable format, such as the immunostrip. PMID:27098957

  3. Biology of Francisella tularensis Subspecies holarctica Live Vaccine Strain in the Tick Vector Dermacentor variabilis

    PubMed Central

    Mani, Rinosh J.; Reichard, Mason V.; Morton, Rebecca J.; Kocan, Katherine M.; Clinkenbeard, Kenneth D.

    2012-01-01

    Background The γ-proteobacterium Francisella tularensis is the etiologic agent of seasonal tick-transmitted tularemia epizootics in rodents and rabbits and of incidental infections in humans. The biology of F. tularensis in its tick vectors has not been fully described, particularly with respect to its quanta and duration of colonization, tissue dissemination, and transovarial transmission. A systematic study of the colonization of Dermacentor variabilis by the F. tularensis subsp. holarctica live vaccine strain (LVS) was undertaken to better understand whether D. variabilis may serve as an inter-epizootic reservoir for F. tularensis. Methodology/Principal Findings Colony-reared larva, nymph, and adult D. variabilis were artificially fed LVS via glass capillary tubes fitted over the tick mouthparts, and the level of colonization determined by microbial culture. Larvae and nymphs were initially colonized with 8.8±0.8×101 and 1.1±0.03×103 CFU/tick, respectively. Post-molting, a significant increase in colonization of both molted nymphs and adults occurred, and LVS persisted in 42% of molted adult ticks at 126 days post-capillary tube feeding. In adult ticks, LVS initially colonized the gut, disseminated to hemolymph and salivary glands by 21 days, and persisted up to 165 days. LVS was detected in the salivary secretions of adult ticks after four days post intra-hemocoelic inoculation, and LVS recovered from salivary gland was infectious to mice with an infectious dose 50% of 3 CFU. LVS in gravid female ticks colonized via the intra-hemocoelic route disseminated to the ovaries and then to the oocytes, but the pathogen was not recovered from the subsequently-hatched larvae. Conclusions/Significance This study demonstrates that D. variabilis can be efficiently colonized with F. tularensis using artificial methods. The persistence of F. tularensis in D. variabilis suggests that this tick species may be involved in the maintenance of enzootic foci of tularemia in the

  4. Francisella tularensis Subspecies holarctica, Tasmania, Australia, 2011

    PubMed Central

    Jackson, Justin; McGregor, Alistair; Cooley, Louise; Ng, Jimmy; Brown, Mitchell; Ong, Chong Wei; Darcy, Catharine

    2012-01-01

    We report a case of ulceroglandular tularemia that developed in a woman after she was bitten by a ringtail possum (Pseudocheirus peregrinus) in a forest in Tasmania, Australia. Francisella tularensis subspecies holarctica was identified. This case indicates the emergence of F. tularensis type B in the Southern Hemisphere. PMID:22931809

  5. Substructure within Salmonella enterica subsp. enterica Isolates from Australian Wildlife▿

    PubMed Central

    Parsons, Sandra K.; Bull, C. Michael; Gordon, David M.

    2011-01-01

    Multilocus sequence typing of 56 Salmonella enterica subsp. enterica strains isolated from Australian wildlife hosts was performed. The results of population assignment algorithms revealed that the 56 strains could be subdivided into two distinct clades. Strains belonging to the two clades were further distinguished phenotypically, genotypically, and with respect to host distribution. PMID:21378038

  6. Paired-End Sequence Mapping Detects Extensive Genomic Rearrangement and Translocation During Divergence of Francisella tularensis Subspecies Tularensis and Francisella tularensis Subspecies holarctica Populations

    DTIC Science & Technology

    2006-03-30

    such as Acanthamoeba castellanii, may also serve as a host for maintenance ofF. tularensis in the aquatic cycle [10]. The species F. tularensis is...and growth of Francisella tularensis in Acanthamoeba castellanii. Appl Environ Microbiol, 2003. 69(1): p. 600-6. 11. Chu, M.C. and R.S. Weyant

  7. Francisella tularensis: No Evidence for Transovarial Transmission in the Tularemia Tick Vectors Dermacentor reticulatus and Ixodes ricinus

    PubMed Central

    Genchi, Marco; Prati, Paola; Vicari, Nadia; Manfredini, Andrea; Sacchi, Luciano; Clementi, Emanuela; Bandi, Claudio; Epis, Sara; Fabbi, Massimo

    2015-01-01

    Background Tularemia is a zoonosis caused by the Francisella tularensis, a highly infectious Gram-negative coccobacillus. Due to easy dissemination, multiple routes of infection, high environmental contamination and morbidity and mortality rates, Francisella is considered a potential bioterrorism threat and classified as a category A select agent by the CDC. Tick bites are among the most prevalent modes of transmission, and ticks have been indicated as a possible reservoir, although their reservoir competence has yet to be defined. Tick-borne transmission of F. tularensis was recognized in 1923, and transstadial transmission has been demonstrated in several tick species. Studies on transovarial transmission, however, have reported conflicting results. Objective The aim of this study was to evaluate the role of ticks as reservoirs for Francisella, assessing the transovarial transmission of F. tularensis subsp. holarctica in ticks, using experimentally-infected females of Dermacentor reticulatus and Ixodes ricinus. Results Transmission electron microscopy and fluorescence in situ hybridization showed F. tularensis within oocytes. However, cultures and bioassays of eggs and larvae were negative; in addition, microscopy techniques revealed bacterial degeneration/death in the oocytes. Conclusions These results suggest that bacterial death might occur in oocytes, preventing the transovarial transmission of Francisella. We can speculate that Francisella does not have a defined reservoir, but that rather various biological niches (e.g. ticks, rodents), that allow the bacterium to persist in the environment. Our results, suggesting that ticks are not competent for the bacterium vertical transmission, are congruent with this view. PMID:26244842

  8. Crystallization of a newly discovered histidine acid phosphatase from Francisella tularensis

    SciTech Connect

    Felts, Richard L.; Reilly, Thomas J.; Calcutt, Michael J.; Tanner, John J.

    2006-01-01

    A histidine acid phosphatase from the CDC Category A pathogen F. tularensis has been crystallized in space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = 61.96, c = 210.78 Å. A 1.75 Å resolution data set was collected at Advanced Light Source beamline 4.2.2. Francisella tularensis is a highly infectious bacterial pathogen that is considered by the Centers for Disease Control and Prevention to be a potential bioterrorism weapon. Here, the crystallization of a 37.2 kDa phosphatase encoded by the genome of F. tularensis subsp. holarctica live vaccine strain is reported. This enzyme shares 41% amino-acid sequence identity with Legionella pneumophila major acid phosphatase and contains the RHGXRXP motif that is characteristic of the histidine acid phosphatase family. Large diffraction-quality crystals were grown in the presence of Tacsimate, HEPES and PEG 3350. The crystals belong to space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = 61.96, c = 210.78 Å. The asymmetric unit is predicted to contain one protein molecule, with a solvent content of 53%. A 1.75 Å resolution native data set was recorded at beamline 4.2.2 of the Lawrence Berkeley National Laboratory Advanced Light Source. Molecular-replacement trials using the human prostatic acid phosphatase structure as the search model (28% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of F. tularensis histidine acid phosphatase will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative.

  9. Targeted gene disruption in Francisella tularensis by group II introns.

    PubMed

    Rodriguez, Stephen A; Davis, Greg; Klose, Karl E

    2009-11-01

    Francisella tularensis is a highly infectious Gram-negative bacterium that is the causative agent of tularemia. Very little is known about the molecular mechanisms responsible for F. tularensis virulence, in part due to the paucity of genetic tools available for the study of F. tularensis. We have developed a gene knockout system for F. tularensis that utilizes retargeted mobile group II introns, or "targetrons". These targetrons disrupt both single and duplicated target genes at high efficiency in three different F. tularensis subspecies. Here we describe in detail the targetron-based method for insertional mutagenesis of F. tularensis genes, which should facilitate a better understanding of F. tularensis pathogenesis. Group II introns can be adapted to inactivate genes in bacteria for which few genetic tools exist, thus providing a powerful tool to study the genetic basis of bacterial pathogenesis.

  10. Environmental Monitoring and Surveillance of Rodents and Vectors for Francisella tularensis Following Outbreaks of Human Tularemia in Georgia.

    PubMed

    Elashvili, Eka; Kracalik, Ian; Burjanadze, Irma; Datukishvili, Sophio; Chanturia, Gvantsa; Tsertsvadze, Nikoloz; Beridze, Levan; Shavishvili, Merab; Dzneladze, Archil; Grdzelidze, Marina; Imnadze, Paata; Pearson, Andrew; Blackburn, Jason K

    2015-10-01

    Tularemia is a re-emerging bacterial zoonosis, broadly distributed across the northern hemisphere. In Georgia, there is a history of human tularemia outbreaks dating back to the 1940s. In response to outbreaks, health officials initiated long-term field surveillance and environmental monitoring. The objective of our study was to obtain information from 57 years of field surveys to identify species that play a role in the occurrence Francisella tularensis subsp. holarctica in the environment in Georgia. We collected historical data on human outbreaks, field collections, population dynamics of the common vole (Microtus arvalis), and conducted surveys on small mammals and vectors from five regions in Georgia during 1956-2012. Bacterial isolation was conducted using standard culturing techniques, and isolation rates for species were obtained for a subset of years. We used a Spearman rank correlation to test for associations between the density of the common vole and isolation rates. From 1956 through 2012, there were four recorded outbreaks of human tularemia (362 cases). A total of 465 bacterial isolates of F. tularensis subsp. holarctica were obtained from 27 species and environmental samples. The number of isolations was highest in the common vole (M. arvalis; 149 isolates; 32%) and Dermacentor marginatus ticks (132 isolates; 28%); isolation rates ranged between 0-0.91% and 0-0.47%, respectively. Population dynamics of the common vole were not correlated with the isolation rate. Given the history of tularemia re-emergence in Georgia, continued field surveys and environmental monitoring may provide an early indication of outbreak risk in humans. In conclusion, our findings provide evidence of long-standing foci of F. tularensis subsp. holarctica that are likely maintained by the common vole-tick cycle.

  11. Environmental Monitoring and Surveillance of Rodents and Vectors for Francisella tularensis Following Outbreaks of Human Tularemia in Georgia

    PubMed Central

    Elashvili, Eka; Kracalik, Ian; Burjanadze, Irma; Datukishvili, Sophio; Chanturia, Gvantsa; Tsertsvadze, Nikoloz; Beridze, Levan; Shavishvili, Merab; Dzneladze, Archil; Grdzelidze, Marina; Imnadze, Paata; Pearson, Andrew

    2015-01-01

    Abstract Tularemia is a re-emerging bacterial zoonosis, broadly distributed across the northern hemisphere. In Georgia, there is a history of human tularemia outbreaks dating back to the 1940s. In response to outbreaks, health officials initiated long-term field surveillance and environmental monitoring. The objective of our study was to obtain information from 57 years of field surveys to identify species that play a role in the occurrence Francisella tularensis subsp. holarctica in the environment in Georgia. We collected historical data on human outbreaks, field collections, population dynamics of the common vole (Microtus arvalis), and conducted surveys on small mammals and vectors from five regions in Georgia during 1956–2012. Bacterial isolation was conducted using standard culturing techniques, and isolation rates for species were obtained for a subset of years. We used a Spearman rank correlation to test for associations between the density of the common vole and isolation rates. From 1956 through 2012, there were four recorded outbreaks of human tularemia (362 cases). A total of 465 bacterial isolates of F. tularensis subsp. holarctica were obtained from 27 species and environmental samples. The number of isolations was highest in the common vole (M. arvalis; 149 isolates; 32%) and Dermacentor marginatus ticks (132 isolates; 28%); isolation rates ranged between 0–0.91% and 0–0.47%, respectively. Population dynamics of the common vole were not correlated with the isolation rate. Given the history of tularemia re-emergence in Georgia, continued field surveys and environmental monitoring may provide an early indication of outbreak risk in humans. In conclusion, our findings provide evidence of long-standing foci of F. tularensis subsp. holarctica that are likely maintained by the common vole–tick cycle. PMID:26394283

  12. Genetic Diversity of Pectobacterium carotovorum subsp. brasiliensis Isolated in Korea

    PubMed Central

    Lee, Dong Hwan; Kim, Jin-Beom; Lim, Jeong-A; Han, Sang-Wook; Heu, Sunggi

    2014-01-01

    The plant pathogenic bacterial genus Pectobacteirum consists of heterogeneous strains. The P. carotovorum species is a complex strain showing divergent characteristics, and a new subspecies named P. carotovorum subsp. brasiliensis has been identified recently. In this paper, we re-identified the P. carotovorum subsp. brasiliensis isolates from those classified under the subspecies carotovorum and newly isolated P. carotovorum subsp. brasiliensis strains. All isolates were able to produce plant cell-wall degrading enzymes such as pectate lyase, polygalacturonase, cellulase and protease. We used genetic and biochemical methods to examine the diversity of P. carotovorum subsp. brasiliensis isolates, and found genetic diversity within the brasiliensis subsp. isolates in Korea. The restriction fragment length polymorphism analysis based on the recA gene revealed a unique pattern for the brasiliensis subspecies. The Korean brasiliensis subsp. isolates were divided into four clades based on pulsed-field gel electrophoresis. However, correlations between clades and isolated hosts or year could not be found, suggesting that diverse brasiliensis subsp. isolates existed. PMID:25288994

  13. Structure-Function Analysis of DipA, a Francisella tularensis Virulence Factor Required for Intracellular Replication

    PubMed Central

    Chong, Audrey; Child, Robert; Wehrly, Tara D.; Rockx-Brouwer, Dedeke; Qin, Aiping; Mann, Barbara J.; Celli, Jean

    2013-01-01

    Francisella tularensis is a highly infectious bacterium whose virulence relies on its ability to rapidly reach the macrophage cytosol and extensively replicate in this compartment. We previously identified a novel Francisella virulence factor, DipA (FTT0369c), which is required for intramacrophage proliferation and survival, and virulence in mice. DipA is a 353 amino acid protein with a Sec-dependent signal peptide, four Sel1-like repeats (SLR), and a C-terminal coiled-coil (CC) domain. Here, we determined through biochemical and localization studies that DipA is a membrane-associated protein exposed on the surface of the prototypical F. tularensis subsp. tularensis strain SchuS4 during macrophage infection. Deletion and substitution mutagenesis showed that the CC domain, but not the SLR motifs, of DipA is required for surface exposure on SchuS4. Complementation of the dipA mutant with either DipA CC or SLR domain mutants did not restore intracellular growth of Francisella, indicating that proper localization and the SLR domains are required for DipA function. Co-immunoprecipitation studies revealed interactions with the Francisella outer membrane protein FopA, suggesting that DipA is part of a membrane-associated complex. Altogether, our findings indicate that DipA is positioned at the host–pathogen interface to influence the intracellular fate of this pathogen. PMID:23840797

  14. Geographic Differentiation of Francisella Tularensis using Molecular Methods

    DTIC Science & Technology

    2006-05-01

    For his extensive global F. tularensis DNA collection, I am grateful to Dr. Anders Johansson from the Division of Infectious Diseases and Clinical...dissemination, its extremely low infectious dose of only ten to fifty organisms when acquired through the inhalation route in humans, and the potential...in this chapter. 14 1.1.3 Ecology of F. tularensis F. tularensis is a widely infectious zoonotic pathogen and has been isolated from as many as 250

  15. IglE Is an Outer Membrane-Associated Lipoprotein Essential for Intracellular Survival and Murine Virulence of Type A Francisella tularensis

    PubMed Central

    Robertson, Gregory T.; Child, Robert; Ingle, Christine; Celli, Jean

    2013-01-01

    IglE is a small, hypothetical protein encoded by the duplicated Francisella pathogenicity island (FPI). Inactivation of both copies of iglE rendered Francisella tularensis subsp. tularensis Schu S4 avirulent and incapable of intracellular replication, owing to an inability to escape the phagosome. This defect was fully reversed following single-copy expression of iglE in trans from attTn7 under the control of the Francisella rpsL promoter, thereby establishing that the loss of iglE, and not polar effects on downstream vgrG gene expression, was responsible for the defect. IglE is exported to the Francisella outer membrane as an ∼13.9-kDa lipoprotein, determined on the basis of a combination of selective Triton X-114 solubilization, radiolabeling with [3H]palmitic acid, and sucrose density gradient membrane partitioning studies. Lastly, a genetic screen using the iglE-null live vaccine strain resulted in the identification of key regions in the carboxyl terminus of IglE that are required for intracellular replication of Francisella tularensis in J774A.1 macrophages. Thus, IglE is essential for Francisella tularensis virulence. Our data support a model that likely includes protein-protein interactions at or near the bacterial cell surface that are unknown at present. PMID:23959721

  16. O-Linked Glycosylation of the PilA Pilin Protein of Francisella tularensis: Identification of the Endogenous Protein-Targeting Oligosaccharyltransferase and Characterization of the Native Oligosaccharide▿†

    PubMed Central

    Egge-Jacobsen, Wolfgang; Salomonsson, Emelie Näslund; Aas, Finn Erik; Forslund, Anna-Lena; Winther-Larsen, Hanne C.; Maier, Josef; Macellaro, Anna; Kuoppa, Kerstin; Oyston, Petra C. F.; Titball, Richard W.; Thomas, Rebecca M.; Forsberg, Åke; Prior, Joann L.; Koomey, Michael

    2011-01-01

    Findings from a number of studies suggest that the PilA pilin proteins may play an important role in the pathogenesis of disease caused by species within the genus Francisella. As such, a thorough understanding of PilA structure and chemistry is warranted. Here, we definitively identified the PglA protein-targeting oligosaccharyltransferase by virtue of its necessity for PilA glycosylation in Francisella tularensis and its sufficiency for PilA glycosylation in Escherichia coli. In addition, we used mass spectrometry to examine PilA affinity purified from Francisella tularensis subsp. tularensis and F. tularensis subsp. holarctica and demonstrated that the protein undergoes multisite, O-linked glycosylation with a pentasaccharide of the structure HexNac-Hex-Hex-HexNac-HexNac. Further analyses revealed microheterogeneity related to forms of the pentasaccharide carrying unusual moieties linked to the distal sugar via a phosphate bridge. Type A and type B strains of Francisella subspecies thus express an O-linked protein glycosylation system utilizing core biosynthetic and assembly pathways conserved in other members of the proteobacteria. As PglA appears to be highly conserved in Francisella species, O-linked protein glycosylation may be a feature common to members of this genus. PMID:21804002

  17. 21 CFR 866.3280 - Francisella tularensis serological reagents.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Francisella tularensis in serum or to identify Francisella tularensis in cultured isolates derived from... provides epidemiological information on this disease. Tularemia is a desease principally of rodents, but... fleas and ticks. The disease takes on several forms depending upon the site of infection, such as...

  18. Identifying Francisella tularensis genes required for growth in host cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Technical Abstract: Francisella tularensis is a highly virulent Gram negative intracellular pathogen capable of infecting a vast diversity of hosts, ranging from amoebae to humans. A hallmark of F. tularensis virulence is its ability to quickly grow to high densities within a diverse set of host cel...

  19. Survival and Growth of Francisella tularensis in Acanthamoeba castellanii

    PubMed Central

    Abd, Hadi; Johansson, Thorsten; Golovliov, Igor; Sandström, Gunnar; Forsman, Mats

    2003-01-01

    Francisella tularensis is a highly infectious, facultative intracellular bacterium which causes epidemics of tularemia in both humans and mammals at regular intervals. The natural reservoir of the bacterium is largely unknown, although it has been speculated that protozoa may harbor it. To test this hypothesis, Acanthamoeba castellanii was cocultured with a strain of F. tularensis engineered to produce green fluorescent protein (GFP) in a nutrient-rich medium. GFP fluorescence within A. castellanii was then monitored by flow cytometry and fluorescence microscopy. In addition, extracellular bacteria were distinguished from intracellular bacteria by targeting with monoclonal antibodies. Electron microscopy was used to determine the intracellular location of F. tularensis in A. castellanii, and viable counts were obtained for both extracellular and intracellular bacteria. The results showed that many F. tularensis cells were located intracellularly in A. castellanii cells. The bacteria multiplied within intracellular vacuoles and eventually killed many of the host cells. F. tularensis was found in intact trophozoites, excreted vesicles, and cysts. Furthermore, F. tularensis grew faster in cocultures with A. castellanii than it did when grown alone in the same medium. This increase in growth was accompanied by a decrease in the number of A. castellanii cells. The interaction between F. tularensis and amoebae demonstrated in this study indicates that ubiquitous protozoa might be an important environmental reservoir for F. tularensis. PMID:12514047

  20. Nucleotide sequences specific to Francisella tularensis and methods for the detection of Francisella tularensis

    DOEpatents

    McCready, Paula M.; Radnedge, Lyndsay; Andersen, Gary L.; Ott, Linda L.; Slezak, Thomas R.; Kuczmarski, Thomas A.; Vitalis, Elizabeth A

    2009-02-24

    Described herein is the identification of nucleotide sequences specific to Francisella tularensis that serves as a marker or signature for identification of this bacterium. In addition, forward and reverse primers and hybridization probes derived from these nucleotide sequences that are used in nucleotide detection methods to detect the presence of the bacterium are disclosed.

  1. Nucleotide sequences specific to Francisella tularensis and methods for the detection of Francisella tularensis

    DOEpatents

    McCready, Paula M.; Radnedge, Lyndsay; Andersen, Gary L.; Ott, Linda L.; Slezak, Thomas R.; Kuczmarski, Thomas A.; Vitalis, Elizabeth A

    2007-02-06

    Described herein is the identification of nucleotide sequences specific to Francisella tularensis that serves as a marker or signature for identification of this bacterium. In addition, forward and reverse primers and hybridization probes derived from these nucleotide sequences that are used in nucleotide detection methods to detect the presence of the bacterium are disclosed.

  2. A novel nanoprobe for the sensitive detection of Francisella tularensis.

    PubMed

    Kim, Ji-eun; Seo, Youngmin; Jeong, Yoon; Hwang, Mintai P; Hwang, Jangsun; Choo, Jaebum; Hong, Jong Wook; Jeon, Jun Ho; Rhie, Gi-eun; Choi, Jonghoon

    2015-11-15

    Francisella tularensis is a human zoonotic pathogen and the causative agent of tularemia, a severe infectious disease. Given the extreme infectivity of F. tularensis and its potential to be used as a biological warfare agent, a fast and sensitive detection method is highly desirable. Herein, we construct a novel detection platform composed of two units: (1) Magnetic beads conjugated with multiple capturing antibodies against F. tularensis for its simple and rapid separation and (2) Genetically-engineered apoferritin protein constructs conjugated with multiple quantum dots and a detection antibody against F. tularensis for the amplification of signal. We demonstrate a 10-fold increase in the sensitivity relative to traditional lateral flow devices that utilize enzyme-based detection methods. We ultimately envision the use of our novel nanoprobe detection platform in future applications that require the highly-sensitive on-site detection of high-risk pathogens.

  3. VIRULENCE AND CITRULLINE UREIDASE ACTIVITY OF PASTEURELLA TULARENSIS12

    PubMed Central

    Marchette, Nyven J.; Nicholes, Paul S.

    1961-01-01

    Marchette, Nyven J. (University of Utah, Salt Lake City), and Paul S. Nicholes. Virulence and citrulline ureidase activity of Pasteurella tularensis. J. Bacteriol. 82:26–32. 1961.—The presence of a citrulline ureidase system in Pasteurella tularensis strains of high virulence, and its absence in avirulent strains and strains of low virulence was confirmed. The presence of this system, however, was shown to be not directly related to virulence. The only wild strain of P. tularensis tested that lacked a citrulline ureidase system was isolated from a rodent. All the strains, isolated from rabbits, rabbit ticks, a human being, and a horse, that were tested possessed this system. The existence of two North American varieties of P. tularensis was postulated on the basis of virulence and citrulline ureidase activity. PMID:13766500

  4. Virulence and citrulline ureidase activity of Pasteurella tularensis.

    PubMed

    MARCHETTE, N J; NICHOLES, P S

    1961-07-01

    Marchette, Nyven J. (University of Utah, Salt Lake City), and Paul S. Nicholes. Virulence and citrulline ureidase activity of Pasteurella tularensis. J. Bacteriol. 82:26-32. 1961.-The presence of a citrulline ureidase system in Pasteurella tularensis strains of high virulence, and its absence in avirulent strains and strains of low virulence was confirmed. The presence of this system, however, was shown to be not directly related to virulence. The only wild strain of P. tularensis tested that lacked a citrulline ureidase system was isolated from a rodent. All the strains, isolated from rabbits, rabbit ticks, a human being, and a horse, that were tested possessed this system. The existence of two North American varieties of P. tularensis was postulated on the basis of virulence and citrulline ureidase activity.

  5. Methods for Enhanced Culture Recovery of Francisella tularensis

    PubMed Central

    Petersen, Jeannine M.; Schriefer, Martin E.; Gage, Kenneth L.; Montenieri, John A.; Carter, Leon G.; Stanley, Miles; Chu, May C.

    2004-01-01

    Francisella tularensis is found in a wide variety of hosts and extrahost environments, making culture recovery a diagnostic challenge. Here we demonstrate improved recovery times and good sensitivity (90%) when cultures were inoculated on the site of an investigation using fresh tissues. For contaminated specimens, antibiotic supplementation of enriched cysteine heart agar blood culture medium improved recovery of F. tularensis by 81.1%. For transport of tissues, immediate freezing yielded culture recovery rates as high as 94%. PMID:15184180

  6. Purification and biophysical characterization of the CapA membrane protein FTT0807 from Francisella tularensis.

    PubMed

    Martin-Garcia, Jose M; Hansen, Debra T; Zook, James; Loskutov, Andrey V; Robida, Mark D; Craciunescu, Felicia M; Sykes, Kathryn F; Wachter, Rebekka M; Fromme, Petra; Allen, James P

    2014-04-01

    The capA gene (FTT0807) from Francisella tularensis subsp. tularensis SCHU S4 encodes a 44.4 kDa integral membrane protein composed of 403 amino acid residues that is part of an apparent operon that encodes at least two other membrane proteins, CapB, and CapC, which together play a critical role in the virulence and pathogenesis of this bacterium. The capA gene was overexpressed in Escherichia coli as a C-terminal His6-tagged fusion with a folding reporter green fluorescent protein (frGFP). Purification procedures using several detergents were developed for the fluorescing and membrane-bound product, yielding approximately 30 mg of pure protein per liter of bacterial culture. Dynamic light scattering indicated that CapA-frGFP was highly monodisperse, with a size that was dependent upon both the concentration and choice of detergent. Circular dichroism showed that CapA-frGFP was stable over the range of 3-9 for the pH, with approximately half of the protein having well-defined α-helical and β-sheet secondary structure. The addition of either sodium chloride or calcium chloride at concentrations producing ionic strengths above 0.1 M resulted in a small increase of the α-helical content and a corresponding decrease in the random-coil content. Secondary-structure predictions on the basis of the analysis of the sequence indicate that the CapA membrane protein has two transmembrane helices with a substantial hydrophilic domain. The hydrophilic domain is predicted to contain a long disordered region of 50-60 residues, suggesting that the increase of α-helical content at high ionic strength could arise because of electrostatic interactions involving the disordered region. CapA is shown to be an inner-membrane protein and is predicted to play a key cellular role in the assembly of polysaccharides.

  7. Identification of Mechanisms for Attenuation of the FSC043 Mutant of Francisella tularensis SCHU S4

    PubMed Central

    Lindgren, Marie; Tancred, Linda; Golovliov, Igor; Conlan, Wayne; Twine, Susan M.

    2014-01-01

    Previously, we identified a spontaneous, essentially avirulent mutant, FSC043, of the highly virulent strain SCHU S4 of Francisella tularensis subsp. tularensis. We have now characterized the phenotype of the mutant and the mechanisms of its attenuation in more detail. Genetic and proteomic analyses revealed that the pdpE gene and most of the pdpC gene were very markedly downregulated and, as previously demonstrated, that the strain expressed partially deleted and fused fupA and fupB genes. FSC043 showed minimal intracellular replication and induced no cell cytotoxicity. The mutant showed delayed phagosomal escape; at 18 h, colocalization with LAMP-1 was 80%, indicating phagosomal localization, whereas the corresponding percentages for SCHU S4 and the ΔfupA mutant were <10%. However, a small subset of the FSC043-infected cells contained up to 100 bacteria with LAMP-1 colocalization of around 30%. The unusual intracellular phenotype was similar to that of the ΔpdpC and ΔpdpC ΔpdpE mutants. Complementation of FSC043 with the intact fupA and fupB genes did not affect the phenotype, whereas complementation with the pdpC and pdpE genes restored intracellular replication and led to marked virulence. Even higher virulence was observed after complementation with both double-gene constructs. After immunization with the FSC043 strain, moderate protection against respiratory challenge with the SCHU S4 strain was observed. In summary, FSC043 showed a highly unusual intracellular phenotype, and based on our findings, we hypothesize that the mutation in the pdpC gene makes an essential contribution to the phenotype. PMID:24935978

  8. Purification and Biophysical Characterization of the CapA Membrane Protein FTT0807 from Francisella tularensis

    PubMed Central

    2015-01-01

    The capA gene (FTT0807) from Francisella tularensis subsp. tularensis SCHU S4 encodes a 44.4 kDa integral membrane protein composed of 403 amino acid residues that is part of an apparent operon that encodes at least two other membrane proteins, CapB, and CapC, which together play a critical role in the virulence and pathogenesis of this bacterium. The capA gene was overexpressed in Escherichia coli as a C-terminal His6-tagged fusion with a folding reporter green fluorescent protein (frGFP). Purification procedures using several detergents were developed for the fluorescing and membrane-bound product, yielding approximately 30 mg of pure protein per liter of bacterial culture. Dynamic light scattering indicated that CapA-frGFP was highly monodisperse, with a size that was dependent upon both the concentration and choice of detergent. Circular dichroism showed that CapA-frGFP was stable over the range of 3–9 for the pH, with approximately half of the protein having well-defined α-helical and β-sheet secondary structure. The addition of either sodium chloride or calcium chloride at concentrations producing ionic strengths above 0.1 M resulted in a small increase of the α-helical content and a corresponding decrease in the random-coil content. Secondary-structure predictions on the basis of the analysis of the sequence indicate that the CapA membrane protein has two transmembrane helices with a substantial hydrophilic domain. The hydrophilic domain is predicted to contain a long disordered region of 50–60 residues, suggesting that the increase of α-helical content at high ionic strength could arise because of electrostatic interactions involving the disordered region. CapA is shown to be an inner-membrane protein and is predicted to play a key cellular role in the assembly of polysaccharides. PMID:24593131

  9. Genome-Wide Identification of Francisella tularensis Virulence Determinants▿

    PubMed Central

    Su, Jingliang; Yang, Jun; Zhao, Daimin; Kawula, Thomas H.; Banas, Jeffrey A.; Zhang, Jing-Ren

    2007-01-01

    Francisella tularensis is a gram-negative pathogen that causes life-threatening infections in humans and has potential for use as a biological weapon. The genetic basis of the F. tularensis virulence is poorly understood. This study screened a total of 3,936 transposon mutants of the live vaccine strain for infection in a mouse model of respiratory tularemia by signature-tagged mutagenesis. We identified 341 mutants attenuated for infection in the lungs. The transposon disruptions were mapped to 95 different genes, virtually all of which are also present in the genomes of other F. tularensis strains, including human pathogenic F. tularensis strain Schu S4. A small subset of these attenuated mutants carried insertions in the genes encoding previously known virulence factors, but the majority of the identified genes have not been previously linked to F. tularensis virulence. Among these are genes encoding putative membrane proteins, proteins associated with stress responses, metabolic proteins, transporter proteins, and proteins with unknown functions. Several attenuated mutants contained disruptions in a putative capsule locus which partially resembles the poly-γ-glutamate capsule biosynthesis locus of Bacillus anthracis, the anthrax agent. Deletional mutation analysis confirmed that this locus is essential for F. tularensis virulence. PMID:17420240

  10. Survey of Francisella tularensis in Wild Animals in Japan in Areas Where Tularemia is Endemic.

    PubMed

    Hotta, Akitoyo; Tanabayashi, Kiyoshi; Fujita, Osamu; Shindo, Junji; Park, Chu-Ho; Kudo, Noboru; Hatai, Hitoshi; Oyamada, Toshifumi; Yamamoto, Yoshie; Takano, Ai; Kawabata, Hiroki; Sharma, Neekun; Uda, Akihiko; Yamada, Akio; Morikawa, Shigeru

    2016-09-21

    Samples taken from 428 wild animals and 126 ticks, collected from a tularemia-endemic area in Japan between 2005 and 2013, were analyzed for the presence of Francisella tularensis. F. tularensis was isolated from a Japanese hare carcass whereas the samples from live animals and ticks were negative for F. tularensis by real-time PCR. Our results suggest that F. tularensis is still present in Japan although its prevalence is considerably low even in areas where tularemia is endemic.

  11. Benzimidazole-Based Antibacterial Agents Against F. tularensis

    PubMed Central

    Kumar, Kunal; Awasthi, Divya; Lee, Seung-Yub; Cummings, Jason E.; Knudson, Susan E.; Slayden, Richard A.; Ojima, Iwao

    2013-01-01

    Francisella tularensis is a highly virulent pathogenic bacterium. In order to identify novel potential antibacterial agents against F. tularensis, libraries of trisubstituted benzimidazoles were screened against F. tularensis LVS strain. In a preliminary screening assay, remarkably, 23 of 2,5,6- and 2,5,7-trisubstituted benzimidazoles showed excellent activity exhibiting greater than 90 % growth inhibition at 1 µg/mL. Among those hits, 21 compounds showed MIC90 values in the range of 0.35–48.6 µg/mL after accurate MIC determination. In ex-vivo efficacy assays, four of these compounds exhibited 2–3 Log reduction in colony forming units (CFU) per mL at concentrations of 10 and 50 µg/mL. PMID:23623254

  12. Symbiosis with Francisella tularensis provides resistance to pathogens in the silkworm

    PubMed Central

    Suzuki, Jin; Uda, Akihiko; Watanabe, Kenta; Shimizu, Takashi; Watarai, Masahisa

    2016-01-01

    Francisella tularensis, the causative agent of tularemia, is a highly virulent facultative intracellular pathogen found in a wide range of animals, including arthropods, and environments. This bacterium has been known for over 100 years, but the lifestyle of F. tularensis in natural reservoirs remains largely unknown. Thus, we established a novel natural host model for F. tularensis using the silkworm (Bombyx mori), which is an insect model for infection by pathogens. F. tularensis established a symbiosis with silkworms, and bacteria were observed in the hemolymph. After infection with F. tularensis, the induction of melanization and nodulation, which are immune responses to bacterial infection, were inhibited in silkworms. Pre-inoculation of silkworms with F. tularensis enhanced the expression of antimicrobial peptides and resistance to infection by pathogenic bacteria. These results suggest that silkworms acquire host resistance via their symbiosis with F. tularensis, which may have important fitness benefits in natural reservoirs. PMID:27507264

  13. Phylogenomic analysis shows that Bacillus amyloliquefaciens subsp. plantarum is a later heterotypic synonym of Bacillus methylotrophicus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The rhizosphere isolated bacteria belonging to the Bacillus amyloliquefaciens subsp. plantarum and Bacillus methylotrophicus clades are an important group of strains that are used as plant growth promoters and antagonists of plant pathogens. These properties have made these strains the focus of comm...

  14. Characterization of Francisella tularensis Outer Membrane Proteins▿ †

    PubMed Central

    Huntley, Jason F.; Conley, Patrick G.; Hagman, Kayla E.; Norgard, Michael V.

    2007-01-01

    Francisella tularensis is a gram-negative coccobacillus that is capable of causing severe, fatal disease in a number of mammalian species, including humans. Little is known about the proteins that are surface exposed on the outer membrane (OM) of F. tularensis, yet identification of such proteins is potentially fundamental to understanding the initial infection process, intracellular survival, virulence, immune evasion and, ultimately, vaccine development. To facilitate the identification of putative F. tularensis outer membrane proteins (OMPs), the genomes of both the type A strain (Schu S4) and type B strain (LVS) were subjected to six bioinformatic analyses for OMP signatures. Compilation of the bioinformatic predictions highlighted 16 putative OMPs, which were cloned and expressed for the generation of polyclonal antisera. Total membranes were extracted from both Schu S4 and LVS by spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation, which separated OMs from cytoplasmic (inner) membrane and other cellular compartments. Validation of OM separation and enrichment was confirmed by probing sucrose gradient fractions with antibodies to putative OMPs and inner membrane proteins. F. tularensis OMs typically migrated in sucrose gradients between densities of 1.17 and 1.20 g/ml, which differed from densities typically observed for other gram-negative bacteria (1.21 to 1.24 g/ml). Finally, the identities of immunogenic proteins were determined by separation on two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analysis. This is the first report of a direct method for F. tularensis OM isolation that, in combination with computational predictions, offers a more comprehensive approach for the characterization of F. tularensis OMPs. PMID:17114266

  15. Recombinant Attenuated Listeria monocytogenes Vaccine Expressing Francisella tularensis IglC Induces Protection in Mice Against Aerosolized Type A F. tularensis

    PubMed Central

    Jia, Qingmei; Lee, Bai-Yu; Clemens, Daniel L.; Bowen, Richard A.; Horwitz, Marcus A.

    2009-01-01

    Fransicella tularensis, the causative agent of tularemia, is in the top category (Category A) of potential agents of bioterrorism. To develop a safer vaccine against aerosolized F. tularensis, we have employed an attenuated Listeria monocytogenes, which shares with F. tularensis an intracellular and extraphagosomal lifestyle, as a delivery vehicle for F. tularensis antigens. We constructed recombinant L. monocytogenes (rLm) vaccines stably expressing 7 F. tularensis proteins including IglC (rLm/iglC), and tested their immunogenicity and protective efficacy against lethal F. tularensis challenge in mice. Mice immunized intradermally with rLm/iglC developed significant cellular immune responses to F. tularensis IglC as evidenced by lymphocyte proliferation and CD4+ and CD8+ T-cell intracellular expression of interferon gamma. Moreover, mice immunized with rLm/iglC were protected against lethal challenge with F. tularensis LVS administered by the intranasal route, a route chosen to mimic airborne infection, and, most importantly, against aerosol challenge with the highly virulent Type A F. tularensis SchuS4 strain. PMID:19126421

  16. 21 CFR 866.3280 - Francisella tularensis serological reagents.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Francisella tularensis serological reagents. 866.3280 Section 866.3280 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  17. 21 CFR 866.3280 - Francisella tularensis serological reagents.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Francisella tularensis serological reagents. 866.3280 Section 866.3280 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  18. 21 CFR 866.3280 - Francisella tularensis serological reagents.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Francisella tularensis serological reagents. 866.3280 Section 866.3280 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  19. 21 CFR 866.3280 - Francisella tularensis serological reagents.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Francisella tularensis serological reagents. 866.3280 Section 866.3280 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  20. Francisella tularensis endocarditis: two case reports and a literature review.

    PubMed

    Gaci, Rostane; Alauzet, Corentine; Selton-Suty, Christine; Lozniewski, Alain; Pulcini, Céline; May, Thierry; Goehringer, François

    2017-02-01

    We report the first two cases of infective endocarditis caused by Francisella tularensis in Europe (two cases have previously been reported outside Europe). We suggest clinicians should consider tularemia as a possible diagnosis in endemic regions in cases of culture-negative endocarditis.

  1. Amblyomma americanum as a Bridging Vector for Human Infection with Francisella tularensis

    PubMed Central

    2015-01-01

    The γ-proteobacterium Francisella tularensis causes seasonal tick-transmitted tularemia outbreaks in natural rabbit hosts and incidental infections in humans in the south-central United States. Although Dermacentor variabilis is considered a primary vector for F. tularensis, Amblyomma americanum is the most abundant tick species in this endemic region. A systematic study of F. tularensis colonization of A. americanum was undertaken to better understand its potential to serve as an overwintering reservoir for F. tularensis and as a bridging vector for human infections. Colony-reared A. americanum were artificially fed F. tularensis subspecies holarctica strain LVS via glass capillaries and colonization levels determined. Capillary-fed larva and nymph were initially infected with 104 CFU/tick which declined prior to molting for both stages, but rebounded post-molting in nymphs and persisted in 53% at 103 to 108 CFU/nymph at 168 days post-capillary feeding (longest sampling time in the study). In contrast, only 18% of adults molted from colonized nymphs maintained LVS colonization at 101 to 105 CFU/adult at 168 days post-capillary feeding (longest sampling time). For adults, LVS initially colonized the gut and disseminated to salivary glands by 24 h and had an ID50 of <5CFU in mice. Francisella tularensis infected the ovaries of gravid females, but transmission to eggs was infrequent and transovarial transmission to hatched larvae was not observed. The prolonged persistence of F. tularensis in A. americanum nymphs supports A. americanum as an overwintering reservoir for F. tularensis from which seasonal epizootics may originate; however, although the rapid dissemination of F. tularensis from gut to salivary glands in adults A. americanum is compatible with intermittent feeding adult males acting as bridging vectors for incidental F. tularensis infections of humans, acquisition of F. tularensis by adults may be unlikely based on adult feeding preference for larger

  2. Role of Glycosylation/Deglycolysation Processes in Francisella tularensis Pathogenesis.

    PubMed

    Barel, Monique; Charbit, Alain

    2017-01-01

    Francisella tularensis is able to invade, survive and replicate inside a variety of cell types. However, in vivo F. tularensis preferentially enters host macrophages where it rapidly escapes to the cytosol to avoid phagosomal stresses and to multiply to high numbers. We previously showed that human monocyte infection by F. tularensis LVS triggered deglycosylation of the glutamine transporter SLC1A5. However, this deglycosylation, specifically induced by Francisella infection, was not restricted to SLC1A5, suggesting that host protein deglycosylation processes in general might contribute to intracellular bacterial adaptation. Indeed, we later found that Francisella infection modulated the transcription of numerous glycosidase and glycosyltransferase genes in human macrophages and analysis of cell extracts revealed an important increase of N and O-protein glycosylation. In eukaryotic cells, glycosylation has significant effects on protein folding, conformation, distribution, stability, and activity and dysfunction of protein glycosylation may lead to development of diseases like cancer and pathogenesis of infectious diseases. Pathogenic bacteria have also evolved dedicated glycosylation machineries and have notably been shown to use these glycoconjugates as ligands to specifically interact with the host. In this review, we will focus on Francisella and summarize our current understanding of the importance of these post-translational modifications on its intracellular niche adaptation.

  3. [Surveillance of Francisella tularensis infection in dogs in Bratislava].

    PubMed

    Gurycová, D; Kopcok, M

    1992-03-01

    Out of 548 serologically investigated dogs from Bratislava and other regions of Slovakia and Moravia, antibodies to F. tularensis were found in 16.4% (Tabs. I, II). In all the investigated groups of dogs from the region of Bratislava the highest seroprevalence by F. tularensis was recorded in watch dogs kept on farms and in cooperatives--37.5% and in rambling dogs--20.7% (Tab. I). The highest seropositivity was found in one to three year old dogs--22.2% (Tab. III). A similar degree of seroprevalence was also observed in one to three years old police dogs which came from the endemic region of tularemia--West Slovakia (19.3%) and East Slovakia (25.6%)--Tab. IV. These facts indicate the persistence of active natural foci in these regions. Serological investigations of the relatively great number of dogs from different regions of Slovakia showed that the presence of F. tularensis antibodies in this animal species, mainly in the watch dogs group, can be taken as a convenient marker or indicator of the existence of active natural foci of tularemia and as a suitable component for surveillance of this diseases.

  4. Seroprevalence study of Francisella tularensis among hunters in Germany.

    PubMed

    Jenzora, Andrea; Jansen, Andreas; Ranisch, Heidrun; Lierz, Michael; Wichmann, Ole; Grunow, Roland

    2008-07-01

    In 2005 and 2006, Francisella tularensis unexpectedly reemerged in western Germany, when several semi-free-living marmosets (Callithrix jacchus) in a research facility died from tularemia and a group of hare hunters became infected. It is believed that hunters may have an elevated risk to be exposed to zoonotic pathogens, including F. tularensis. A previous cross-sectional study of the German population (n=6883) revealed a prevalence of 0.2%. Here, we investigated 286 sera from individuals mainly hunting in districts with emerging tularemia cases (group 1) and 84 sera from a region currently not conspicuous for tularemia (group 2). Methods included standard enzyme-linked immunosorbent assay (ELISA), Western blot analysis and indirect immunofluorescence assay. We found five out of the 286 hunters (1.7%; 95% CI 0.6-4.0%) in group 1 positive with standard ELISA and Western blot, but none in the Berlin area (group 2; 95% CI 0-0.04%). Group 1 showed an elevated risk for hunters to be seropositive for F. tularensis compared with the cross-sectional study (OR=7.7; P<0.001). This indicates a higher prevalence for tularemia in hunters of a suspected endemic region of Germany.

  5. Role of Glycosylation/Deglycolysation Processes in Francisella tularensis Pathogenesis

    PubMed Central

    Barel, Monique; Charbit, Alain

    2017-01-01

    Francisella tularensis is able to invade, survive and replicate inside a variety of cell types. However, in vivo F. tularensis preferentially enters host macrophages where it rapidly escapes to the cytosol to avoid phagosomal stresses and to multiply to high numbers. We previously showed that human monocyte infection by F. tularensis LVS triggered deglycosylation of the glutamine transporter SLC1A5. However, this deglycosylation, specifically induced by Francisella infection, was not restricted to SLC1A5, suggesting that host protein deglycosylation processes in general might contribute to intracellular bacterial adaptation. Indeed, we later found that Francisella infection modulated the transcription of numerous glycosidase and glycosyltransferase genes in human macrophages and analysis of cell extracts revealed an important increase of N and O-protein glycosylation. In eukaryotic cells, glycosylation has significant effects on protein folding, conformation, distribution, stability, and activity and dysfunction of protein glycosylation may lead to development of diseases like cancer and pathogenesis of infectious diseases. Pathogenic bacteria have also evolved dedicated glycosylation machineries and have notably been shown to use these glycoconjugates as ligands to specifically interact with the host. In this review, we will focus on Francisella and summarize our current understanding of the importance of these post-translational modifications on its intracellular niche adaptation. PMID:28377902

  6. UV-C Inactivation of Francisella tularensis Utah-112 on agar surfaces, stainless steel, and foods

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Francisella tularensis has been identified as a microorganism of concern in the field of food security. There is currently very little information on the ability to inactivate F. tularensis on foods using non-thermal processing technologies. The ability of ultraviolet light (UV-C) to inactivate F....

  7. Large Direct Repeats Flank Genomic Rearrangements between a New Clinical Isolate of Francisella tularensis subsp. tularensis A1 and Schu S4

    DTIC Science & Technology

    2010-02-03

    Silver Spring, Maryland, United States of America, 7 Oak Ridge National Laboratory, Oak Ridge, Tennessee , United States of America Abstract Francisella...Interpolated variable order motifs for identification of horizontally acquired DNA: revisiting the Salmonella pathoge- nicity islands. Bioinformatics

  8. Genetic identification of unique immunological responses in mice infected with virulent and attenuated Francisella tularensis

    PubMed Central

    Kingry, Luke C.; Troyer, Ryan M.; Marlenee, Nicole L.; Bielefeldt-Ohmann, Helle; Bowen, Richard A.; Schenkel, Alan R.; Dow, Steven W.; Slayden, Richard A.

    2010-01-01

    Francisella tularensis is a category A select agent based on its infectivity and virulence but disease mechanisms in Francisella tularensis infection remain poorly understood. Murine pulmonary models of infection were therefore employed to assess and compare dissemination and pathology and to elucidate the host immune response to infection with the highly virulent Type A F. tularensis strain Schu4 versus the less virulent Type B live vaccine strain (LVS). We found that dissemination and pathology in the spleen was significantly greater in mice infected with F. tularensis Schu4 compared to mice infected with F. tularensis LVS. Using gene expression profiling to compare the response to infection with the two F. tularensis strains, we found that there were significant differences in the expression of genes involved in the apoptosis pathway, antigen processing and presentation pathways, and inflammatory response pathways in mice infected with Schu4 when compared to LVS. These transcriptional differences coincided with marked differences in dissemination and severity of organ lesions in mice infected with the Schu4 and LVS strains. Therefore, these findings indicate that altered apoptosis, antigen presentation and production of inflammatory mediators explain the differences in pathogenicity of F. tularensis Schu4 and LVS. PMID:21070859

  9. Recombinase Polymerase Amplification Assay for Rapid Detection of Francisella tularensis

    PubMed Central

    Euler, Milena; Wang, Yongjie; Otto, Peter; Tomaso, Herbert; Escudero, Raquel; Anda, Pedro; Hufert, Frank T.

    2012-01-01

    Several real-time PCR approaches to develop field detection for Francisella tularensis, the infectious agent causing tularemia, have been explored. We report the development of a novel qualitative real-time isothermal recombinase polymerase amplification (RPA) assay for use on a small ESEQuant Tube Scanner device. The analytical sensitivity and specificity were tested using a plasmid standard and DNA extracts from infected rabbit tissues. The assay showed a performance comparable to real-time PCR but reduced the assay time to 10 min. The rapid RPA method has great application potential for field use or point-of-care diagnostics. PMID:22518861

  10. Recombinase polymerase amplification assay for rapid detection of Francisella tularensis.

    PubMed

    Euler, Milena; Wang, Yongjie; Otto, Peter; Tomaso, Herbert; Escudero, Raquel; Anda, Pedro; Hufert, Frank T; Weidmann, Manfred

    2012-07-01

    Several real-time PCR approaches to develop field detection for Francisella tularensis, the infectious agent causing tularemia, have been explored. We report the development of a novel qualitative real-time isothermal recombinase polymerase amplification (RPA) assay for use on a small ESEQuant Tube Scanner device. The analytical sensitivity and specificity were tested using a plasmid standard and DNA extracts from infected rabbit tissues. The assay showed a performance comparable to real-time PCR but reduced the assay time to 10 min. The rapid RPA method has great application potential for field use or point-of-care diagnostics.

  11. Francisella tularensis infection in a stone marten (Martes foina) without classic pathological lesions consistent with tularemia.

    PubMed

    Origgi, Francesco C; Wu, Natacha; Pilo, Paola

    2013-07-01

    The current report describes the isolation and typing of a strain of Francisella tularensis, the causative agent of tularemia, from the spleen of a stone marten (Martes foina) showing no classic lesions consistent with the disease. The identification of this bacterium, belonging to the World Health Organization risk 3 category and considered to have a low infectious dose, could be performed only because of an ongoing project screening F. tularensis in the environment sensu lato. The findings described herein should alert diagnostic laboratories of the possible presence of F. tularensis in clinical samples in countries where tularemia is endemic even in cases with no consistent anamnesis and from unsuspected animal species.

  12. Identifying Francisella tularensis genes required for growth in host cells.

    PubMed

    Brunton, J; Steele, S; Miller, C; Lovullo, E; Taft-Benz, S; Kawula, T

    2015-08-01

    Francisella tularensis is a highly virulent Gram-negative intracellular pathogen capable of infecting a vast diversity of hosts, ranging from amoebae to humans. A hallmark of F. tularensis virulence is its ability to quickly grow to high densities within a diverse set of host cells, including, but not limited to, macrophages and epithelial cells. We developed a luminescence reporter system to facilitate a large-scale transposon mutagenesis screen to identify genes required for growth in macrophage and epithelial cell lines. We screened 7,454 individual mutants, 269 of which exhibited reduced intracellular growth. Transposon insertions in the 269 growth-defective strains mapped to 68 different genes. FTT_0924, a gene of unknown function but highly conserved among Francisella species, was identified in this screen to be defective for intracellular growth within both macrophage and epithelial cell lines. FTT_0924 was required for full Schu S4 virulence in a murine pulmonary infection model. The ΔFTT_0924 mutant bacterial membrane is permeable when replicating in hypotonic solution and within macrophages, resulting in strongly reduced viability. The permeability and reduced viability were rescued when the mutant was grown in a hypertonic solution, indicating that FTT_0924 is required for resisting osmotic stress. The ΔFTT_0924 mutant was also significantly more sensitive to β-lactam antibiotics than Schu S4. Taken together, the data strongly suggest that FTT_0924 is required for maintaining peptidoglycan integrity and virulence.

  13. Identifying Francisella tularensis Genes Required for Growth in Host Cells

    PubMed Central

    Brunton, J.; Steele, S.; Miller, C.; Lovullo, E.; Taft-Benz, S.

    2015-01-01

    Francisella tularensis is a highly virulent Gram-negative intracellular pathogen capable of infecting a vast diversity of hosts, ranging from amoebae to humans. A hallmark of F. tularensis virulence is its ability to quickly grow to high densities within a diverse set of host cells, including, but not limited to, macrophages and epithelial cells. We developed a luminescence reporter system to facilitate a large-scale transposon mutagenesis screen to identify genes required for growth in macrophage and epithelial cell lines. We screened 7,454 individual mutants, 269 of which exhibited reduced intracellular growth. Transposon insertions in the 269 growth-defective strains mapped to 68 different genes. FTT_0924, a gene of unknown function but highly conserved among Francisella species, was identified in this screen to be defective for intracellular growth within both macrophage and epithelial cell lines. FTT_0924 was required for full Schu S4 virulence in a murine pulmonary infection model. The ΔFTT_0924 mutant bacterial membrane is permeable when replicating in hypotonic solution and within macrophages, resulting in strongly reduced viability. The permeability and reduced viability were rescued when the mutant was grown in a hypertonic solution, indicating that FTT_0924 is required for resisting osmotic stress. The ΔFTT_0924 mutant was also significantly more sensitive to β-lactam antibiotics than Schu S4. Taken together, the data strongly suggest that FTT_0924 is required for maintaining peptidoglycan integrity and virulence. PMID:25987704

  14. Bacillus velezensis is not a later heterotypic synonym of Bacillus amyloliquefaciens; Bacillus methylotrophicus, Bacillus amyloliquefaciens subsp plantarum and ‘Bacillus oryzicola’ are later heterotypic synonyms of Bacillus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The rhizosphere isolated bacteria belonging to the Bacillus amyloliquefaciens subsp. plantarum and Bacillus methylotrophicus clades are an important group of strains that are used as plant growth promoters and antagonists of plant pathogens. These properties have made these strains the focus of comm...

  15. CpG oligodeoxyribonucleotides protect mice from Burholderia pseudomallei but not Francisella tularensis Schu 54 aersols

    DTIC Science & Technology

    2010-01-01

    live vaccine strain (LVS), when administered before parenteral challenge. Given the potential to develop CpG ODN as a pre-treatment for multiple...1] have been successfully developed as adjuvants for a broad array of bacterial subunit vaccines and are currently undergoing multiple clinical...tularensis live vaccine strain (LVS) [3] suggest that CpG ODN may also protect against human-virulent F. tularensis Schu S4 infection. Significantly

  16. ECO-EPIZOOTIOLOGIC STUDY OF FRANCISELLA TULARENSIS, THE AGENT OF TULAREMIA, IN QUÉBEC WILDLIFE.

    PubMed

    Gabriele-Rivet, Vanessa; Ogden, Nicholas; Massé, Ariane; Antonation, Kym; Corbett, Cindi; Dibernardo, Antonia; Lindsay, L Robbin; Leighton, Patrick A; Arsenault, Julie

    2016-04-28

    In Canada, Francisella tularensis , the zoonotic bacterial agent of tularemia, affects mostly snowshoe hares ( Lepus americanus ), muskrats ( Ondatra zibethicus ), and beavers ( Castor canadensis ). Despite numerous studies, the ecologic cycle and natural reservoirs of F. tularensis are not clearly defined. We conducted a cross-sectional study to estimate the prevalence of F. tularensis in snowshoe hares, muskrats, and coyotes ( Canis latrans ) in four regions of Québec, Canada, and to describe the risk of infection in relation to host and environmental characteristics at three spatial scales. Between October 2012 and April 2013, trappers captured 345 snowshoe hares, 411 muskrats, and 385 coyotes. Blood samples were tested by microagglutination tests, and DNA extracts of liver, kidney, lung, and spleen of snowshoe hares and muskrats were tested by real-time PCR to detect past and active infection to F. tularensis , respectively. Individual host characteristics, including body condition, age, and sex, were evaluated as risk factors of infection, along with ecologic characteristics of the location of capture extracted from geographic databases. Prevalences of antibody to F. tularensis and 95% confidence intervals were 2.9% (1.4-5.1%) in coyotes, 0.6% (0.1-2.1%) in hares, and 0% (0.0-0.9%) in muskrats. Francisella tularensis DNA was not detected by real-time PCR in the pools of four organs from muskrats and hares, but F. tularensis type AI was detected during testing of the individual organs of two antibody-positive hares. Exact logistic regression analyses showed that age was a significant predictor of antibody detection in coyotes, as were the proportion of forest and the proportion of area considered as suitable habitat for hares in the environment around the location of capture of the coyotes. Our results suggest a terrestrial cycle of F. tularensis in the regions studied.

  17. Components of the type six secretion system are substrates of Francisella tularensis Schu S4 DsbA-like FipB protein

    PubMed Central

    Qin, Aiping; Zhang, Yan; Clark, Melinda E.; Moore, Emily A.; Rabideau, Meaghan M.; Moreau, G. Brett; Mann, Barbara J.

    2016-01-01

    ABSTRACT FipB, an essential virulence factor in the highly virulent Schu S4 strain of F. tularensis subsp. tularensis, shares sequence similarity with Disulfide Bond formation (Dsb) proteins, which can have oxidoreductase, isomerase, or chaperone activity. To further explore FipB's role in virulence potential substrates were identified by co-purification and 2D gel electrophoresis, followed by protein sequencing using mass spectrometry. A total of 119 potential substrates were identified. Proteins with predicted enzymatic activity were prevalent, and there were 19 proteins that had been previously identified as impacting virulence. Among the potential substrates were IglC, IglB, and PdpB, three components of the Francisella Type Six Secretion System (T6SS), which is also essential for virulence. T6SS are widespread in Gram-negative pathogens, but have not been reported to be dependent on Dsb-like proteins for assembly or function. The presented results suggest that FipB affects IglB and IglC substrates differently. In a fipB mutant there were differences in free sulfhydryl accessibility of IglC, but not IglB, when compared to wild-type bacteria. However, for both proteins FipB appears to act as a chaperone that facilitates proper folding and conformation. Understanding the role FipB plays the assembly and structure in this T6SS may reveal critical aspects of assembly that are common and novel among this widely distributed class of secretion systems. PMID:27028889

  18. Enterococcus saccharolyticus subsp. taiwanensis subsp. nov., isolated from broccoli.

    PubMed

    Chen, Yi-sheng; Lin, Yu-hsuan; Pan, Shwu-fen; Ji, Si-hua; Chang, Yu-chung; Yu, Chi-rong; Liou, Min-shiuan; Wu, Hui-chung; Otoguro, Misa; Yanagida, Fujitoshi; Liao, Chen-chung; Chiu, Chi-ming; Huang, Bi-qiang

    2013-12-01

    A coccal strain isolated from fresh broccoli was initially identified as Enterococcus saccharolyticus; however, molecular identification and phenotypic traits did not support this identification. DNA-DNA hybridization with the type strain of E. saccharolyticus (76.4 % relatedness), DNA G+C content (35.7 mol%), phylogenetic analysis based on 16S rRNA, pheS and rpoA gene sequences, rep-PCR fingerprinting and profiles of cellular fatty acids, whole-cell proteins and enzyme activities, together with carbohydrate metabolism characteristics, indicated that this strain is distinct and represents a novel subspecies, for which the name Enterococcus saccharolyticus subsp. taiwanensis subsp. nov. is proposed. The type strain is 812(T) ( = NBRC 109476(T) = BCRC 80575(T)). Furthermore, we present an emended description of Enterococcus saccharolyticus and proposal of Enterococcus saccharolyticus subsp. saccharolyticus subsp. nov. (type strain ATCC 43076(T) = CCUG 27643(T) = CCUG 33311(T) = CIP 103246(T) = DSM 20726(T) = JCM 8734(T) = LMG 11427(T) = NBRC 100493(T) = NCIMB 702594(T)).

  19. Evidence that clade A and clade B head lice live in sympatry and recombine in Algeria.

    PubMed

    Boutellis, A; Bitam, I; Fekir, K; Mana, N; Raoult, D

    2015-03-01

    Pediculus humanus L. (Psocodea: Pediculidae) can be characterized into three deeply divergent lineages (clades) based on mitochondrial DNA. Clade A consists of both head lice and clothing lice and is distributed worldwide. Clade B consists of head lice only and is mainly found in North and Central America, and in western Europe and Australia. Clade C, which consists only of head lice, is found in Ethiopia, Nepal and Senegal. Twenty-six head lice collected from pupils at different elementary schools in two localities in Algiers (Algeria) were analysed using molecular methods for genotyping lice (cytochrome b and the multi-spacer typing (MST) method. For the first time, we found clade B head lice in Africa living in sympatry with clade A head lice. The phylogenetic analysis of the concatenated sequences of these populations of head lice showed that clade A and clade B head lice had recombined, suggesting that interbreeding occurs when lice live in sympatry.

  20. Structure and Function of REP34 Implicates Carboxypeptidase Activity in Francisella tularensis Host Cell Invasion*

    PubMed Central

    Feld, Geoffrey K.; El-Etr, Sahar; Corzett, Michele H.; Hunter, Mark S.; Belhocine, Kamila; Monack, Denise M.; Frank, Matthias; Segelke, Brent W.; Rasley, Amy

    2014-01-01

    Francisella tularensis is the etiological agent of tularemia, or rabbit fever. Although F. tularensis is a recognized biothreat agent with broad and expanding geographical range, its mechanism of infection and environmental persistence remain poorly understood. Previously, we identified seven F. tularensis proteins that induce a rapid encystment phenotype (REP) in the free-living amoeba, Acanthamoeba castellanii. Encystment is essential to the pathogen's long term intracellular survival in the amoeba. Here, we characterize the cellular and molecular function of REP34, a REP protein with a mass of 34 kDa. A REP34 knock-out strain of F. tularensis has a reduced ability to both induce encystment in A. castellanii and invade human macrophages. We determined the crystal structure of REP34 to 2.05-Å resolution and demonstrate robust carboxypeptidase B-like activity for the enzyme. REP34 is a zinc-containing monomeric protein with close structural homology to the metallocarboxypeptidase family of peptidases. REP34 possesses a novel topology and substrate binding pocket that deviates from the canonical funnelin structure of carboxypeptidases, putatively resulting in a catalytic role for a conserved tyrosine and distinct S1′ recognition site. Taken together, these results identify REP34 as an active carboxypeptidase, implicate the enzyme as a potential key F. tularensis effector protein, and may help elucidate a mechanistic understanding of F. tularensis infection of phagocytic cells. PMID:25231992

  1. Antioxidant Defenses of Francisella tularensis Modulate Macrophage Function and Production of Proinflammatory Cytokines.

    PubMed

    Rabadi, Seham M; Sanchez, Belkys C; Varanat, Mrudula; Ma, Zhuo; Catlett, Sally V; Melendez, Juan Andres; Malik, Meenakshi; Bakshi, Chandra Shekhar

    2016-03-04

    Francisella tularensis, the causative agent of a fatal human disease known as tularemia, has been used in the bioweapon programs of several countries in the past, and now it is considered a potential bioterror agent. Extreme infectivity and virulence of F. tularensis is due to its ability to evade immune detection and to suppress the host's innate immune responses. However, Francisella-encoded factors and mechanisms responsible for causing immune suppression are not completely understood. Macrophages and neutrophils generate reactive oxygen species (ROS)/reactive nitrogen species as a defense mechanism for the clearance of phagocytosed microorganisms. ROS serve a dual role; at high concentrations they act as microbicidal effector molecules that destroy intracellular pathogens, and at low concentrations they serve as secondary signaling messengers that regulate the expression of various inflammatory mediators. We hypothesized that the antioxidant defenses of F. tularensis maintain redox homeostasis in infected macrophages to prevent activation of redox-sensitive signaling components that ultimately result in suppression of pro-inflammatory cytokine production and macrophage microbicidal activity. We demonstrate that antioxidant enzymes of F. tularensis prevent the activation of redox-sensitive MAPK signaling components, NF-κB signaling, and the production of pro-inflammatory cytokines by inhibiting the accumulation of ROS in infected macrophages. We also report that F. tularensis inhibits ROS-dependent autophagy to promote its intramacrophage survival. Collectively, this study reveals novel pathogenic mechanisms adopted by F. tularensis to modulate macrophage innate immune functions to create an environment permissive for its intracellular survival and growth.

  2. From the Outside-In: The Francisella tularensis Envelope and Virulence

    PubMed Central

    Rowe, Hannah M.; Huntley, Jason F.

    2015-01-01

    Francisella tularensis is a highly-infectious bacterium that causes the rapid, and often lethal disease, tularemia. Many studies have been performed to identify and characterize the virulence factors that F. tularensis uses to infect a wide variety of hosts and host cell types, evade immune defenses, and induce severe disease and death. This review focuses on the virulence factors that are present in the F. tularensis envelope, including capsule, LPS, outer membrane, periplasm, inner membrane, secretion systems, and various molecules in each of aforementioned sub-compartments. Whereas, no single bacterial molecule or molecular complex single-handedly controls F. tularensis virulence, we review here how diverse bacterial systems work in conjunction to subvert the immune system, attach to and invade host cells, alter phagosome/lysosome maturation pathways, replicate in host cells without being detected, inhibit apoptosis, and induce host cell death for bacterial release and infection of adjacent cells. Given that the F. tularensis envelope is the outermost layer of the bacterium, we highlight herein how many of these molecules directly interact with the host to promote infection and disease. These and future envelope studies are important to advance our collective understanding of F. tularensis virulence mechanisms and offer targets for future vaccine development efforts. PMID:26779445

  3. What limits the morphological disparity of clades?

    PubMed Central

    Oyston, Jack W.; Hughes, Martin; Wagner, Peter J.; Gerber, Sylvain; Wills, Matthew A.

    2015-01-01

    The morphological disparity of species within major clades shows a variety of trajectory patterns through evolutionary time. However, there is a significant tendency for groups to reach their maximum disparity relatively early in their histories, even while their species richness or diversity is comparatively low. This pattern of early high-disparity suggests that there are internal constraints (e.g. developmental pleiotropy) or external restrictions (e.g. ecological competition) upon the variety of morphologies that can subsequently evolve. It has also been demonstrated that the rate of evolution of new character states decreases in most clades through time (character saturation), as does the rate of origination of novel bodyplans and higher taxa. Here, we tested whether there was a simple relationship between the level or rate of character state exhaustion and the shape of a clade's disparity profile: specifically, its centre of gravity (CG). In a sample of 93 extinct major clades, most showed some degree of exhaustion, but all continued to evolve new states up until their extinction. Projection of states/steps curves suggested that clades realized an average of 60% of their inferred maximum numbers of states. Despite a weak but significant correlation between overall levels of homoplasy and the CG of clade disparity profiles, there were no significant relationships between any of our indices of exhaustion curve shape and the clade disparity CG. Clades showing early high-disparity were no more likely to have early character saturation than those with maximum disparity late in their evolution. PMID:26640649

  4. What limits the morphological disparity of clades?

    PubMed

    Oyston, Jack W; Hughes, Martin; Wagner, Peter J; Gerber, Sylvain; Wills, Matthew A

    2015-12-06

    The morphological disparity of species within major clades shows a variety of trajectory patterns through evolutionary time. However, there is a significant tendency for groups to reach their maximum disparity relatively early in their histories, even while their species richness or diversity is comparatively low. This pattern of early high-disparity suggests that there are internal constraints (e.g. developmental pleiotropy) or external restrictions (e.g. ecological competition) upon the variety of morphologies that can subsequently evolve. It has also been demonstrated that the rate of evolution of new character states decreases in most clades through time (character saturation), as does the rate of origination of novel bodyplans and higher taxa. Here, we tested whether there was a simple relationship between the level or rate of character state exhaustion and the shape of a clade's disparity profile: specifically, its centre of gravity (CG). In a sample of 93 extinct major clades, most showed some degree of exhaustion, but all continued to evolve new states up until their extinction. Projection of states/steps curves suggested that clades realized an average of 60% of their inferred maximum numbers of states. Despite a weak but significant correlation between overall levels of homoplasy and the CG of clade disparity profiles, there were no significant relationships between any of our indices of exhaustion curve shape and the clade disparity CG. Clades showing early high-disparity were no more likely to have early character saturation than those with maximum disparity late in their evolution.

  5. Cranial base evolution within the hominin clade

    PubMed Central

    Nevell, L; Wood, B

    2008-01-01

    The base of the cranium (i.e. the basioccipital, the sphenoid and the temporal bones) is of particular interest because it undergoes significant morphological change within the hominin clade, and because basicranial morphology features in several hominin species diagnoses. We use a parsimony analysis of published cranial and dental data to predict the cranial base morphology expected in the hypothetical last common ancestor of the Pan–Homo clade. We also predict the primitive condition of the cranial base for the hominin clade, and document the evolution of the cranial base within the major subclades within the hominin clade. This analysis suggests that cranial base morphology has continued to evolve in the hominin clade, both before and after the emergence of the genus Homo. PMID:18380865

  6. Gallium Potentiates the Antibacterial Effect of Gentamicin against Francisella tularensis.

    PubMed

    Lindgren, Helena; Sjöstedt, Anders

    2015-10-26

    The reasons why aminoglycosides are bactericidal have not been not fully elucidated, and evidence indicates that the cidal effects are at least partly dependent on iron. We demonstrate that availability of iron markedly affects the susceptibility of the facultative intracellular bacterium Francisella tularensis strain SCHU S4 to the aminoglycoside gentamicin. Specifically, the intracellular depots of iron were inversely correlated to gentamicin susceptibility, whereas the extracellular iron concentrations were directly correlated to the susceptibility. Further proof of the intimate link between iron availability and antibiotic susceptibility were the findings that a ΔfslA mutant, which is defective for siderophore-dependent uptake of ferric iron, showed enhanced gentamicin susceptibility and that a ΔfeoB mutant, which is defective for uptake of ferrous iron, displayed complete growth arrest in the presence of gentamicin. Based on the aforementioned findings, it was hypothesized that gallium could potentiate the effect of gentamicin, since gallium is sequestered by iron uptake systems. The ferrozine assay demonstrated that the presence of gallium inhibited >70% of the iron uptake. Addition of gentamicin and/or gallium to infected bone marrow-derived macrophages showed that both 100 μM gallium and 10 μg/ml of gentamicin inhibited intracellular growth of SCHU S4 and that the combined treatment acted synergistically. Moreover, treatment of F. tularensis-infected mice with gentamicin and gallium showed an additive effect. Collectively, the data demonstrate that SCHU S4 is dependent on iron to minimize the effects of gentamicin and that gallium, by inhibiting the iron uptake, potentiates the bactericidal effect of gentamicin in vitro and in vivo.

  7. Gallium Potentiates the Antibacterial Effect of Gentamicin against Francisella tularensis

    PubMed Central

    Lindgren, Helena

    2015-01-01

    The reasons why aminoglycosides are bactericidal have not been not fully elucidated, and evidence indicates that the cidal effects are at least partly dependent on iron. We demonstrate that availability of iron markedly affects the susceptibility of the facultative intracellular bacterium Francisella tularensis strain SCHU S4 to the aminoglycoside gentamicin. Specifically, the intracellular depots of iron were inversely correlated to gentamicin susceptibility, whereas the extracellular iron concentrations were directly correlated to the susceptibility. Further proof of the intimate link between iron availability and antibiotic susceptibility were the findings that a ΔfslA mutant, which is defective for siderophore-dependent uptake of ferric iron, showed enhanced gentamicin susceptibility and that a ΔfeoB mutant, which is defective for uptake of ferrous iron, displayed complete growth arrest in the presence of gentamicin. Based on the aforementioned findings, it was hypothesized that gallium could potentiate the effect of gentamicin, since gallium is sequestered by iron uptake systems. The ferrozine assay demonstrated that the presence of gallium inhibited >70% of the iron uptake. Addition of gentamicin and/or gallium to infected bone marrow-derived macrophages showed that both 100 μM gallium and 10 μg/ml of gentamicin inhibited intracellular growth of SCHU S4 and that the combined treatment acted synergistically. Moreover, treatment of F. tularensis-infected mice with gentamicin and gallium showed an additive effect. Collectively, the data demonstrate that SCHU S4 is dependent on iron to minimize the effects of gentamicin and that gallium, by inhibiting the iron uptake, potentiates the bactericidal effect of gentamicin in vitro and in vivo. PMID:26503658

  8. Genome sequence and phenotypic analysis of a first German Francisella sp. isolate (W12-1067) not belonging to the species Francisella tularensis

    PubMed Central

    2014-01-01

    Background Francisella isolates from patients suffering from tularemia in Germany are generally strains of the species F. tularensis subsp. holarctica. To our knowledge, no other Francisella species are known for Germany. Recently, a new Francisella species could be isolated from a water reservoir of a cooling tower in Germany. Results We identified a Francisella sp. (isolate W12-1067) whose 16S rDNA is 99% identical to the respective nucleotide sequence of the recently published strain F. guangzhouensis. The overall sequence identity of the fopA, gyrA, rpoA, groEL, sdhA and dnaK genes is only 89%, indicating that strain W12-1067 is not identical to F. guangzhouensis. W12-1067 was isolated from a water reservoir of a cooling tower of a hospital in Germany. The growth optimum of the isolate is approximately 30°C, it can grow in the presence of 4–5% NaCl (halotolerant) and is able to grow without additional cysteine within the medium. The strain was able to replicate within a mouse-derived macrophage-like cell line. The whole genome of the strain was sequenced (~1.7 mbp, 32.2% G + C content) and the draft genome was annotated. Various virulence genes common to the genus Francisella are present, but the Francisella pathogenicity island (FPI) is missing. However, another putative type-VI secretion system is present within the genome of strain W12-1067. Conclusions Isolate W12-1067 is closely related to the recently described F. guangzhouensis species and it replicates within eukaryotic host cells. Since W12-1067 exhibits a putative new type-VI secretion system and F. tularensis subsp. holarctica was found not to be the sole species in Germany, the new isolate is an interesting species to be analyzed in more detail. Further research is needed to investigate the epidemiology, ecology and pathogenicity of Francisella species present in Germany. PMID:24961323

  9. Performance of a Handheld PCR Instrument in the Detection of Bacillus anthracis, Francisella tularensis, and Yersinia pestis: Sensitivity, Specificity, and Effect of Interferents on Assay Results

    DTIC Science & Technology

    2004-12-01

    1 PERFORMANCE OF A HANDHELD PCR INSTRUMENT IN THE DETECTION OF BACILLUS ANTHRACIS, FRANCISELLA TULARENSIS, AND YERSINIA PESTIS: SENSITIVITY...fluorogenic PCR assay reagents for the detection of three biological threat agents, Bacillus anthracis (BA), Francisella tularensis (FT), and Yersinia...TITLE AND SUBTITLE Performance Of A Handheld Pcr Instrument In The Detection Of Bacillus Anthracis, Francisella Tularensis, And Yersinia Pestis

  10. Genetic recombination events between sympatric Clade A and Clade C lice in Africa.

    PubMed

    Veracx, Aurélie; Boutellis, Amina; Raoult, Didier

    2013-09-01

    Human head and body lice have been classified into three phylogenetic clades (Clades A, B, and C) based on mitochondrial DNA. Based on nuclear markers (the 18S rRNA gene and the PM2 spacer), two genotypes of Clade A head and body lice, including one that is specifically African (Clade A2), have been described. In this study, we sequenced the PM2 spacer of Clade C head lice from Ethiopia and compared these sequences with sequences from previous works. Trees were drawn, and an analysis of genetic diversity based on the cytochrome b gene and the PM2 spacer was performed for African and non-African lice. In the tree drawn based on the PM2 spacer, the African and non-African lice formed separate clusters. However, Clade C lice from Ethiopia were placed within the African Clade A subcluster (Clade A2). This result suggests that recombination events have occurred between Clade A2 lice and Clade C lice, reflecting the sympatric nature of African lice. Finally, the PM2 spacer and cytochrome b gene sequences of human lice revealed a higher level of genetic diversity in Africa than in other regions.

  11. The Protease Locus of Francisella tularensis LVS Is Required for Stress Tolerance and Infection in the Mammalian Host

    PubMed Central

    He, Lihong; Nair, Manoj Kumar Mohan; Chen, Yuling; Liu, Xue; Zhang, Mengyun; Hazlett, Karsten R. O.

    2016-01-01

    Francisella tularensis is the causative agent of tularemia and a category A potential agent of bioterrorism, but the pathogenic mechanisms of F. tularensis are largely unknown. Our previous transposon mutagenesis screen identified 95 lung infectivity-associated F. tularensis genes, including those encoding the Lon and ClpP proteases. The present study validates the importance of Lon and ClpP in intramacrophage growth and infection of the mammalian host by using unmarked deletion mutants of the F. tularensis live vaccine strain (LVS). Further experiments revealed that lon and clpP are also required for F. tularensis tolerance to stressful conditions. A quantitative proteomic comparison between heat-stressed LVS and the isogenic Lon-deficient mutant identified 29 putative Lon substrate proteins. The follow-up protein degradation experiments identified five substrates of the F. tularensis Lon protease (FTL578, FTL663, FTL1217, FTL1228, and FTL1957). FTL578 (ornithine cyclodeaminase), FTL663 (heat shock protein), and FTL1228 (iron-sulfur activator complex subunit SufD) have been previously described as virulence-associated factors in F. tularensis. Identification of these Lon substrates has thus provided important clues for further understanding of the F. tularensis stress response and pathogenesis. The high-throughput approach developed in this study can be used for systematic identification of the Lon substrates in other prokaryotic and eukaryotic organisms. PMID:26902724

  12. Proteomic analysis of bronchoalveolar lavage fluid proteins from mice infected with Francisella tularensis ssp novicida

    SciTech Connect

    Varnum, Susan M.; Webb-Robertson, Bobbie-Jo M.; Pounds, Joel G.; Moore, Ronald J.; Smith, Richard D.; Frevert, Charles; Skerret, Shawn J.; Wunschel, David S.

    2012-07-06

    Francisella tularensis causes the zoonosis tularemia in humans and is one of the most virulent bacterial pathogens. We utilized a global proteomic approach to characterize protein changes in bronchoalveolar lavage fluid from mice exposed to one of three organisms, F. tularensis ssp. novicida, an avirulent mutant of F. tularensis ssp. novicida (F.t. novicida-ΔmglA); and Pseudomonas aeruginosa. The composition of BALF proteins was altered following infection, including proteins involved in neutrophil activation, oxidative stress and inflammatory responses. Components of the innate immune response were induced including the acute phase response and the complement system, however the timing of their induction varied. Francisella tularensis ssp. novicida infected mice do not appear to have an effective innate immune response in the first hours of infection, however within 24 hours they show an upregulation of innate immune response proteins. This delayed response is in contrast to P. aeruginosa infected animals which show an early innate immune response. Likewise, F.t. novicida-ΔmglA infection initiates an early innate immune response, however this response is dimished by 24 hours. Finally, this study identifies several candidate biomarkers, including Chitinase 3-like-1 (CHI3L1 or YKL-40) and peroxiredoxin 1, that are associated with F. tularensis ssp. novicida but not P. aeruginosa infection.

  13. Mast cell TLR2 signaling is crucial for effective killing of Francisella tularensis.

    PubMed

    Rodriguez, Annette R; Yu, Jieh-Juen; Guentzel, M Neal; Navara, Christopher S; Klose, Karl E; Forsthuber, Thomas G; Chambers, James P; Berton, Michael T; Arulanandam, Bernard P

    2012-06-01

    TLR signaling is critical for early host defense against pathogens, but the contributions of mast cell TLR-mediated mechanisms and subsequent effector functions during pulmonary infection are largely unknown. We have previously demonstrated that mast cells, through the production of IL-4, effectively control Francisella tularensis replication. In this study, the highly human virulent strain of F. tularensis SCHU S4 and the live vaccine strain were used to investigate the contribution of mast cell/TLR regulation of Francisella. Mast cells required TLR2 for effective bacterial killing, regulation of the hydrolytic enzyme cathepsin L, and for coordination and trafficking of MHC class II and lysosomal-associated membrane protein 2. Infected TLR2(-/-) mast cells, in contrast to wild-type and TLR4(-/-) cells, lacked detectable IL-4 and displayed increased cell death with a 2-3 log increase of F. tularensis replication, but could be rescued with rIL-4 treatment. Importantly, MHC class II and lysosomal-associated membrane protein 2 localization with labeled F. tularensis in the lungs was greater in wild-type than in TLR2(-/-) mice. These results provide evidence for the important effector contribution of mast cells and TLR2-mediated signaling on early innate processes in the lung following pulmonary F. tularensis infection and provide additional insight into possible mechanisms by which intracellular pathogens modulate respiratory immune defenses.

  14. Monophosphoryl Lipid A Enhances Efficacy of a Francisella tularensis LVS-Catanionic Nanoparticle Subunit Vaccine against F. tularensis Schu S4 Challenge by Augmenting both Humoral and Cellular Immunity.

    PubMed

    Richard, Katharina; Mann, Barbara J; Qin, Aiping; Barry, Eileen M; Ernst, Robert K; Vogel, Stefanie N

    2017-03-01

    Francisella tularensis, a bacterial biothreat agent, has no approved vaccine in the United States. Previously, we showed that incorporating lysates from partially attenuated F. tularensis LVS or fully virulent F. tularensis Schu S4 strains into catanionic surfactant vesicle (V) nanoparticles (LVS-V and Schu S4-V, respectively) protected fully against F. tularensis LVS intraperitoneal (i.p.) challenge in mice. However, we achieved only partial protection against F. tularensis Schu S4 intranasal (i.n.) challenge, even when employing heterologous prime-boost immunization strategies. We now extend these findings to show that both LVS-V and Schu S4-V immunization (i.p./i.p.) elicited similarly high titers of anti-F. tularensis IgG and that the titers could be further increased by adding monophosphoryl lipid A (MPL), a nontoxic Toll-like receptor 4 (TLR4) adjuvant that is included in several U.S. FDA-approved vaccines. LVS-V+MPL immune sera also detected more F. tularensis antigens than LVS-V immune sera and, after passive transfer to naive mice, significantly delayed the time to death against F. tularensis Schu S4 subcutaneous (s.c.) but not i.n. challenge. Active immunization with LVS-V+MPL (i.p./i.p.) also increased the frequency of gamma interferon (IFN-γ)-secreting activated helper T cells, IFN-γ production, and the ability of splenocytes to control intramacrophage F. tularensis LVS replication ex vivo Active LVS-V+MPL immunization via heterologous routes (i.p./i.n.) significantly elevated IgA and IgG levels in bronchoalveolar lavage fluid and significantly enhanced protection against i.n. F. tularensis Schu S4 challenge (to ∼60%). These data represent a significant step in the development of a subunit vaccine against the highly virulent type A strains.

  15. Experimental Infection of voles with Francisella tularensis indicates their amplification role in tularemia outbreaks.

    PubMed

    Rossow, Heidi; Forbes, Kristian M; Tarkka, Eveliina; Kinnunen, Paula M; Hemmilä, Heidi; Huitu, Otso; Nikkari, Simo; Henttonen, Heikki; Kipar, Anja; Vapalahti, Olli

    2014-01-01

    Tularemia outbreaks in humans have been linked to fluctuations in rodent population density, but the mode of bacterial maintenance in nature is unclear. Here we report on an experiment to investigate the pathogenesis of Francisella tularensis infection in wild rodents, and thereby assess their potential to spread the bacterium. We infected 20 field voles (Microtus agrestis) and 12 bank voles (Myodes glareolus) with a strain of F. tularensis ssp. holarctica isolated from a human patient. Upon euthanasia or death, voles were necropsied and specimens collected for histological assessment and identification of bacteria by immunohistology and PCR. Bacterial excretion and a rapid lethal clinical course with pathological changes consistent with bacteremia and tissue necrosis were observed in infected animals. The results support a role for voles as an amplification host of F. tularensis, as excreta and, in particular, carcasses with high bacterial burden could serve as a source for environmental contamination.

  16. Delayed presence of alternatively activated macrophages during a Francisella tularensis infection.

    PubMed

    D'Elia, Riccardo V; Laws, Thomas R; Núñez, Alejandro; Taylor, Christopher; Clark, Graeme C

    2015-01-01

    Francisella tularensis is an intracellular bacterium that has the ability to multiply within the macrophage. The phenotype of a macrophage can determine whether the infection is cleared or the host succumbs to disease. Previously published data has suggested that F. tularensis LVS actively induces the alternative phenotype as a survival mechanism. In these studies we demonstrate that this is not the case for the more virulent strain of F. tularensis SCHU-S4. During an intranasal mouse model of infection, immuno-histochemistry identified that iNOS positive ("classical") macrophages are present at 72 h post-infection and remain high (supported by CCL-5 release) in numbers. In contrast, arginase/FIZZ-1 positive ("alternative") cells appear later and in low numbers during the development of the disease tularemia.

  17. Experimental Infection of Voles with Francisella tularensis Indicates Their Amplification Role in Tularemia Outbreaks

    PubMed Central

    Rossow, Heidi; Forbes, Kristian M.; Tarkka, Eveliina; Kinnunen, Paula M.; Hemmilä, Heidi; Huitu, Otso; Nikkari, Simo; Henttonen, Heikki; Kipar, Anja; Vapalahti, Olli

    2014-01-01

    Tularemia outbreaks in humans have been linked to fluctuations in rodent population density, but the mode of bacterial maintenance in nature is unclear. Here we report on an experiment to investigate the pathogenesis of Francisella tularensis infection in wild rodents, and thereby assess their potential to spread the bacterium. We infected 20 field voles (Microtus agrestis) and 12 bank voles (Myodes glareolus) with a strain of F. tularensis ssp. holarctica isolated from a human patient. Upon euthanasia or death, voles were necropsied and specimens collected for histological assessment and identification of bacteria by immunohistology and PCR. Bacterial excretion and a rapid lethal clinical course with pathological changes consistent with bacteremia and tissue necrosis were observed in infected animals. The results support a role for voles as an amplification host of F. tularensis, as excreta and, in particular, carcasses with high bacterial burden could serve as a source for environmental contamination. PMID:25271640

  18. Open and compressed conformations of Francisella tularensis ClpP

    PubMed Central

    Díaz‐Sáez, Laura; Pankov, Genady

    2016-01-01

    ABSTRACT Caseinolytic proteases are large oligomeric assemblies responsible for maintaining protein homeostasis in bacteria and in so doing influence a wide range of biological processes. The functional assembly involves three chaperones together with the oligomeric caseinolytic protease catalytic subunit P (ClpP). This protease represents a potential target for therapeutic intervention in pathogenic bacteria. Here, we detail an efficient protocol for production of recombinant ClpP from Francisella tularensis, and the structural characterization of three crystal forms which grow under similar conditions. One crystal form reveals a compressed state of the ClpP tetradecamer and two forms an open state. A comparison of the two types of structure infers that differences at the enzyme active site result from a conformational change involving a highly localized disorder‐order transition of a β‐strand α‐helix combination. This transition occurs at a subunit‐subunit interface. Our study may now underpin future efforts in a structure‐based approach to target ClpP for inhibitor or activator development. Proteins 2016; 85:188–194. © 2016 Wiley Periodicals, Inc. PMID:27802578

  19. Early Interactions of Murine Macrophages with Francisella tularensis Map to Mouse Chromosome 19

    PubMed Central

    Fink, Avner; Hassan, Musa A.; Okan, Nihal A.; Sheffer, Michal; Camejo, Ana; Saeij, Jeroen P. J.

    2016-01-01

    ABSTRACT Differences among individuals in susceptibility to infectious diseases can be modulated by host genetics. Much of the research in this field has aimed to identify loci within the host genome that are associated with these differences. In mice, A/J (AJ) and C57BL/6J (B6) mice show differential susceptibilities to various pathogens, including the intracellular pathogen Francisella tularensis. Because macrophages are the main initial target during F. tularensis infection, we explored early interactions of macrophages from these two mouse strains with F. tularensis as well as the genetic factors underlying these interactions. Our results indicate that bacterial interactions with bone marrow-derived macrophages (BMDMs) during early stages of infection are different in the AJ and B6 strains. During these early stages, bacteria are more numerous in B6 than in AJ macrophages and display differences in trafficking and early transcriptional response within these macrophages. To determine the genetic basis for these differences, we infected BMDMs isolated from recombinant inbred (RI) mice derived from reciprocal crosses between AJ and B6, and we followed early bacterial counts within these macrophages. Quantitative trait locus (QTL) analysis revealed a locus on chromosome 19 that is associated with early differences in bacterial counts in AJ versus B6 macrophages. QTL analysis of published data that measured the differential susceptibilities of the same RI mice to an in vivo challenge with F. tularensis confirmed the F. tularensis susceptibility QTL on chromosome 19. Overall, our results show that early interactions of macrophages with F. tularensis are dependent on the macrophage genetic background. PMID:26980837

  20. Inhibitors of Ribosome Rescue Arrest Growth of Francisella tularensis at All Stages of Intracellular Replication

    PubMed Central

    Goralski, Tyler D. P.; Dewan, Kalyan K.; Alumasa, John N.; Avanzato, Victoria; Place, David E.; Markley, Rachel L.; Katkere, Bhuvana; Rabadi, Seham M.; Bakshi, Chandra Shekhar

    2016-01-01

    Bacteria require at least one pathway to rescue ribosomes stalled at the ends of mRNAs. The primary pathway for ribosome rescue is trans-translation, which is conserved in >99% of sequenced bacterial genomes. Some species also have backup systems, such as ArfA or ArfB, which can rescue ribosomes in the absence of sufficient trans-translation activity. Small-molecule inhibitors of ribosome rescue have broad-spectrum antimicrobial activity against bacteria grown in liquid culture. These compounds were tested against the tier 1 select agent Francisella tularensis to determine if they can limit bacterial proliferation during infection of eukaryotic cells. The inhibitors KKL-10 and KKL-40 exhibited exceptional antimicrobial activity against both attenuated and fully virulent strains of F. tularensis in vitro and during ex vivo infection. Addition of KKL-10 or KKL-40 to macrophages or liver cells at any time after infection by F. tularensis prevented further bacterial proliferation. When macrophages were stimulated with the proinflammatory cytokine gamma interferon before being infected by F. tularensis, addition of KKL-10 or KKL-40 reduced intracellular bacteria by >99%, indicating that the combination of cytokine-induced stress and a nonfunctional ribosome rescue pathway is fatal to F. tularensis. Neither KKL-10 nor KKL-40 was cytotoxic to eukaryotic cells in culture. These results demonstrate that ribosome rescue is required for F. tularensis growth at all stages of its infection cycle and suggest that KKL-10 and KKL-40 are good lead compounds for antibiotic development. PMID:26953190

  1. Francisella tularensis detection using magnetic labels and a magnetic biosensor based on frequency mixing

    NASA Astrophysics Data System (ADS)

    Meyer, Martin H. F.; Krause, Hans-Joachim; Hartmann, Markus; Miethe, Peter; Oster, Jürgen; Keusgen, Michael

    2007-04-01

    A biosensor that uses resonant coils with a special frequency-mixing technique and magnetic beads as detectable labels has been established for the detection of Francisella tularensis, the causative agent for tularemia. The detection principle is based on a sandwich immunoassay using an anti-Ft antibody for immunofiltration immobilized to ABICAP ® polyethylene filters, and biotinylated with streptavidin-coated magnetic beads as labels. The linear detection range of this biosensor was found to be 10 4-10 6 cfu F. tularensis lipopolysaccharide (LPS) per ml. Tested sample matrices were physiological PBS buffer and rabbit serum.

  2. Keep an Ear Out for Francisella tularensis: Otomastoiditis Cases after Canyoneering.

    PubMed

    Guerpillon, Brice; Boibieux, Andre; Guenne, Clemence; Ploton, Christine; Ferry, Tristan; Maurin, Max; Forestier, Emmanuel; Dauwalder, Olivier; Manipoud, Patrick; Ltaïef-Boudrigua, Aicha; Gürkov, Robert; Vandenesch, Francois; Bouchiat, Coralie

    2016-01-01

    We report here three unusual cases of otomastoiditis due to Francisella tularensis, complicated by cervical abscesses and persistent hearing loss, plus facial paralysis for one patient. Intriguingly, the three patients had practiced canyoneering independently in the same French river, between 2009 and 2014, several days before clinical symptoms onset. The results point out that fresh water exposure may be a potential contamination route for tularemia. Besides, due to the frequent complications and sequelae, we believe that F. tularensis should be considered as a possible etiology in case of otitis media, failure of the conventional antibiotic treatment, and suspicious exposure of the bacteria.

  3. Keep an Ear Out for Francisella tularensis: Otomastoiditis Cases after Canyoneering

    PubMed Central

    Guerpillon, Brice; Boibieux, Andre; Guenne, Clemence; Ploton, Christine; Ferry, Tristan; Maurin, Max; Forestier, Emmanuel; Dauwalder, Olivier; Manipoud, Patrick; Ltaïef-Boudrigua, Aicha; Gürkov, Robert; Vandenesch, Francois; Bouchiat, Coralie

    2016-01-01

    We report here three unusual cases of otomastoiditis due to Francisella tularensis, complicated by cervical abscesses and persistent hearing loss, plus facial paralysis for one patient. Intriguingly, the three patients had practiced canyoneering independently in the same French river, between 2009 and 2014, several days before clinical symptoms onset. The results point out that fresh water exposure may be a potential contamination route for tularemia. Besides, due to the frequent complications and sequelae, we believe that F. tularensis should be considered as a possible etiology in case of otitis media, failure of the conventional antibiotic treatment, and suspicious exposure of the bacteria. PMID:26973838

  4. Identification of Mycobacterium avium subsp. hominissuis Isolated From Drinking Water

    EPA Science Inventory

    Mycobacterium avium (MA) is divided into four subspecies based primarily on host-range and consists of MA subsp. avium (birds), MA subsp. silvaticum (wood pigeons), MA subsp. paratuberculosis (broad, poorly-defined host range), and the recently described MA subsp. hominissuis (hu...

  5. Optimal swab processing recovery method for detection of bioterrorism-related Francisella tularensis by real-time PCR.

    PubMed

    Walker, Roblena E; Petersen, Jeannine M; Stephens, Kenyatta W; Dauphin, Leslie A

    2010-10-01

    Francisella tularensis, the etiological agent of tularemia, is regarded as a potential bioterrorism agent. The advent of bioterrorism has heightened awareness of the need for validated methods for processing environmental samples. In this study we determined the optimal method for processing environmental swabs for the recovery and subsequent detection of F. tularensis by the use of real-time PCR assays. Four swab processing recovery methods were compared: heat, sonication, vortexing, and the Swab Extraction Tube System (SETS). These methods were evaluated using cotton, foam, polyester and rayon swabs spiked with six pathogenic strains of F. tularensis. Real-time PCR analysis using a multi-target 5'nuclease assay for F. tularensis showed that the use of the SETS method resulted in the best limit of detection when evaluated using multiple strains of F. tularensis. We demonstrated also that the efficiency of F. tularensis recovery from swab specimens was not equivalent for all swab processing methodologies and, thus, that this variable can affect real-time PCR assay sensitivity. The effectiveness of the SETS method was independent of the automated DNA extraction method and real-time PCR platforms used. In conclusion, diagnostic laboratories can now potentially incorporate the SETS method into specimen processing protocols for the rapid and efficient detection of F. tularensis by real-time PCR during laboratory bioterrorism-related investigations.

  6. A method for functional trans-complementation of intracellular Francisella tularensis.

    PubMed

    Steele, Shaun; Taft-Benz, Sharon; Kawula, Thomas

    2014-01-01

    Francisella tularensis is a highly infectious bacterial pathogen that invades and replicates within numerous host cell types. After uptake, F. tularensis bacteria escape the phagosome, replicate within the cytosol, and suppress cytokine responses. However, the mechanisms employed by F. tularensis to thrive within host cells are mostly unknown. Potential F. tularensis mutants involved in host-pathogen interactions are typically discovered by negative selection screens for intracellular replication or virulence. Mutants that fulfill these criteria fall into two categories: mutants with intrinsic intracellular growth defects and mutants that fail to modify detrimental host cell processes. It is often difficult and time consuming to discriminate between these two possibilities. We devised a method to functionally trans-complement and thus identify mutants that fail to modify the host response. In this assay, host cells are consistently and reproducibly infected with two different F. tularensis strains by physically tethering the bacteria to antibody-coated beads. To examine the efficacy of this protocol, we tested phagosomal escape, cytokine suppression, and intracellular replication for F. tularensis ΔripA and ΔpdpC. ΔripA has an intracellular growth defect that is likely due to an intrinsic defect and fails to suppress IL-1β secretion. In the co-infection model, ΔripA was unable to replicate in the host cell when wild-type bacteria infected the same cell, but cytokine suppression was rescued. Therefore, ΔripA intracellular growth is due to an intrinsic bacterial defect while cytokine secretion results from a failed host-pathogen interaction. Likewise, ΔpdpC is deficient for phagosomal escape, intracellular survival and suppression of IL-1β secretion. Wild-type bacteria that entered through the same phagosome as ΔpdpC rescued all of these phenotypes, indicating that ΔpdpC failed to properly manipulate the host. In summary, functional trans

  7. Evolutionary Ecology of the Marine Roseobacter Clade

    PubMed Central

    Luo, Haiwei

    2014-01-01

    SUMMARY Members of the Roseobacter clade are equipped with a tremendous diversity of metabolic capabilities, which in part explains their success in so many different marine habitats. Ideas on how this diversity evolved and is maintained are reviewed, focusing on recent evolutionary studies exploring the timing and mechanisms of Roseobacter ecological diversification. PMID:25428935

  8. Phylogenetic analysis of the lux operon distinguishes two evolutionarily distinct clades of Photobacterium leiognathi.

    PubMed

    Ast, Jennifer C; Dunlap, Paul V

    2004-05-01

    The luminous marine bacterium Photobacterium mandapamensis was synonymized several years ago with Photobacterium leiognathi based on a high degree of phenotypic and genetic similarity. To test the possibility that P. leiognathi as now formulated, however, actually contains two distinct bacterial groups reflecting the earlier identification of P. mandapamensis and P. leiognathi as separate species, we compared P. leiognathi strains isolated from light-organ symbiosis with leiognathid fishes (i.e., ATCC 25521(T), ATCC 25587, lequu.1.1 and lleuc.1.1) with strains from seawater originally described as P. mandapamensis and later synonymized as P. leiognathi (i.e., ATCC 27561(T) and ATCC 33981) and certain strains initially identified as P. leiognathi (i.e., PL-721, PL-741, 554). Analysis of the 16S rRNA and gyrB genes did not resolve distinct clades, affirming a close relationship among these strains. However, strains ATCC 27561(T), ATCC 33981, PL-721, PL-741 and 554 were found to bear a luxF gene in the lux operon ( luxABFE), whereas ATCC 25521(T), ATCC 25587, lequu.1.1 and lleuc.1.1 lack this gene ( luxABE). Phylogenetic analysis of the luxAB(F)E region confirmed this distinction. Furthermore, ATCC 27561(T), ATCC 33981, PL-721, PL-741 and 554 all produced a higher level of luminescence on high-salt medium, as previously described for PL-721, whereas ATCC 25521(T), ATCC 25587, lequu.1.1 and lleuc.1.1 all produced a higher level of luminescence on low-salt medium, a characteristic of P. leiognathi from leiognathid fish light organs. These results demonstrate that P. leiognathi contains two evolutionarily and phenotypically distinct clades, P. leiognathi subsp. leiognathi (strains ATCC 25521(T), ATCC 25587, lequu.1.1 and lleuc.1.1), and P. leiognathi subsp. mandapamensis (strains ATCC 27561(T), ATCC 33981, PL-721, PL-741 and 554).

  9. [Real time PCR hybridization for the rapid and specific identification of Francisella tularensis].

    PubMed

    Bielawska-Drózd, Agata; Niemcewicz, Marcin; Gaweł, Jerzy; Bartoszcze, Michał; Graniak, Grzegorz; Joniec, Justyna; Kołodziej, Marcin

    2010-01-01

    Tularemia is highly infectious and fatal zoonotic disease caused by Gram negative bacteria Francisella tularensis. The necessity to undergo medical treatment in early phase of illness in humans and possibility of making use of bacterial aerosol by terrorists in an attack create an urgent need to implement a rapid and effective method which enables to identify the agent. In our study two primers FopA F/R and hybridization probes FopA S1/S2 designed from fopA gene sequence, were tested for their potential applicability to identify F. tularensis. In this research 50 strains of F. tularensis were used and the test gave positive results. Reaction specificity was confirmed by using of non-Francisella tularensis bacterial species. The results obtained in the real-time PCR reaction with primers Tul4 F/R and hybridization probes Tul4 S1/S2, designed from tul4 gene, were comparable to the results from previous experiment with fopA - primers set. Investigation of fopA and tul4 primers and hybridization probes properties revealed characteristic Tm (melting temperature) value of the products--61 degrees C and 60 degrees C, respectively. Detection sensitivity was remarkably higher when fopA primers set was used 1 fg/microl, and for tul4 primers set, minimal detectable concentration is 10 fg/microl.

  10. Comparison of Francisella tularensis genomes reveals evolutionary events associated with the emergence of human pathogenic strains

    PubMed Central

    Rohmer, Laurence; Fong, Christine; Abmayr, Simone; Wasnick, Michael; Larson Freeman, Theodore J; Radey, Matthew; Guina, Tina; Svensson, Kerstin; Hayden, Hillary S; Jacobs, Michael; Gallagher, Larry A; Manoil, Colin; Ernst, Robert K; Drees, Becky; Buckley, Danielle; Haugen, Eric; Bovee, Donald; Zhou, Yang; Chang, Jean; Levy, Ruth; Lim, Regina; Gillett, Will; Guenthener, Don; Kang, Allison; Shaffer, Scott A; Taylor, Greg; Chen, Jinzhi; Gallis, Byron; D'Argenio, David A; Forsman, Mats; Olson, Maynard V; Goodlett, David R; Kaul, Rajinder; Miller, Samuel I; Brittnacher, Mitchell J

    2007-01-01

    Background Francisella tularensis subspecies tularensis and holarctica are pathogenic to humans, whereas the two other subspecies, novicida and mediasiatica, rarely cause disease. To uncover the factors that allow subspecies tularensis and holarctica to be pathogenic to humans, we compared their genome sequences with the genome sequence of Francisella tularensis subspecies novicida U112, which is nonpathogenic to humans. Results Comparison of the genomes of human pathogenic Francisella strains with the genome of U112 identifies genes specific to the human pathogenic strains and reveals pseudogenes that previously were unidentified. In addition, this analysis provides a coarse chronology of the evolutionary events that took place during the emergence of the human pathogenic strains. Genomic rearrangements at the level of insertion sequences (IS elements), point mutations, and small indels took place in the human pathogenic strains during and after differentiation from the nonpathogenic strain, resulting in gene inactivation. Conclusion The chronology of events suggests a substantial role for genetic drift in the formation of pseudogenes in Francisella genomes. Mutations that occurred early in the evolution, however, might have been fixed in the population either because of evolutionary bottlenecks or because they were pathoadaptive (beneficial in the context of infection). Because the structure of Francisella genomes is similar to that of the genomes of other emerging or highly pathogenic bacteria, this evolutionary scenario may be shared by pathogens from other species. PMID:17550600

  11. Glutathione provides a source of cysteine essential for intracellular multiplication of Francisella tularensis.

    PubMed

    Alkhuder, Khaled; Meibom, Karin L; Dubail, Iharilalao; Dupuis, Marion; Charbit, Alain

    2009-01-01

    Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularemia. Its ability to multiply and survive in macrophages is critical for its virulence. By screening a bank of HimarFT transposon mutants of the F. tularensis live vaccine strain (LVS) to isolate intracellular growth-deficient mutants, we selected one mutant in a gene encoding a putative gamma-glutamyl transpeptidase (GGT). This gene (FTL_0766) was hence designated ggt. The mutant strain showed impaired intracellular multiplication and was strongly attenuated for virulence in mice. Here we present evidence that the GGT activity of F. tularensis allows utilization of glutathione (GSH, gamma-glutamyl-cysteinyl-glycine) and gamma-glutamyl-cysteine dipeptide as cysteine sources to ensure intracellular growth. This is the first demonstration of the essential role of a nutrient acquisition system in the intracellular multiplication of F. tularensis. GSH is the most abundant source of cysteine in the host cytosol. Thus, the capacity this intracellular bacterial pathogen has evolved to utilize the available GSH, as a source of cysteine in the host cytosol, constitutes a paradigm of bacteria-host adaptation.

  12. Inactivation of F.tularensis Utah-112 on food and food contact surfaces by ultraviolet light

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Francisella tularensis is the causative agent of tularemia, a plague-like illness that affects animals and humans, and has caused large illness pandemics in the last century. It has also been used as a biological warfare agent, and tularemia can be contracted through consumption of contaminated food...

  13. Iron Acquisition in Mycobacterium avium subsp. paratuberculosis

    PubMed Central

    Wang, Joyce; Moolji, Jalal; Dufort, Alex; Staffa, Alfredo; Domenech, Pilar; Reed, Michael B.

    2015-01-01

    ABSTRACT Mycobacterium avium subsp. paratuberculosis is a host-adapted pathogen that evolved from the environmental bacterium M. avium subsp. hominissuis through gene loss and gene acquisition. Growth of M. avium subsp. paratuberculosis in the laboratory is enhanced by supplementation of the media with the iron-binding siderophore mycobactin J. Here we examined the production of mycobactins by related organisms and searched for an alternative iron uptake system in M. avium subsp. paratuberculosis. Through thin-layer chromatography and radiolabeled iron-uptake studies, we showed that M. avium subsp. paratuberculosis is impaired for both mycobactin synthesis and iron acquisition. Consistent with these observations, we identified several mutations, including deletions, in M. avium subsp. paratuberculosis genes coding for mycobactin synthesis. Using a transposon-mediated mutagenesis screen conditional on growth without myobactin, we identified a potential mycobactin-independent iron uptake system on a M. avium subsp. paratuberculosis-specific genomic island, LSPP15. We obtained a transposon (Tn) mutant with a disruption in the LSPP15 gene MAP3776c for targeted study. The mutant manifests increased iron uptake as well as intracellular iron content, with genes downstream of the transposon insertion (MAP3775c to MAP3772c [MAP3775-2c]) upregulated as the result of a polar effect. As an independent confirmation, we observed the same iron uptake phenotypes by overexpressing MAP3775-2c in wild-type M. avium subsp. paratuberculosis. These data indicate that the horizontally acquired LSPP15 genes contribute to iron acquisition by M. avium subsp. paratuberculosis, potentially allowing the subsequent loss of siderophore production by this pathogen. IMPORTANCE Many microbes are able to scavenge iron from their surroundings by producing iron-chelating siderophores. One exception is Mycobacterium avium subsp. paratuberculosis, a fastidious, slow-growing animal pathogen whose growth

  14. Nucleolin, a Shuttle Protein Promoting Infection of Human Monocytes by Francisella tularensis

    PubMed Central

    Barel, Monique; Meibom, Karin; Charbit, Alain

    2010-01-01

    Background Francisella tularensis is a highly virulent facultative intracellular bacterium, disseminating in vivo mainly within host mononuclear phagocytes. After entry into macrophages, F. tularensis initially resides in a phagosomal compartment, whose maturation is then arrested. Bacteria escape rapidly into the cytoplasm, where they replicate freely. We recently demonstrated that nucleolin, an eukaryotic protein able to traffic from the nucleus to the cell surface, acted as a surface receptor for F. tularensis LVS on human monocyte-like THP-1 cells. Methodology/Principal Findings Here, we followed the fate of nucleolin once F. tularensis has been endocytosed. We first confirmed by siRNA silencing experiments that expression of nucleolin protein was essential for binding of LVS on human macrophage-type THP-1 cells. We then showed that nucleolin co-localized with intracellular bacteria in the phagosomal compartment. Strikingly, in that compartment, nucleolin also co-localized with LAMP-1, a late endosomal marker. Co-immunoprecipation assays further demonstrated an interaction of nucleolin with LAMP-1. Co-localization of nucleolin with LVS was no longer detectable at 24 h when bacteria were multiplying in the cytoplasm. In contrast, with an iglC mutant of LVS, which remains trapped into the phagosomal compartment, or with inert particles, nucleolin/bacteria co-localization remained almost constant. Conclusions/Significance We herein confirm the importance of nucleolin expression for LVS binding and its specificity as nucleolin is not involved in binding of another intracellular pathogen as L. monocytogenes or an inert particle. Association of nucleolin with F. tularensis during infection continues intracellularly after endocytosis of the bacteria. The present work therefore unravels for the first time the presence of nucleolin in the phagosomal compartment of macrophages. PMID:21152024

  15. PATHOGENESIS AND IMMUNE RESPONSES OF FRANCISELLA TULARENSIS STRAINS IN WILD-CAUGHT COTTONTAIL RABBITS (SYLVILAGUS SPP.).

    PubMed

    Brown, Vienna R; Adney, Danielle R; Bielefeldt-Ohmann, Helle; Gordy, Paul W; Felix, Todd A; Olea-Popelka, Francisco J; Bowen, Richard A

    2015-07-01

    Francisella tularensis is a highly virulent, zoonotic bacterium that causes significant natural disease and is of concern as an organism for bioterrorism. Serologic testing of wildlife is frequently used to monitor spatial patterns of infection and to quantify exposure. Cottontail rabbits (Sylvilagus spp.) are a natural reservoir for F. tularensis in the US, although very little work has been done experimentally to determine how these animals respond to infection; thus, information gathered from field samples can be difficult to interpret. We characterized clinical disease, bacteremia, pathology, and antibody kinetics of North American cottontail rabbits experimentally infected with five strains of F. tularensis. Rabbits were infected with four field strains, including MA00-2987 (type A1b), WY96-3418 (type A2), KY99-3387, and OR96-0246 (type B), and with SchuS4 (type A1a), a widely used, virulent laboratory strain. Infection with the different strains of the bacterium resulted in varied patterns of clinical disease, gross pathology, and histopathology. Each of the type A strains were highly virulent, with rabbits succumbing to infection 3-13 d after infection. At necropsy, numerous microabscesses were observed in the livers and spleens of most rabbits, associated with high bacterial organ burdens. In contrast, most rabbits infected with type B strains developed mild fever and became lethargic, but the disease was infrequently lethal. Those rabbits infected with type B strains that survived past 14 d developed a robust humoral immune response, and F. tularensis was not isolated from liver, spleen, or lung of those animals. Understanding F. tularensis infection in a natural reservoir species can guide serosurveillance and generate new insights into environmental maintenance of this pathogen.

  16. Nasal Acai Polysaccharides Potentiate Innate Immunity to Protect against Pulmonary Francisella tularensis and Burkholderia pseudomallei Infections

    PubMed Central

    Skyberg, Jerod A.; Rollins, MaryClare F.; Holderness, Jeff S.; Marlenee, Nicole L.; Schepetkin, Igor A.; Goodyear, Andrew; Dow, Steven W.; Jutila, Mark A.; Pascual, David W.

    2012-01-01

    Pulmonary Francisella tularensis and Burkholderia pseudomallei infections are highly lethal in untreated patients, and current antibiotic regimens are not always effective. Activating the innate immune system provides an alternative means of treating infection and can also complement antibiotic therapies. Several natural agonists were screened for their ability to enhance host resistance to infection, and polysaccharides derived from the Acai berry (Acai PS) were found to have potent abilities as an immunotherapeutic to treat F. tularensis and B. pseudomallei infections. In vitro, Acai PS impaired replication of Francisella in primary human macrophages co-cultured with autologous NK cells via augmentation of NK cell IFN-γ. Furthermore, Acai PS administered nasally before or after infection protected mice against type A F. tularensis aerosol challenge with survival rates up to 80%, and protection was still observed, albeit reduced, when mice were treated two days post-infection. Nasal Acai PS administration augmented intracellular expression of IFN-γ by NK cells in the lungs of F. tularensis-infected mice, and neutralization of IFN-γ ablated the protective effect of Acai PS. Likewise, nasal Acai PS treatment conferred protection against pulmonary infection with B. pseudomallei strain 1026b. Acai PS dramatically reduced the replication of B. pseudomallei in the lung and blocked bacterial dissemination to the spleen and liver. Nasal administration of Acai PS enhanced IFN-γ responses by NK and γδ T cells in the lungs, while neutralization of IFN-γ totally abrogated the protective effect of Acai PS against pulmonary B. pseudomallei infection. Collectively, these results demonstrate Acai PS is a potent innate immune agonist that can resolve F. tularensis and B. pseudomallei infections, suggesting this innate immune agonist has broad-spectrum activity against virulent intracellular pathogens. PMID:22438809

  17. Protective B-cell epitopes of Francisella tularensis O-polysaccharide in a mouse model of respiratory tularaemia

    PubMed Central

    Lu, Zhaohua; Madico, Guillermo; Roche, Marly I; Wang, Qi; Hui, Julia H; Perkins, Hillary M; Zaia, Joseph; Costello, Catherine E; Sharon, Jacqueline

    2012-01-01

    Antibodies to the lipopolysaccharide (LPS) of Francisella tularensis have been shown to be protective against respiratory tularaemia in mouse models, and we have previously described mouse monoclonal antibodies (mAbs) to non-overlapping terminal and internal epitopes of the F. tularensis LPS O-polysaccharide (OAg). In the current study, we used F. tularensis LPS oligosaccharides of defined OAg repeat length as molecular rulers in competition ELISA to demonstrate that the epitope targeted by the terminal OAg-binding mAb FB11 is contained within one tetrasaccharide repeat whereas the epitope targeted by the internal OAg-binding mAb Ab52 spans two tetrasaccharide repeats. Both mAbs conferred survival to BALB/c mice infected intranasally with the F. tularensis type B live vaccine strain and prolonged survival of BALB/c mice infected intranasally with the highly virulent F. tularensis type A strain SchuS4. The protective effects correlated with reduced bacterial burden in mAb-treated infected mice. These results indicate that an oligosaccharide with two OAg tetrasaccharide repeats covers both terminal and internal protective OAg epitopes, which may inform the design of vaccines for tularaemia. Furthermore, the FB11 and Ab52 mAbs could serve as reporters to monitor the response of vaccine recipients to protective B-cell epitopes of F. tularensis OAg. PMID:22486311

  18. Francisella tularensis type A strains cause the rapid encystment of Acanthamoeba castellanii and survive in amoebal cysts for three weeks postinfection.

    PubMed

    El-Etr, Sahar H; Margolis, Jeffrey J; Monack, Denise; Robison, Richard A; Cohen, Marissa; Moore, Emily; Rasley, Amy

    2009-12-01

    Francisella tularensis, the causative agent of the zoonotic disease tularemia, has recently gained increased attention due to the emergence of tularemia in geographical areas where the disease has been previously unknown and to the organism's potential as a bioterrorism agent. Although F. tularensis has an extremely broad host range, the bacterial reservoir in nature has not been conclusively identified. In this study, the ability of virulent F. tularensis strains to survive and replicate in the amoeba Acanthamoeba castellanii was explored. We observe that A. castellanii trophozoites rapidly encyst in response to F. tularensis infection and that this rapid encystment phenotype is caused by factor(s) secreted by amoebae and/or F. tularensis into the coculture medium. Further, our results indicate that in contrast to the live vaccine strain LVS, virulent strains of F. tularensis can survive in A. castellanii cysts for at least 3 weeks postinfection and that the induction of rapid amoeba encystment is essential for survival. In addition, our data indicate that pathogenic F. tularensis strains block lysosomal fusion in A. castellanii. Taken together, these data suggest that interactions between F. tularensis strains and amoebae may play a role in the environmental persistence of F. tularensis.

  19. Francisella tularensis type A Strains Cause the Rapid Encystment of Acanthamoeba castellanii and Survive in Amoebal Cysts for Three Weeks post Infection

    SciTech Connect

    El-Etr, S H; Margolis, J; Monack, D; Robison, R; Cohen, M; Moore, E; Rasley, A

    2009-07-28

    Francisella tularensis, the causative agent of the zoonotic disease tularemia, has recently gained increased attention due to the emergence of tularemia in geographical areas where the disease has been previously unknown, and the organism's potential as a bioterrorism agent. Although F. tularensis has an extremely broad host range, the bacterial reservoir in nature has not been conclusively identified. In this study, the ability of virulent F. tularensis strains to survive and replicate in the amoeba Acanthamoeba castellanii was explored. We observe that A. castellanii trophozoites rapidly encyst in response to F. tularensis infection and that this rapid encystment phenotype (REP) is caused by factor(s) secreted by amoebae and/or F. tularensis into the co-culture media. Further, our results indicate that in contrast to LVS, virulent strains of F. tularensis can survive in A. castellanii cysts for at least 3 weeks post infection and that induction of rapid amoeba encystment is essential for survival. In addition, our data indicate that pathogenic F. tularensis strains block lysosomal fusion in A. castellanii. Taken together, these data suggest that the interactions between F. tularensis strains and amoeba may play a role in the environmental persistence of F. tularensis.

  20. Pullulanase Is Necessary for the Efficient Intracellular Growth of Francisella tularensis

    PubMed Central

    Takimoto, Kazuhiro; Deyu, Tian; Koyama, Yuuki; Park, Eun-sil; Fujita, Osamu; Hotta, Akitoyo; Morikawa, Shigeru

    2016-01-01

    Pullulanase, an enzyme that catalyzes the hydrolysis of polysaccharides, has been identified in a broad range of organisms, including bacteria, yeasts, fungi, and animals. The pullulanase (pulB; FTT_0412c) of F. tularensis subspecies tularensis Schu S4 is considered to be a homologue of the type I pullulanase (pulA) of the other Francisella subspecies. The significance of Francisella pullulanase has been obscure until now. In the present study, we characterized a recombinant PulB of F. tularensis SCHU P9, which was expressed as a his-tagged protein in Escherichia coli. The recombinant PulB was confirmed to be a type I pullulanase by its enzymatic activity in vitro. A pulB gene knockout mutant of F. tularensis SCHU P9 (ΔpulB) was constructed using the TargeTron Knockout system and plasmid pKEK1140 to clarify the function of PulB during the growth of F. tularensis in macrophages. The intracellular growth of the ΔpulB mutant in murine macrophage J774.1 cells was significantly reduced compared with that of the parental strain SCHU P9. Expression of PulB in ΔpulB, using an expression plasmid, resulted in the complementation of the reduced growth in macrophages, suggesting that PulB is necessary for the efficient growth of F. tularensis in macrophages. To assess the role of PulB in virulence, the knockout and parent bacterial strains were used to infect C57BL/6J mice. Histopathological analyses showed that tissues from ΔpulB-infected mice showed milder lesions compared to those from SCHU P9-infected mice. However, all mice infected with SCHU P9 and ΔpulB showed the similar levels of bacterial loads in their tissues. The results suggest that PulB plays a significant role in bacterial growth within murine macrophage but does not contribute to bacterial virulence in vivo. PMID:27448164

  1. Molecular Epidemiology of Mycobacterium avium subsp. paratuberculosis Isolates Recovered from Wild Animal Species

    PubMed Central

    Motiwala, Alifiya S.; Amonsin, Alongkorn; Strother, Megan; Manning, Elizabeth J. B.; Kapur, Vivek; Sreevatsan, Srinand

    2004-01-01

    Mycobacterial isolates were obtained by radiometric culture from 33 different species of captive or free-ranging animals (n = 106) and environmental sources (n = 3) from six geographic zones within the United States. The identities of all 109 isolates were confirmed by using mycobactin J dependence and characterization of five well-defined molecular markers, including two integration loci of IS900 (loci L1 and L9), one Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis)-specific sequence (locus 251), and one M. avium subsp. avium-specific marker (IS1245), as well as hsp65 and IS1311 restriction endonuclease analyses. Seventy-six acid-fast isolates were identified as M. paratuberculosis, 15 were identified as belonging to the M. avium-M. intracellulare complex (but not M. paratuberculosis), and the remaining 18 were identified as mycobacteria outside the M. avium-M. intracellulare complex. Fingerprinting by multiplex PCR for IS900 integration loci clustered 67 of the 76 M. paratuberculosis strains into a single clade (designated clade A18) and had a Simpson's diversity index (D) of 0.53. In contrast, sequence-based characterization of a recently identified M. paratuberculosis short sequence repeat (SSR) region enabled the differentiation of the M. paratuberculosis isolates in clade A18 into seven distinct alleles (D = 0.75). The analysis revealed eight subtypes among the 33 species of animals, suggesting the interspecies transmission of specific strains. Taken together, the results of our analyses demonstrate that SSR analysis enables the genetic characterization of M. paratuberculosis isolates from different host species and provide evidence for the host specificity of some M. paratuberculosis strains as well as sharing of strains between wild and domesticated animal species. PMID:15071028

  2. Simultaneous real-time PCR detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis.

    PubMed

    Skottman, T; Piiparinen, H; Hyytiäinen, H; Myllys, V; Skurnik, M; Nikkari, S

    2007-03-01

    This report describes the development of in-house real-time PCR assays using minor groove binding probes for simultaneous detection of the Bacillus anthracis pag and cap genes, the Francisella tularensis 23 KDa gene, as well as the Yersinia pestis pla gene. The sensitivities of these assays were at least 1 fg, except for the assay targeting the Bacillus anthracis cap gene, which showed a sensitivity of 10 fg when total DNA was used as a template in a serial dilution. The clinical value of the Bacillus anthracis- and Francisella tularensis-specific assays was demonstrated by successful amplification of DNA from cases of cow anthrax and hare tularemia, respectively. No cross-reactivity between these species-specific assays or with 39 other bacterial species was noted. These assays may provide a rapid tool for the simultaneous detection and identification of the three category A bacterial species listed as biological threats by the Centers for Disease Control and Prevention.

  3. Francisella tularensis Catalase Restricts Immune Function by Impairing TRPM2 Channel Activity.

    PubMed

    Shakerley, Nicole L; Chandrasekaran, Akshaya; Trebak, Mohamed; Miller, Barbara A; Melendez, J Andrés

    2016-02-19

    As an innate defense mechanism, macrophages produce reactive oxygen species that weaken pathogens and serve as secondary messengers involved in immune function. The Gram-negative bacterium Francisella tularensis utilizes its antioxidant armature to limit the host immune response, but the mechanism behind this suppression is not defined. Here we establish that F. tularensis limits Ca(2+) entry in macrophages, thereby limiting actin reorganization and IL-6 production in a redox-dependent fashion. Wild type (live vaccine strain) or catalase-deficient F. tularensis (ΔkatG) show distinct profiles in their H2O2 scavenging rates, 1 and 0.015 pm/s, respectively. Murine alveolar macrophages infected with ΔkatG display abnormally high basal intracellular Ca(2+) concentration that did not increase further in response to H2O2. Additionally, ΔkatG-infected macrophages displayed limited Ca(2+) influx in response to ionomycin, as a result of ionophore H2O2 sensitivity. Exogenously added H2O2 or H2O2 generated by ΔkatG likely oxidizes ionomycin and alters its ability to transport Ca(2+). Basal increases in cytosolic Ca(2+) and insensitivity to H2O2-mediated Ca(2+) entry in ΔkatG-infected cells are reversed by the Ca(2+) channel inhibitors 2-aminoethyl diphenylborinate and SKF-96365. 2-Aminoethyl diphenylborinate but not SKF-96365 abrogated ΔkatG-dependent increases in macrophage actin remodeling and IL-6 secretion, suggesting a role for H2O2-mediated Ca(2+) entry through the transient receptor potential melastatin 2 (TRPM2) channel in macrophages. Indeed, increases in basal Ca(2+), actin polymerization, and IL-6 production are reversed in TRPM2-null macrophages infected with ΔkatG. Together, our findings provide compelling evidence that F. tularensis catalase restricts reactive oxygen species to temper macrophage TRPM2-mediated Ca(2+) signaling and limit host immune function.

  4. Modulation of virulence factors in Francisella tularensis determines human macrophage responses

    PubMed Central

    Carlson, Paul E.; Carroll, James A.; O’Dee, Dawn M.; Nau, Gerard J.

    2009-01-01

    Francisella tularensis, the causative agent of tularemia and Category A biodefense agent, is known to replicate within host macrophages, though the pathogenesis of this organism is incompletely understood. We have isolated a variant of F. tularensis Live Vaccine Strain (LVS) based on colony morphology and its effect on macrophages. Human monocyte-derived macrophages produced more tumor necrosis factor α (TNFα), interleukin (IL)-1β, IL-6, and IL-12 p40 following exposure to the variant, designated the activating variant (ACV). The immunoreactivity of the lipopolysaccharide (LPS) from both LVS and ACV was comparable to the previously described blue variant and was distinct from the gray variant of LVS. We found, however, the soluble protein fractions of LVS and ACV differed. Further investigation using two-dimensional gel electrophoresis demonstrated higher levels of several proteins in the parental LVS isolate. The differentially-expressed proteins featured several associated with virulence in F. tularensis and other pathogens, including intracellular growth locus C (IglC), a σ54 modulation protein family member (YhbH), and aconitase. ACV reverted to the LVS phenotype, indicated by low cytokine induction and high IglC expression, after growth in a chemically-defined media. These data provide evidence that the levels of virulence factors in F. tularensis are modulated based on culture conditions and that this modulation impacts host responses. This work provides a basis for investigation of Francisella virulence factor regulation and the identification of additional factors, co-regulated with IglC, that affect macrophage responses. PMID:17369012

  5. GroEL and Lipopolysaccharide from Francisella tularensis Live Vaccine Strain Synergistically Activate Human Macrophages ▿

    PubMed Central

    Noah, Courtney E.; Malik, Meenakshi; Bublitz, DeAnna C.; Camenares, Devin; Sellati, Timothy J.; Benach, Jorge L.; Furie, Martha B.

    2010-01-01

    Francisella tularensis, the causative agent of tularemia, interacts with host cells of innate immunity in an atypical manner. For most Gram-negative bacteria, the release of lipopolysaccharide (LPS) from their outer membranes stimulates an inflammatory response. When LPS from the attenuated live vaccine strain (LVS) or the highly virulent Schu S4 strain of F. tularensis was incubated with human umbilical vein endothelial cells, neither species of LPS induced expression of the adhesion molecule E-selectin or secretion of the chemokine CCL2. Moreover, a high concentration (10 μg/ml) of LVS or Schu S4 LPS was required to stimulate production of CCL2 by human monocyte-derived macrophages (huMDM). A screen for alternative proinflammatory factors of F. tularensis LVS identified the heat shock protein GroEL as a potential candidate. Recombinant LVS GroEL at a concentration of 10 μg/ml elicited secretion of CXCL8 and CCL2 by huMDM through a TLR4-dependent mechanism. When 1 μg of LVS GroEL/ml was added to an equivalent amount of LVS LPS, the two components synergistically activated the huMDM to produce CXCL8. Schu S4 GroEL was less stimulatory than LVS GroEL and showed a lesser degree of synergy when combined with Schu S4 LPS. These findings suggest that the intrinsically low proinflammatory activity of F. tularensis LPS may be increased in the infected human host through interactions with other components of the bacterium. PMID:20123721

  6. Isolation of Francisella tularensis and Yersinia pestis from Blood Cultures by Plasma Purification and Immunomagnetic Separation Accelerates Antibiotic Susceptibility Determination

    PubMed Central

    Aloni-Grinstein, Ronit; Schuster, Ofir; Yitzhaki, Shmuel; Aftalion, Moshe; Maoz, Sharon; Steinberger-Levy, Ida; Ber, Raphael

    2017-01-01

    The early symptoms of tularemia and plague, which are caused by Francisella tularensis and Yersinia pestis infection, respectively, are common to other illnesses, resulting in a low index of suspicion among clinicians. Moreover, because these diseases can be treated only with antibiotics, rapid isolation of the bacteria and antibiotic susceptibility testing (AST) are preferable. Blood cultures of patients may serve as a source for bacteria isolation. However, due to the slow growth rates of F. tularensis and Y. pestis on solid media, isolation by plating blood culture samples on proper agar plates may require several days. Thus, improving the isolation procedure prior to antibiotic susceptibility determination is a major clinically relevant need. In this study, we developed a rapid, selective procedure for the isolation of F. tularensis and Y. pestis from blood cultures. We examined drop-plating and plasma purification followed by immunomagnetic separation (IMS) as alternative isolation methods. We determined that replacing the classical isolation method with drop-plating is advantageous with respect to time at the expense of specificity. Hence, we also examined isolation by IMS. Sub-localization of F. tularensis within blood cultures of infected mice has revealed that the majority of the bacteria are located within the extracellular fraction, in the plasma. Y. pestis also resides within the plasma. Therefore, the plasma fraction was isolated from blood cultures and subjected to an IMS procedure using polyclonal anti-F. tularensis live vaccine strain (LVS) or anti-Y. pestis antibodies conjugated to 50-nm nano-beads. The time required to reach an inoculum of sufficient bacteria for AST was shortest when using the plasma and IMSs for both bacteria, saving up to 2 days of incubation for F. tularensis and 1 day for Y. pestis. Our isolation procedure provides a proof of concept for the clinical relevance of rapid isolation for AST from F. tularensis- and Y. pestis

  7. Decontamination of a hospital room using gaseous chlorine dioxide: Bacillus anthracis, Francisella tularensis, and Yersinia pestis.

    PubMed

    Lowe, John J; Gibbs, Shawn G; Iwen, Peter C; Smith, Philip W; Hewlett, Angela L

    2013-01-01

    This study assessed the efficacy of gaseous chlorine dioxide for inactivation of Bacillus anthracis, Francisella tularensis, and Yersinia pestis in a hospital patient care suite. Spore and vegetative cells of Bacillus anthracis Sterne 34F2, spores of Bacillus atrophaeus ATCC 9372 and vegetative cells of both Francisella tularensis ATCC 6223 and Yersinia pestis A1122 were exposed to gaseous chlorine dioxide in a patient care suite. Organism inactivation was then assessed by log reduction in viable organisms postexposure to chlorine dioxide gas compared to non-exposed control organism. Hospital room decontamination protocols utilizing chlorine dioxide gas concentrations of 377 to 385 ppm maintained to exposures of 767 ppm-hours with 65% relative humidity consistently achieved complete inactivation of B. anthracis and B. atrophaeus spores, as well as vegetative cells of B. anthracis, F. tularensis, and Y. pestis. Decrease in exposure (ppm-hours) and relative humidity (<65%) or restricting airflow reduced inactivation but achieved >8 log reductions in organisms. Up to 10-log reductions were achieved in a hospital room with limited impact on adjacent areas, indicating chlorine dioxide concentrations needed for decontamination of highly concentrated (>6 logs) organisms can be achieved throughout a hospital room. This study translates laboratory chlorine dioxide fumigation studies applied in a complex clinical environment.

  8. Francisella tularensis Antioxidants Harness Reactive Oxygen Species to Restrict Macrophage Signaling and Cytokine Production*

    PubMed Central

    Melillo, Amanda A.; Bakshi, Chandra Shekhar; Melendez, J. Andrés

    2010-01-01

    Francisella tularensis is the etiologic agent of the highly infectious animal and human disease tularemia. Its extreme infectivity and virulence are associated with its ability to evade immune detection, which we now link to its robust reactive oxygen species-scavenging capacity. Infection of primary human monocyte-derived macrophages with virulent F. tularensis SchuS4 prevented proinflammatory cytokine production in the presence or absence of IFN-γ compared with infection with the attenuated live vaccine strain. SchuS4 infection also blocked signals required for macrophage cytokine production, including Akt phosphorylation, IκBα degradation, and NF-κB nuclear localization and activation. Concomitant with SchuS4-mediated suppression of Akt phosphorylation was an increase in the levels of the Akt antagonist PTEN. Moreover, SchuS4 prevented the H2O2-dependent oxidative inactivation of PTEN compared with a virulent live vaccine strain. Mutation of catalase (katG) sensitized F. tularensis to H2O2 and enhanced PTEN oxidation, Akt phosphorylation, NF-κB activation, and inflammatory cytokine production. Together, these findings suggest a novel role for bacterial antioxidants in restricting macrophage activation through their ability to preserve phosphatases that temper kinase signaling and proinflammatory cytokine production. PMID:20558723

  9. Natural History of Francisella tularensis in Aerosol-Challenged BALB/c Mice

    PubMed Central

    Chuvala, Lara; Riggins, Renaldo; Cirz, Ryan; Cass, Robert; Louie, Arnold; Drusano, G. L.

    2016-01-01

    The objective of this study was to evaluate the natural history and pathogenesis of Francisella tularensis in a murine model of inhalational tularemia with the SchuS4 strain. Before the efficacy of antimicrobials could be assessed in this model, further model development was required to determine the optimal time to start therapy. This study helped define the time course of infection after aerosol challenge by quantifying the presence of bacteria in lung, blood, and spleen at multiple harvest points. In this study, mice were infected via a targeted inhaled dose of 100 50% lethal doses (LD50s) (LD50 = 300 CFU) of F. tularensis by whole-body aerosol. At 1, 24, 36, 48, 60, 72, 75, 78, 81, 84, 87, and 90 h postchallenge, groups of 15 animals were sacrificed and blood, lung, and splenic tissue samples were harvested, homogenized, plated, and incubated to evaluate the bacterial load in those tissues. It was determined that of the 3 sample types harvested, splenic tissue provided the most consistent bacterial counts, which steadily increased with the progressing infection. Further, it was determined that lung samples from all (15/15) animals were positive for infection at 75 h postaerosolization and that 14/15 animals had positive splenic tissue counts. Bacterial levels in blood were not predictive of treatment initiation. For future therapeutic evaluation studies in this model using F. tularensis (SchuS4), it was determined that therapy should be initiated at 75 h postchallenge and validated by spleen involvement. PMID:26824958

  10. Natural History of Francisella tularensis in Aerosol-Challenged BALB/c Mice.

    PubMed

    Heine, Henry S; Chuvala, Lara; Riggins, Renaldo; Cirz, Ryan; Cass, Robert; Louie, Arnold; Drusano, G L

    2016-01-11

    The objective of this study was to evaluate the natural history and pathogenesis of Francisella tularensis in a murine model of inhalational tularemia with the SchuS4 strain. Before the efficacy of antimicrobials could be assessed in this model, further model development was required to determine the optimal time to start therapy. This study helped define the time course of infection after aerosol challenge by quantifying the presence of bacteria in lung, blood, and spleen at multiple harvest points. In this study, mice were infected via a targeted inhaled dose of 100 50% lethal doses (LD50s) (LD50 = 300 CFU) of F. tularensis by whole-body aerosol. At 1, 24, 36, 48, 60, 72, 75, 78, 81, 84, 87, and 90 h postchallenge, groups of 15 animals were sacrificed and blood, lung, and splenic tissue samples were harvested, homogenized, plated, and incubated to evaluate the bacterial load in those tissues. It was determined that of the 3 sample types harvested, splenic tissue provided the most consistent bacterial counts, which steadily increased with the progressing infection. Further, it was determined that lung samples from all (15/15) animals were positive for infection at 75 h postaerosolization and that 14/15 animals had positive splenic tissue counts. Bacterial levels in blood were not predictive of treatment initiation. For future therapeutic evaluation studies in this model using F. tularensis (SchuS4), it was determined that therapy should be initiated at 75 h postchallenge and validated by spleen involvement.

  11. Characterization of pathogenic vibrios isolated from bivalve hatcheries in Galicia, NW Atlantic coast of Spain. Description of Vibrio tubiashii subsp. europaeus [corrected] subsp. nov.

    PubMed

    Prado, Susana; Dubert, Javier; Barja, Juan L

    2015-02-01

    The taxonomic position of the bivalve pathogen PP-638 was studied together with five similar isolates. The strains were isolated from flat oyster (Ostrea edulis) and Manila clam (Venerupis philippinarum) cultures during outbreaks of disease in two shellfish hatcheries (Galicia, NW Spain). The pathogenicity, previously established for PP-638, was demonstrated with all isolates and for several bivalve species, including the original hosts. On the basis of phenotypic characterization and 16S rRNA gene sequences, a tight group was defined within the genus Vibrio. Multilocus sequence analysis (MLSA) based on concatenated sequences of the 16S rRNA gene and the five housekeeping genes recA, rpoA, pyrH, gyrB and ftsZ revealed that these strains form a cluster within the Orientalis clade, close to the species Vibrio tubiashii. The results of MLSA, the DDH rate and the phenotypic differences with the type strain of V. tubiashii supported the differentiation of the Galician isolates as a new subspecies within V. tubiashii, for which the name V. tubiashii subsp. europaeus [corrected] subsp. nov. is proposed (type strain PP-638(T)=CECT 8136(T)=DSM 7349(T)) The emended description of V. tubiashii is included. The pathogenicity assays widen the host range of V. tubiashii to add two unreported species, Venerupis decussata and Donax trunculus, and the described as relatively resistant species V. philippinarum.

  12. Campylobacter fetus subsp. testudinum subsp. nov., isolated from humans and reptiles.

    PubMed

    Fitzgerald, Collette; Tu, Zheng Chao; Patrick, Mary; Stiles, Tracy; Lawson, Andy J; Santovenia, Monica; Gilbert, Maarten J; van Bergen, Marcel; Joyce, Kevin; Pruckler, Janet; Stroika, Steven; Duim, Birgitta; Miller, William G; Loparev, Vladimir; Sinnige, Jan C; Fields, Patricia I; Tauxe, Robert V; Blaser, Martin J; Wagenaar, Jaap A

    2014-09-01

    A polyphasic study was undertaken to determine the taxonomic position of 13 Campylobacter fetus-like strains from humans (n = 8) and reptiles (n = 5). The results of matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS and genomic data from sap analysis, 16S rRNA gene and hsp60 sequence comparison, pulsed-field gel electrophoresis, amplified fragment length polymorphism analysis, DNA-DNA hybridization and whole genome sequencing demonstrated that these strains are closely related to C. fetus but clearly differentiated from recognized subspecies of C. fetus. Therefore, this unique cluster of 13 strains represents a novel subspecies within the species C. fetus, for which the name Campylobacter fetus subsp. testudinum subsp. nov. is proposed, with strain 03-427(T) ( = ATCC BAA-2539(T) = LMG 27499(T)) as the type strain. Although this novel taxon could not be differentiated from C. fetus subsp. fetus and C. fetus subsp. venerealis using conventional phenotypic tests, MALDI-TOF MS revealed the presence of multiple phenotypic biomarkers which distinguish Campylobacter fetus subsp. testudinum subsp. nov. from recognized subspecies of C. fetus.

  13. Staphylococcus petrasii sp. nov. including S. petrasii subsp. petrasii subsp. nov. and S. petrasii subsp. croceilyticus subsp. nov., isolated from human clinical specimens and human ear infections.

    PubMed

    Pantůček, Roman; Švec, Pavel; Dajcs, Joseph J; Machová, Ivana; Černohlávková, Jitka; Šedo, Ondrej; Gelbíčová, Tereza; Mašlaňová, Ivana; Doškař, Jiří; Zdráhal, Zbyněk; Růžičková, Vladislava; Sedláček, Ivo

    2013-03-01

    Thirteen coagulase-negative, oxidase-negative, and novobiocin-susceptible staphylococci were isolated from human clinical specimens. The isolates were differentiated from known staphylococcal species on the basis of 16S rRNA, hsp60, rpoB, dnaJ, tuf, and gap gene sequencing, automated ribotyping, (GTG)5-PCR fingerprinting, and MALDI-TOF MS analysis. Phylogenetic analysis based on the 16S rRNA gene sequence indicated phylogenetic relatedness of the analyzed strains to Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus devriesei, and Staphylococcus lugdunensis. DNA-DNA hybridization experiments between representative strains CCM 8418(T), CCM 8421(T), and the closest phylogenetic neighbors confirmed that the isolates represent novel Staphylococcus species, for which the name Staphylococcus petrasii sp. nov. is proposed. Genotypic and phenotypic analyses unambiguously split the strains into two closely related subclusters. Based on the results, two novel subspecies S. petrasii subsp. petrasii subsp. nov. and S. petrasii subsp. croceilyticus subsp. nov. are proposed, with type strains CCM 8418(T) (=CCUG 62727(T)) and CCM 8421(T) (=CCUG 62728(T)), respectively.

  14. Legionella pneumophila serogroup Lansing 3 isolated from a patient with fatal pneumonia, and descriptions of L. pneumophila subsp. pneumophila subsp. nov., L. pneumophila subsp. fraseri subsp. nov., and L. pneumophila subsp. pascullei subsp. nov.

    PubMed Central

    Brenner, D J; Steigerwalt, A G; Epple, P; Bibb, W F; McKinney, R M; Starnes, R W; Colville, J M; Selander, R K; Edelstein, P H; Moss, C W

    1988-01-01

    Previous DNA relatedness and enzyme electrophoretic mobility studies indicated heterogeneity among strains of Legionella pneumophila serogroups 1, 4, 5, and Lansing 3 (a new, as yet unnumbered serogroup). In this study 60 L. pneumophila strains were studied by DNA hybridization (hydroxyapatite method) to assess their genomic relatedness. These strains were also studied biochemically and serologically to determine whether they formed one or more phenotypic groups. DNA relatedness studies identified three groups. DNA group 1 contained the type strain Philadelphia 1 and strains from serogroups 1 through 14 of L. pneumophila. The average relatedness of DNA group 1 strains was 88% at 60 degrees C with 1.1% divergence in related sequences and 85% at 75 degrees C. DNA group 2 contained strain Los Angeles 1, the reference strain of serogroup 4, and strains of serogroups 1, 4, 5, and Lansing 3, an unnumbered serogroup. Average relatedness of DNA group 2 strains was 84% at 60 degrees C with 0.7% divergence and 87% at 75 degrees C. Reciprocal relatedness of DNA groups 1 and 2 was approximately 67% at 60 degrees C with 6.0% divergence and 48% at 75 degrees C. DNA group 3 strains were in serogroup 5. They were 98% related at 60 degrees C with 0.5% divergence and 97% related at 75 degrees C. Reciprocal relatedness of DNA group 3 and DNA group 1 was approximately 74% at 60 degrees C with 5.3% divergence and 43% at 75 degrees C, and reciprocal relatedness of DNA groups 3 and 2 was 66% at 60 degrees C with 5.7% divergence and 55% at 75 degrees C. The DNA groups could not be separated biochemically or serologically or by cell wall fatty acid and isoprenoid quinone composition. Three subspecies of L. pneumophila are proposed to accommodate the three DNA groups: L. pneumophila subsp. pneumophila subsp. nov. for DNA group 1, L. pneumophila subsp. fraseri subsp. nov. for DNA group 2, and pneumophila subsp. pascullei subsp. nov. for DNA group 3. PMID:3053773

  15. Identification of intrinsically metronidazole-resistant clades of Gardnerella vaginalis.

    PubMed

    Schuyler, Jessica A; Mordechai, Eli; Adelson, Martin E; Sobel, Jack D; Gygax, Scott E; Hilbert, David W

    2016-01-01

    Gardnerella vaginalis is associated with bacterial vaginosis (BV), the most common cause of vaginal discharge. Metronidazole is a front-line therapy for BV, and treatment failure and recurrent disease are common problems. Whole-genome sequencing studies have revealed that G. vaginalis has a population structure that consists of 4 clades: clades 1 and 3 are associated with BV, whereas clades 2 and 4 are not. To determine if metronidazole susceptibility is associated with population structure, we analyzed 87 clinical isolates and found that metronidazole resistance (MIC ≥32 μg/mL) was highly associated with clade (P<0.0001), as 14/14 clade 3 isolates (100%) and 22/22 clade 4 isolates (100%) exhibited resistance, compared to only 16/37 clade 1 isolates (35%) and 1/14 clade 2 isolates (7.1%). The identification of intrinsically metronidazole-resistant G. vaginalis clades will facilitate future studies on the relationship between metronidazole resistance and BV treatment failure.

  16. Two new species of Lactarius associated with Alnus acuminata subsp. arguta in Mexico.

    PubMed

    Montoya, Leticia; Bandala, Victor M; Garay, Edith

    2014-01-01

    In pure stands of Alnus acuminata subsp. arguta trees from Sierra Norte de Puebla (central Mexico) two undescribed ectomycorrhizal species of Lactarius were discovered. Distinction of the two new species is based on morphological characters and supported with phylogenetic analyses of the nuclear ribosomal DNA ITS region and part of the gene that encodes for the second largest subunit of RNA polymerase II (rpb2). The phylogenies inferred recovered the two species in different clades strongly supported by posterior probabilities and bootstrap values. The new Lactarius species are recognized as part of the assemblage of ectomycorrhizal fungi associated with Alnus acuminata. Information about these taxa includes the morphological variation achieved along 16 monitories 2010-2013. Descriptions are provided. They are accompanied by photos including SEM photomicrographs of basidiospores and information on differences between them and other related taxa from Europe and the United States.

  17. Phylogenetic Analysis and Polyphasic Characterization of Clavibacter michiganensis Strains Isolated from Tomato Seeds Reveal that Nonpathogenic Strains Are Distinct from C. michiganensis subsp. michiganensis

    PubMed Central

    Durand, Karine; Orgeur, Geoffrey; Balidas, Samuel; Fricot, Céline; Bonneau, Sophie; Quillévéré, Anne; Audusseau, Corinne; Olivier, Valérie; Grimault, Valérie; Mathis, René

    2012-01-01

    The genus Clavibacter comprises one species and five subspecies of plant-pathogenic bacteria, four of which are classified as quarantine organisms due to the high economic threat they pose. Clavibacter michiganensis subsp. michiganensis is one of the most important pathogens of tomato, but the recommended diagnostic tools are not satisfactory due to false-negative and/or -positive results. To provide a robust analysis of the genetic relatedness among a worldwide collection of C. michiganensis subsp. michiganensis strains, relatives (strains from the four other C. michiganensis subspecies), and nonpathogenic Clavibacter-like strains isolated from tomato, we performed multilocus sequence-based analysis and typing (MLSA and MLST) based on six housekeeping genes (atpD, dnaK, gyrB, ppK, recA, and rpoB). We compared this “framework” with phenotypic and genotypic characteristics such as pathogenicity on tomato, reaction to two antisera by immunofluorescence and to five PCR identification tests, and the presence of four genes encoding the main C. michiganensis subsp. michiganensis pathogenicity determinants. We showed that C. michiganensis subsp. michiganensis is monophyletic and is distinct from its closest taxonomic neighbors. The nonpathogenic Clavibacter-like strains were identified as C. michiganensis using 16S rRNA gene sequencing. These strains, while cross-reacting with C. michiganensis subsp. michiganensis identification tools, are phylogenetically distinct from the pathogenic strains but belong to the C. michiganensis clade. C. michiganensis subsp. michiganensis clonal complexes linked strains from highly diverse geographical origins and also strains isolated over long periods of time in the same location. This illustrates the importance of seed transmission in the worldwide dispersion of this pathogen and its survival and adaptation abilities in a new environment once introduced. PMID:23001675

  18. Evidence of sympatry of clade a and clade B head lice in a pre-Columbian Chilean mummy from Camarones.

    PubMed

    Boutellis, Amina; Drali, Rezak; Rivera, Mario A; Mumcuoglu, Kosta Y; Raoult, Didier

    2013-01-01

    Three different lineages of head lice are known to parasitize humans. Clade A, which is currently worldwide in distribution, was previously demonstrated to be present in the Americas before the time of Columbus. The two other types of head lice are geographically restricted to America and Australia for clade B and to Africa and Asia for clade C. In this study, we tested two operculated nits from a 4,000-year-old Chilean mummy of Camarones for the presence of the partial Cytb mitochondrial gene (270 bp). Our finding shows that clade B head lice were present in America before the arrival of the European colonists.

  19. Evidence of Sympatry of Clade A and Clade B Head Lice in a Pre-Columbian Chilean Mummy from Camarones

    PubMed Central

    Boutellis, Amina; Drali, Rezak; Rivera, Mario A.; Mumcuoglu, Kosta Y.; Raoult, Didier

    2013-01-01

    Three different lineages of head lice are known to parasitize humans. Clade A, which is currently worldwide in distribution, was previously demonstrated to be present in the Americas before the time of Columbus. The two other types of head lice are geographically restricted to America and Australia for clade B and to Africa and Asia for clade C. In this study, we tested two operculated nits from a 4,000-year-old Chilean mummy of Camarones for the presence of the partial Cytb mitochondrial gene (270 bp). Our finding shows that clade B head lice were present in America before the arrival of the European colonists. PMID:24204678

  20. Nitrous Oxide Reduction Kinetics Distinguish Bacteria Harboring Clade I NosZ from Those Harboring Clade II NosZ

    PubMed Central

    Nissen, Silke; Park, Doyoung; Sanford, Robert A.

    2016-01-01

    ABSTRACT Bacteria capable of reduction of nitrous oxide (N2O) to N2 separate into clade I and clade II organisms on the basis of nos operon structures and nosZ sequence features. To explore the possible ecological consequences of distinct nos clusters, the growth of bacterial isolates with either clade I (Pseudomonas stutzeri strain DCP-Ps1, Shewanella loihica strain PV-4) or clade II (Dechloromonas aromatica strain RCB, Anaeromyxobacter dehalogenans strain 2CP-C) nosZ with N2O was examined. Growth curves did not reveal trends distinguishing the clade I and clade II organisms tested; however, the growth yields of clade II organisms exceeded those of clade I organisms by 1.5- to 1.8-fold. Further, whole-cell half-saturation constants (Kss) for N2O distinguished clade I from clade II organisms. The apparent Ks values of 0.324 ± 0.078 μM for D. aromatica and 1.34 ± 0.35 μM for A. dehalogenans were significantly lower than the values measured for P. stutzeri (35.5 ± 9.3 μM) and S. loihica (7.07 ± 1.13 μM). Genome sequencing demonstrated that Dechloromonas denitrificans possessed a clade II nosZ gene, and a measured Ks of 1.01 ± 0.18 μM for N2O was consistent with the values determined for the other clade II organisms tested. These observations provide a plausible mechanistic basis for why the relative activity of bacteria with clade I nos operons compared to that of bacteria with clade II nos operons may control N2O emissions and determine a soil's N2O sink capacity. IMPORTANCE Anthropogenic activities, in particular fertilizer application for agricultural production, increase N2O emissions to the atmosphere. N2O is a strong greenhouse gas with ozone destruction potential, and there is concern that nitrogen may become the major driver of climate change. Microbial N2O reductase (NosZ) catalyzes N2O reduction to environmentally benign dinitrogen gas and represents the major N2O sink process. The observation that bacterial groups with clade I nosZ versus those

  1. Automated microfluidically controlled electrochemical biosensor for the rapid and highly sensitive detection of Francisella tularensis.

    PubMed

    Dulay, Samuel B; Gransee, Rainer; Julich, Sandra; Tomaso, Herbert; O'Sullivan, Ciara K

    2014-09-15

    Tularemia is a highly infectious zoonotic disease caused by a Gram-negative coccoid rod bacterium, Francisella tularensis. Tularemia is considered as a life-threatening potential biological warfare agent due to its high virulence, transmission, mortality and simplicity of cultivation. In the work reported here, different electrochemical immunosensor formats for the detection of whole F. tularensis bacteria were developed and their performance compared. An anti-Francisella antibody (FB11) was used for the detection that recognises the lipopolysaccharide found in the outer membrane of the bacteria. In the first approach, gold-supported self-assembled monolayers of a carboxyl terminated bipodal alkanethiol were used to covalently cross-link with the FB11 antibody. In an alternative second approach F(ab) fragments of the FB11 antibody were generated and directly chemisorbed onto the gold electrode surface. The second approach resulted in an increased capture efficiency and higher sensitivity. Detection limits of 4.5 ng/mL for the lipopolysaccharide antigen and 31 bacteria/mL for the F. tularensis bacteria were achieved. Having demonstrated the functionality of the immunosensor, an electrode array was functionalised with the antibody fragment and integrated with microfluidics and housed in a tester set-up that facilitated complete automation of the assay. The only end-user intervention is sample addition, requiring less than one-minute hands-on time. The use of the automated microfluidic set-up not only required much lower reagent volumes but also the required incubation time was considerably reduced and a notable increase of 3-fold in assay sensitivity was achieved with a total assay time from sample addition to read-out of less than 20 min.

  2. Long-range dispersal moved Francisella tularensis into Western Europe from the East.

    PubMed

    Dwibedi, Chinmay; Birdsell, Dawn; Lärkeryd, Adrian; Myrtennäs, Kerstin; Öhrman, Caroline; Nilsson, Elin; Karlsson, Edvin; Hochhalter, Christian; Rivera, Andrew; Maltinsky, Sara; Bayer, Brittany; Keim, Paul; Scholz, Holger C; Tomaso, Herbert; Wittwer, Matthias; Beuret, Christian; Schuerch, Nadia; Pilo, Paola; Hernández Pérez, Marta; Rodriguez-Lazaro, David; Escudero, Raquel; Anda, Pedro; Forsman, Mats; Wagner, David M; Larsson, Pär; Johansson, Anders

    2016-12-01

    For many infections transmitting to humans from reservoirs in nature, disease dispersal patterns over space and time are largely unknown. Here, a reversed genomics approach helped us understand disease dispersal and yielded insight into evolution and biological properties of Francisella tularensis, the bacterium causing tularemia. We whole-genome sequenced 67 strains and characterized by single-nucleotide polymorphism assays 138 strains, collected from individuals infected 1947-2012 across Western Europe. We used the data for phylogenetic, population genetic and geographical network analyses. All strains (n=205) belonged to a monophyletic population of recent ancestry not found outside Western Europe. Most strains (n=195) throughout the study area were assigned to a star-like phylogenetic pattern indicating that colonization of Western Europe occurred via clonal expansion. In the East of the study area, strains were more diverse, consistent with a founder population spreading from east to west. The relationship of genetic and geographic distance within the F. tularensis population was complex and indicated multiple long-distance dispersal events. Mutation rate estimates based on year of isolation indicated null rates; in outbreak hotspots only, there was a rate of 0.4 mutations/genome/year. Patterns of nucleotide substitution showed marked AT mutational bias suggestive of genetic drift. These results demonstrate that tularemia has moved from east to west in Europe and that F. tularensis has a biology characterized by long-range geographical dispersal events and mostly slow, but variable, replication rates. The results indicate that mutation-driven evolution, a resting survival phase, genetic drift and long-distance geographical dispersal events have interacted to generate genetic diversity within this species.

  3. Long-range dispersal moved Francisella tularensis into Western Europe from the East

    PubMed Central

    Dwibedi, Chinmay; Birdsell, Dawn; Lärkeryd, Adrian; Myrtennäs, Kerstin; Öhrman, Caroline; Nilsson, Elin; Karlsson, Edvin; Hochhalter, Christian; Rivera, Andrew; Maltinsky, Sara; Bayer, Brittany; Keim, Paul; Scholz, Holger C.; Tomaso, Herbert; Wittwer, Matthias; Beuret, Christian; Schuerch, Nadia; Pilo, Paola; Hernández Pérez, Marta; Rodriguez-Lazaro, David; Escudero, Raquel; Anda, Pedro; Forsman, Mats; Wagner, David M.; Larsson, Pär

    2016-01-01

    For many infections transmitting to humans from reservoirs in nature, disease dispersal patterns over space and time are largely unknown. Here, a reversed genomics approach helped us understand disease dispersal and yielded insight into evolution and biological properties of Francisella tularensis, the bacterium causing tularemia. We whole-genome sequenced 67 strains and characterized by single-nucleotide polymorphism assays 138 strains, collected from individuals infected 1947-2012 across Western Europe. We used the data for phylogenetic, population genetic and geographical network analyses. All strains (n=205) belonged to a monophyletic population of recent ancestry not found outside Western Europe. Most strains (n=195) throughout the study area were assigned to a star-like phylogenetic pattern indicating that colonization of Western Europe occurred via clonal expansion. In the East of the study area, strains were more diverse, consistent with a founder population spreading from east to west. The relationship of genetic and geographic distance within the F. tularensis population was complex and indicated multiple long-distance dispersal events. Mutation rate estimates based on year of isolation indicated null rates; in outbreak hotspots only, there was a rate of 0.4 mutations/genome/year. Patterns of nucleotide substitution showed marked AT mutational bias suggestive of genetic drift. These results demonstrate that tularemia has moved from east to west in Europe and that F. tularensis has a biology characterized by long-range geographical dispersal events and mostly slow, but variable, replication rates. The results indicate that mutation-driven evolution, a resting survival phase, genetic drift and long-distance geographical dispersal events have interacted to generate genetic diversity within this species. PMID:28348839

  4. Aged mice display an altered pulmonary host response to Francisella tularensis live vaccine strain (LVS) infections

    PubMed Central

    CA, Mares; SS, Ojeda; Q, Li; EG, Morris; JJ, Coalson; JM, Teale

    2012-01-01

    Aging is a complex phenomenon that has been shown to affect many organ systems including the innate and adaptive immune systems. The current study was designed to examine the potential effect of immunosenescence on the pulmonary immune response using a Francisella tularensis live vaccine strain (LVS) inhalation infection model. F. tularensis is a gram-negative intracellular pathogen that can cause a severe pneumonia.In this study both young (8-12 week old) and aged (20-24 month old) mice were infected intranasally with LVS. Lung tissues from young and aged mice were used to assess pathology, recruitment of immune cell types and cytokine expression levels at various times post infection. Bacterial burdens were also assessed. Interestingly, the lungs of aged animals harbored fewer organisms at early time points of infection (day 1, day 3) compared with their younger counterparts. In addition, only aged animals displayed small perivascular aggregates at these early time points that appeared mostly mononuclear in nature. However, the kinetics of infiltrating polymorphonuclear neutrophils (PMNs) and increased cytokine levels measured in the bronchial alveolar lavage fluid (BALF) were delayed in infected aged animals relative to young infected animals with neutrophils appearing at day 5 post infection (PI) in the aged animals as opposed to day 3 PI in the young infected animals. Also evident were alterations in the ratios of mononuclear to PMNs at distinct post infection times. The above evidence indicates that aged mice elicit an altered immune response in the lung to respiratory Francisella tularensis LVS infections compared to their younger counterparts. PMID:19825409

  5. Nonrandom Distribution of Vector Ticks (Dermacentor variabilis) Infected by Francisella tularensis

    PubMed Central

    Goethert, Heidi K.; Telford, Sam R.

    2009-01-01

    The island of Martha's Vineyard, Massachusetts, is the site of a sustained outbreak of tularemia due to Francisella tularensis tularensis. Dog ticks, Dermacentor variabilis, appear to be critical in the perpetuation of the agent there. Tularemia has long been characterized as an agent of natural focality, stably persisting in characteristic sites of transmission, but this suggestion has never been rigorously tested. Accordingly, we sought to identify a natural focus of transmission of the agent of tularemia by mapping the distribution of PCR-positive ticks. From 2004 to 2007, questing D. variabilis were collected from 85 individual waypoints along a 1.5 km transect in a field site on Martha's Vineyard. The positions of PCR-positive ticks were then mapped using ArcGIS. Cluster analysis identified an area approximately 290 meters in diameter, 9 waypoints, that was significantly more likely to yield PCR-positive ticks (relative risk 3.3, P = 0.001) than the rest of the field site. Genotyping of F. tularensis using variable number tandem repeat (VNTR) analysis on PCR-positive ticks yielded 13 different haplotypes, the vast majority of which was one dominant haplotype. Positive ticks collected in the cluster were 3.4 times (relative risk = 3.4, P<0.0001) more likely to have an uncommon haplotype than those collected elsewhere from the transect. We conclude that we have identified a microfocus where the agent of tularemia stably perpetuates and that this area is where genetic diversity is generated. PMID:19247435

  6. Bacillus anthracis, Francisella tularensis and Yersinia pestis. The most important bacterial warfare agents - review.

    PubMed

    Pohanka, M; Skládal, P

    2009-01-01

    There are three most important bacterial causative agents of serious infections that could be misused for warfare purposes: Bacillus anthracis (the causative agent of anthrax) is the most frequently mentioned one; however, Fracisella tularensis (causing tularemia) and Yersinia pestis (the causative agent of plague) are further bacterial agents enlisted by Centers for Disease Control and Prevention into the category A of potential biological weapons. This review intends to summarize basic information about these bacterial agents. Military aspects of their pathogenesis and the detection techniques suitable for field use are discussed.

  7. The Longibrachiatum Clade of Trichoderma: a revision with new species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Longibrachiatum Clade of Trichoderma is revised. Eight new species are described (T. aethiopicum, T. capillare, T. flagellatum, T. gillesii, T. gracile, T. pinnatum, T. saturnisporopsis, T. solani). The twenty-one species known to belong to the Longibrachiatum Clade are included in a synoptic ke...

  8. The first closed genome sequence of Campylobacter fetus subsp. venerealis biovar intermedius

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Campylobacter fetus venerealis biovar intermedius is a variant of Campylobacter fetus subsp. venerealis, the causative agent of Bovine Genital Campylobacteriosis. In contrast to Campylobacter fetus subsp. venerealis which is restricted to the genital tract of cattle, Campylobacter fetus subsp. vener...

  9. Major clades of Agaricales: a multilocus phylogenetic overview.

    PubMed

    Matheny, P Brandon; Curtis, Judd M; Hofstetter, Valérie; Aime, M Catherine; Moncalvo, Jean-Marc; Ge, Zai-Wei; Slot, Jason C; Ammirati, Joseph F; Baroni, Timothy J; Bougher, Neale L; Hughes, Karen W; Lodge, D Jean; Kerrigan, Richard W; Seidl, Michelle T; Aanen, Duur K; DeNitis, Matthew; Daniele, Graciela M; Desjardin, Dennis E; Kropp, Bradley R; Norvell, Lorelei L; Parker, Andrew; Vellinga, Else C; Vilgalys, Rytas; Hibbett, David S

    2006-01-01

    An overview of the phylogeny of the Agaricales is presented based on a multilocus analysis of a six-gene region supermatrix. Bayesian analyses of 5611 nucleotide characters of rpb1, rpb1-intron 2, rpb2 and 18S, 25S, and 5.8S ribosomal RNA genes recovered six major clades, which are recognized informally and labeled the Agaricoid, Tricholomatoid, Marasmioid, Pluteoid, Hygrophoroid and Plicaturopsidoid clades. Each clade is discussed in terms of key morphological and ecological traits. At least 11 origins of the ectomycorrhizal habit appear to have evolved in the Agaricales, with possibly as many as nine origins in the Agaricoid plus Tricholomatoid clade alone. A family-based phylogenetic classification is sketched for the Agaricales, in which 30 families, four unplaced tribes and two informally named clades are recognized.

  10. An easy, simple inexpensive test for the specific detection of Pectobacterium carotovorum subsp. carotovorum based on sequence analysis of the pmrA gene

    PubMed Central

    2013-01-01

    Background The species Pectobacterium carotovorum includes a diverse subspecies of bacteria that cause disease on a wide variety of plants. In Morocco, approximately 95% of the P. carotovorum isolates from potato plants with tuber soft rot are P. carotovorum subsp. carotovorum. However, identification of this pathogen is not always related to visual disease symptoms. This is especially true when different pathogen cause similar diseases on potato, citing as an example, P. carotovorum, P. atrosepticum and P. wasabiae. Numerous conventional methods were used to characterize Pectobacterium spp., including biochemical assays, specific PCR-based tests, and construction of phylogenetic trees by using gene sequences. In this study, an alternative method is presented using a gene linked to pathogenicity, in order to allow accuracy at subspecies level. The pmrA gene (response regulator) has been used for identification and analysis of the relationships among twenty nine Pectobacterium carotovorum subsp. carotovorum and other Pectobacterium subspecies. Results Phylogenetic analyses of pmrA sequences compared to ERIC-PCR and 16S rDNA sequencing, demonstrated that there is considerable genetic diversity in P. carotovorum subsp. carotovorum strains, which can be divided into two distinct groups within the same clade. Conclusions pmrA sequence analysis is likely to be a reliable tool to identify the subspecies Pectobacterium carotovorum subsp. carotovorum and estimate their genetic diversity. PMID:23890050

  11. Staphylococcus petrasii subsp. pragensis subsp. nov., occurring in human clinical material.

    PubMed

    Švec, Pavel; De Bel, Annelies; Sedláček, Ivo; Petráš, Petr; Gelbíčová, Tereza; Černohlávková, Jitka; Mašlanˇová, Ivana; Cnockaert, Margo; Varbanovová, Ivana; Echahidi, Fedoua; Vandamme, Peter; Pantuček, Roman

    2015-07-01

    Seven coagulase-negative, oxidase-negative and novobiocin-susceptible staphylococci assigned tentatively as Staphylococcus petrasii were investigated in this study in order to elucidate their taxonomic position. All strains were initially shown to form a genetically homogeneous group separated from remaining species of the genus Staphylococcus by using a repetitive sequence-based PCR fingerprinting with the (GTG)5 primer. Phylogenetic analysis based on 16S rRNA gene, hsp60, rpoB, dnaJ, gap and tuf sequences showed that the group is closely related to Staphylococcus petrasii but separated from the three hitherto known subspecies, S. petrasii subsp. petrasii, S. petrasii subsp. croceilyticus and S. petrasii subsp. jettensis. Further investigation using automated ribotyping, MALDI-TOF mass spectrometry, fatty acid methyl ester analysis, DNA-DNA hybridization and extensive biotyping confirmed that the analysed group represents a novel subspecies within S. petrasii, for which the name Staphylococcus petrasii subsp. pragensis subsp. nov. is proposed. The type strain is NRL/St 12/356(T) ( = CCM 8529(T) = LMG 28327(T)).

  12. Antibacterial Activity of Alkyl Gallates against Xanthomonas citri subsp. citri

    PubMed Central

    Silva, I. C.; Regasini, L. O.; Petrônio, M. S.; Silva, D. H. S.; Bolzani, V. S.; Belasque, J.; Sacramento, L. V. S.

    2013-01-01

    The plant-pathogenic bacterium Xanthomonas citri subsp. citri is the causal agent of Asiatic citrus canker, a serious disease that affects all the cultivars of citrus in subtropical citrus-producing areas worldwide. There is no curative treatment for citrus canker; thus, the eradication of infected plants constitutes the only effective control of the spread of X. citri subsp. citri. Since the eradication program in the state of São Paulo, Brazil, is under threat, there is a clear risk of X. citri subsp. citri becoming endemic in the main orange-producing area in the world. Here we evaluated the potential use of alkyl gallates to prevent X. citri subsp. citri growth. These esters displayed a potent anti-X. citri subsp. citri activity similar to that of kanamycin (positive control), as evaluated by the resazurin microtiter assay (REMA). The treatment of X. citri subsp. citri cells with these compounds induced altered cell morphology, and investigations of the possible intracellular targets using X. citri subsp. citri strains labeled for the septum and centromere pointed to a common target involved in chromosome segregation and cell division. Finally, the artificial inoculation of citrus with X. citri subsp. citri cells pretreated with alkyl gallates showed that the bacterium loses the ability to colonize its host, which indicates the potential of these esters to protect citrus plants against X. citri subsp. citri infection. PMID:23104804

  13. Fragrance components of Platanthera bifolia subsp. osca.

    PubMed

    D'Auria, Maurizio; Lorenz, Richard; Racioppi, Rocco; Romano, Vito Antonio

    2017-02-10

    SPME-GC-MS analysis of the scent of Platanthera bifolia subsp. osca collected during the night showed as main components lilac alcohols B, C and D and lilac aldehydes A, B and C. Other significant chemical components were linalool and caryophyllene. Some differences were found in comparison with previously reported analyses of the scent of P. bifolia and Platanthera chlorantha. The most important difference found was in the composition of the ester fraction.

  14. Sensitivity of Francisella tularensis to ultrapure water and deoxycholate: implications for bacterial intracellular growth assay in macrophages

    PubMed Central

    Chalabaev, Sabina; Anderson, Christine A.; Onderdonk, Andrew B.; Kasper, Dennis L.

    2011-01-01

    The ability of Francisella tularensis to replicate in macrophages is critical for its pathogenesis, therefore intracellular growth assays are important tools for assessing virulence. We show that two lysis solutions commonly used in these assays, deionized water and deoxycholate in PBS, lead to highly inaccurate measurements of intracellular bacterial survival. PMID:21420447

  15. CpG Oligodeoxyribonucleotides Protect Mice from Burkholderia Pseudomallei but not Francisella Tularensis Schu S4 Aerosols

    DTIC Science & Technology

    2010-01-01

    David A Rozak1*, Herbert C Gelhaus1,3, Mark Smith2, Mojgan Zadeh1,4, Louis Huzella2, David Waag1, Jeffrey J Adamovicz1,5 Abstract Studies have shown...tularensis LPS, and contributes to F. novicida murine pathogenesis. Microbes and infection/ Institut Pasteur 2003, 5(5):397-403. 12. Forsman M, Sandstrom G

  16. NMR Structure of Francisella tularensis Virulence Determinant Reveals Structural Homology to Bet v1 Allergen Proteins.

    PubMed

    Zook, James; Mo, Gina; Sisco, Nicholas J; Craciunescu, Felicia M; Hansen, Debra T; Baravati, Bobby; Cherry, Brian R; Sykes, Kathryn; Wachter, Rebekka; Van Horn, Wade D; Fromme, Petra

    2015-06-02

    Tularemia is a potentially fatal bacterial infection caused by Francisella tularensis, and is endemic to North America and many parts of northern Europe and Asia. The outer membrane lipoprotein, Flpp3, has been identified as a virulence determinant as well as a potential subunit template for vaccine development. Here we present the first structure for the soluble domain of Flpp3 from the highly infectious Type A SCHU S4 strain, derived through high-resolution solution nuclear magnetic resonance (NMR) spectroscopy; the first structure of a lipoprotein from the genus Francisella. The Flpp3 structure demonstrates a globular protein with an electrostatically polarized surface containing an internal cavity-a putative binding site based on the structurally homologous Bet v1 protein family of allergens. NMR-based relaxation studies suggest loop regions that potentially modulate access to the internal cavity. The Flpp3 structure may add to the understanding of F. tularensis virulence and contribute to the development of effective vaccines.

  17. Characterization of Francisella tularensis Schu S4 defined mutants as live-attenuated vaccine candidates.

    PubMed

    Santiago, Araceli E; Mann, Barbara J; Qin, Aiping; Cunningham, Aimee L; Cole, Leah E; Grassel, Christen; Vogel, Stefanie N; Levine, Myron M; Barry, Eileen M

    2015-08-01

    Francisella tularensis (Ft), the etiological agent of tularemia and a Tier 1 select agent, has been previously weaponized and remains a high priority for vaccine development. Ft tularensis (type A) and Ft holarctica (type B) cause most human disease. We selected six attenuating genes from the live vaccine strain (LVS; type B), F. novicida and other intracellular bacteria: FTT0507, FTT0584, FTT0742, FTT1019c (guaA), FTT1043 (mip) and FTT1317c (guaB) and created unmarked deletion mutants of each in the highly human virulent Ft strain Schu S4 (Type A) background. FTT0507, FTT0584, FTT0742 and FTT1043 Schu S4 mutants were not attenuated for virulence in vitro or in vivo. In contrast, Schu S4 gua mutants were unable to replicate in murine macrophages and were attenuated in vivo, with an i.n. LD50 > 10(5) CFU in C57BL/6 mice. However, the gua mutants failed to protect mice against lethal challenge with WT Schu S4, despite demonstrating partial protection in rabbits in a previous study. These results contrast with the highly protective capacity of LVS gua mutants against a lethal LVS challenge in mice, and underscore differences between these strains and the animal models in which they are evaluated, and therefore have important implications for vaccine development.

  18. Serosurveillance for Francisella tularensis among wild animals in Japan using a newly developed competitive enzyme-linked immunosorbent assay.

    PubMed

    Sharma, Neekun; Hotta, Akitoyo; Yamamoto, Yoshie; Uda, Akihiko; Fujita, Osamu; Mizoguchi, Toshio; Shindo, Junji; Park, Chun-Ho; Kudo, Noboru; Hatai, Hitoshi; Oyamada, Toshifumi; Yamada, Akio; Morikawa, Shigeru; Tanabayashi, Kiyoshi

    2014-04-01

    Tularemia, a highly infectious zoonotic disease caused by Francisella tularensis, occurs sporadically in Japan. However, little is known about the prevalence of the disease in wild animals. A total of 632 samples obtained from 150 Japanese black bears, 142 Japanese hares, 120 small rodents, 97 rats, 53 raptors, 26 Japanese monkeys, 21 Japanese raccoon dogs, 20 masked palm civets, and three Japanese red foxes between 2002 and 2010 were investigated for the presence of antibodies to F. tularensis by competitive enzyme-linked immunosorbent assay (cELISA) and the commonly used microagglutination (MA) test. Seropositive cELISA and MA results were obtained in 23 and 18 Japanese black bears, three and two Japanese raccoon dogs, and two and one small rodents, respectively. All MA-positive samples (n=21) were also positive by cELISA. Six of seven samples that were only positive by cELISA were confirmed to be antibody-positive by western blot analysis. These findings suggest that cELISA is a highly sensitive and useful test for serosurveillance of tularemia among various species of wild animals. Because this is the first study to detect F. tularensis-seropositive Japanese raccoon dogs, these could join Japanese black bears as sentinel animals for tularemia in the wild in Japan. Further continuous serosurveillance for F. tularensis in various species of wild animals using appropriate methods such as cELISA is important to assess the risks of human exposure and to improve our understanding of the ecology of F. tularensis in the wild.

  19. EmrA1 Membrane Fusion Protein of Francisella tularensis LVS is required for Resistance to Oxidative Stress, Intramacrophage Survival and Virulence in Mice

    PubMed Central

    Ma, Zhuo; Banik, Sukalyani; Rane, Harshita; Mora, Vanessa T.; Rabadi, Seham M.; Doyle, Christopher R.; Thanassi, David G.; Bakshi, Chandra Shekhar; Malik, Meenakshi

    2014-01-01

    Francisella tularensis is a Category A Biodefense agent that causes a fatal human disease known as tularemia. The pathogenicity of F. tularensis depends on its ability to persist inside host immune cells primarily by resisting an attack from host-generated reactive oxygen and nitrogen species (ROS/RNS). Based on the ability of F. tularensis to resist high ROS/RNS levels, we have hypothesized that additional unknown factors act in conjunction with known antioxidant defenses to render ROS resistance. By screening a transposon insertion library of F. tularensis LVS in the presence of hydrogen peroxide, we have identified an oxidant sensitive mutant in putative EmrA1 (FTL_0687) secretion protein. The results demonstrate that the emrA1 mutant is highly sensitive to oxidants and several antimicrobial agents, and exhibits diminished intramacrophage growth that can be restored to wild type F. tularensis LVS levels either by transcomplementation, inhibition of ROS generation, or infection in NADPH oxidase deficient (gp91Phox−/−) macrophages. The emrA1 mutant is attenuated for virulence, which is restored by infection in gp91Phox−/− mice. Further, EmrA1 contributes to oxidative stress resistance by affecting secretion of Francisella antioxidant enzymes SodB and KatG. This study exposes unique links between transporter activity and the antioxidant defense mechanisms of F. tularensis. PMID:24397487

  20. Seroprevalence for Coxiella burnetii, Francisella tularensis, Brucella abortus and Brucella melitensis in Austrian adults: a cross-sectional survey among military personnel and civilians.

    PubMed

    Tobudic, Selma; Nedomansky, Klara; Poeppl, Wolfgang; Müller, Maria; Faas, Angelus; Mooseder, Gerhard; Allerberger, Franz; Stanek, Gerold; Burgmann, Heinz

    2014-04-01

    The prevalence of Coxiella burnetii, Francisella tularensis, Brucella abortus, and Brucella melitensis infections in Austria and the exposure risk of military personnel were assessed in an exploratory nationwide cross-sectional seroprevalence survey in 526 healthy adult individuals, 222 of which were soldiers and 304 were civilians. Screening for IgA/IgG antibodies to C. burnetii (Phase I) and IgG/IgM antibodies to C. burnetii (Phase II), and to F. tularensis was done with commercial enzyme-linked immunosorbent assays. To detect antibodies against B. abortus and B. melitensis, an in-house complement fixation test was used. Overall, 11 individuals (2.0%) showed antibodies to C. burnetii, 3 individuals (0.5%) were seropositive for F. tularensis, and one (0.3%) individual was borderline positive. All individuals positive or borderline for F. tularensis tested negative for antibodies against C. burnetii. All individuals tested negative for antibodies against B. melitensis/B. abortus. There were no significant differences between the seroprevalence of C. burnetii and F. tularensis among military personnel and civilians. Our data demonstrate serological evidence of a low rate of exposure to C. burnetii and F. tularensis among the Austrian adult population and military personnel.

  1. Hymenochaetales: a molecular phylogeny for the hymenochaetoid clade.

    PubMed

    Larsson, Karl-Henrik; Parmasto, Erast; Fischer, Michael; Langer, Ewald; Nakasone, Karen K; Redhead, Scott A

    2006-01-01

    The hymenochaetoid clade is dominated by wood-decaying species previously classified in the artificial families Corticiaceae, Polyporaceae and Stereaceae. The majority of these species cause a white rot. The polypore Bridgeoporus and several corticicoid species with inconspicuous basidiomata live in association with brown-rotted wood, but their nutritional strategy is not known. Mycorrhizal habit is reported for Coltricia perennis but needs confirmttion. A surprising element in the hymenochaetoid clade is a group of small white to brightly pigmented agarics earlier classified in Omphalina. They form a subclade together with some similarly colored stipitate stereoid and corticioid species. Several are associated with living mosses or one-celled green algae. Hyphoderma pratermissum and some related corticioid species have specialized organs for trapping and killing nematodes as a source of nitrogen. There are no unequivocal morphological synapomorphies known for the hymenochaetoid clade. However almost all species examined ultrastructurally have dolipore septa with continuous parenthesomes while perforate parenthesomes is the normal condition for other homobasidiomycete clades. The agaricoid Hymenochaetales have not been examined. Within Hymenochaetales the Hymenochaetaceae forms a distinct clade but unfortunately all morphological characters supporting Hymenochaetaceae also are found in species outside the clade. Other subclades recovered by the molecular phylogenetic analyses are less uniform, and the overall resolution within the nuclear LSU tree presented here is still unsatisfactory.

  2. The hominin fossil record: taxa, grades and clades

    PubMed Central

    Wood, Bernard; Lonergan, Nicholas

    2008-01-01

    This paper begins by reviewing the fossil evidence for human evolution. It presents summaries of each of the taxa recognized in a relatively speciose hominin taxonomy. These taxa are grouped in grades, namely possible and probable hominins, archaic hominins, megadont archaic hominins, transitional hominins, pre-modern Homo and anatomically modern Homo. The second part of this contribution considers some of the controversies that surround hominin taxonomy and systematics. The first is the vexed question of how you tell an early hominin from an early panin, or from taxa belonging to an extinct clade closely related to the Pan-Homo clade. Secondly, we consider how many species should be recognized within the hominin fossil record, and review the philosophies and methods used to identify taxa within the hominin fossil record. Thirdly, we examine how relationships within the hominin clade are investigated, including descriptions of the methods used to break down an integrated structure into tractable analytical units, and then how cladograms are generated and compared. We then review the internal structure of the hominin clade, including the problem of how many subclades should be recognized within the hominin clade, and we examine the reliability of hominin cladistic hypotheses. The last part of the paper reviews the concepts of a genus, including the criteria that should be used for recognizing genera within the hominin clade. PMID:18380861

  3. FmvB: A Francisella tularensis Magnesium-Responsive Outer Membrane Protein that Plays a Role in Virulence

    PubMed Central

    Wu, Xiaojun; Ren, Guoping; Gunning, William T.; Weaver, David A.; Kalinoski, Andrea L.; Khuder, Sadik A.; Huntley, Jason F.

    2016-01-01

    Francisella tularensis is the causative agent of the lethal disease tularemia. Despite decades of research, little is understood about why F. tularensis is so virulent. Bacterial outer membrane proteins (OMPs) are involved in various virulence processes, including protein secretion, host cell attachment, and intracellular survival. Many pathogenic bacteria require metals for intracellular survival and OMPs often play important roles in metal uptake. Previous studies identified three F. tularensis OMPs that play roles in iron acquisition. In this study, we examined two previously uncharacterized proteins, FTT0267 (named fmvA, for Francisella metal and virulence) and FTT0602c (fmvB), which are homologs of the previously studied F. tularensis iron acquisition genes and are predicted OMPs. To study the potential roles of FmvA and FmvB in metal acquisition and virulence, we first examined fmvA and fmvB expression following pulmonary infection of mice, finding that fmvB was upregulated up to 5-fold during F. tularensis infection of mice. Despite sequence homology to previously-characterized iron-acquisition genes, FmvA and FmvB do not appear to be involved iron uptake, as neither fmvA nor fmvB were upregulated in iron-limiting media and neither ΔfmvA nor ΔfmvB exhibited growth defects in iron limitation. However, when other metals were examined in this study, magnesium-limitation significantly induced fmvB expression, ΔfmvB was found to express significantly higher levels of lipopolysaccharide (LPS) in magnesium-limiting medium, and increased numbers of surface protrusions were observed on ΔfmvB in magnesium-limiting medium, compared to wild-type F. tularensis grown in magnesium-limiting medium. RNA sequencing analysis of ΔfmvB revealed the potential mechanism for increased LPS expression, as LPS synthesis genes kdtA and wbtA were significantly upregulated in ΔfmvB, compared with wild-type F. tularensis. To provide further evidence for the potential role of FmvB in

  4. Complete genome sequences of Campylobacter hyointestinalis subsp. hyointestinalis strain LMG9260 and Campylobacter hyointestinalis subsp. lawsonii strain LMG15993

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Campylobacter hyointestinalis is isolated primarily from ruminants and swine, but is also occasionally isolated from humans. C. hyointestinalis is currently divided into two subspecies: subsps. hyointestinalis and lawsonii. This study describes the first closed whole-genome sequences of the subsp. h...

  5. Flavonoids from Aconitum napellus subsp. neomontanum.

    PubMed

    Fico, G; Braca, A; De Tommasi, N; Tomè, F; Morelli, I

    2001-06-01

    Three flavonol glycosides quercetin 7-O-(6-trans-caffeoyl)-beta-glucopyranosyl-(1-->3)-alpha-rhamnopyranoside-3-O-beta-glucopyranoside (1), kaempferol 7-O-(6-trans-caffeoyl)-beta-glucopyranosyl-(1-->3)-alpha-rhamnopyranoside-3-O-beta-glucopyranoside (2), and kaempferol 7-O-(6-trans-p-coumaroyl)-beta-glucopyranosyl-(1-->3)-alpha-rhamnopyranoside-3-O-beta-glucopyranoside (3), together with the known beta-3,4-dihydroxyphenethyl beta-glucopyranoside, were isolated from the flowers of Aconitum napellus subsp. neomontanum. Their structures were elucidated by spectroscopic methods, including 2D NMR spectral techniques.

  6. Update on Streptococcus equi subsp equi infections.

    PubMed

    Mallicote, Martha

    2015-04-01

    There are few diseases that ignite as much fervor among horse owners as strangles. Streptococcus equi subsp equi (strangles) infections frequently require the treating veterinarian to manage not only the clinical cases but also the biosecurity and provision of information to all involved parties. Although the disease is typically characterized by low mortality and high morbidity, restrictions of horse movement that result from appropriate quarantine procedures often frustrate the involved parties. The aims of this article are to provide clinically relevant information for diagnosis, treatment, and biosecurity management of strangles infection.

  7. Proposal to rename Carnobacterium inhibens as Carnobacterium inhibens subsp. inhibens subsp. nov. and description of Carnobacterium inhibens subsp. gilichinskyi subsp. nov., a psychrotolerant bacterium isolated from Siberian permafrost.

    PubMed

    Nicholson, Wayne L; Zhalnina, Kateryna; de Oliveira, Rafael R; Triplett, Eric W

    2015-02-01

    A novel, psychrotolerant facultative anaerobe, strain WN1359(T), was isolated from a permafrost borehole sample collected at the right bank of the Kolyma River in Siberia, Russia. Gram-positive-staining, non-motile, rod-shaped cells were observed with sizes of 1-2 µm long and 0.4-0.5 µm wide. Growth occurred in the range of pH 5.8-9.0 with optimal growth at pH 7.8-8.6 (pH optimum 8.2). The novel isolate grew at temperatures from 0-37 °C and optimal growth occurred at 25 °C. The novel isolate does not require NaCl; growth was observed between 0 and 8.8 % (1.5 M) NaCl with optimal growth at 0.5 % (w/v) NaCl. The isolate was a catalase-negative, facultatively anaerobic chemo-organoheterotroph that used sugars but not several single amino acids or dipeptides as substrates. The major metabolic end-product was lactic acid in the ratio of 86 % l-lactate : 14 % d-lactate. Strain WN1359(T) was sensitive to ampicillin, chloramphenicol, fusidic acid, lincomycin, monocycline, rifampicin, rifamycin SV, spectinomycin, streptomycin, troleandomycin and vancomycin, and resistant to nalidixic acid and aztreonam. The fatty acid content was predominantly unsaturated (70.2 %), branched-chain unsaturated (11.7 %) and saturated (12.5 %). The DNA G+C content was 35.3 mol% by whole genome sequence analysis. 16S rRNA gene sequence analysis showed 98.7 % sequence identity between strain WN1359(T) and Carnobacterium inhibens. Genome relatedness was computed using both Genome-to-Genome Distance Analysis (GGDA) and Average Nucleotide Identity (ANI), which both strongly supported strain WN1359(T) belonging to the species C. inhibens. On the basis of these results, the permafrost isolate WN1359(T) represents a novel subspecies of C. inhibens, for which the name Carnobacterium inhibens subsp. gilichinskyi subsp. nov. is proposed. The type strain is WN1359(T) ( = ATCC BAA-2557(T) = DSM 27470(T)). The subspecies Carnobacterium inhibens subsp. inhibens subsp. nov. is created automatically. An

  8. Francisella philomiragia comb. nov. (formerly Yersinia philomiragia) and Francisella tularensis biogroup novicida (formerly Francisella novicida) associated with human disease.

    PubMed Central

    Hollis, D G; Weaver, R E; Steigerwalt, A G; Wenger, J D; Moss, C W; Brenner, D J

    1989-01-01

    Over a 12-year period, 16 human strains of a gram-negative, catalase-positive, halophilic, aerobic, nonmotile, small coccoid bacterium were received for identification. On the bases of biochemical characteristics and cellular fatty acid profiles, 14 of these strains were similar to the "Philomiragia" bacterium (Yersinia philomiragia, species incertae sedis). Additional characteristics were growth on Thayer-Martin agar but no growth or sparse, delayed growth on MacConkey agar; oxidase positive; acid production, often weak and delayed, from D-glucose, sucrose, and maltose; urease negative; no reduction of nitrates; and H2S produced but often delayed in triple sugar iron agar. Both the human isolates and the "Philomiragia" bacterium contained C10:0, C14:0, C16:0, C18:1 omega 9c, C18:0, 3-OH C18:0, C22:0, and C24:1 as major cellular fatty acids and ubiquinone eight (Q8) as the major isoprenoid quinone. These cellular acids in these relative amounts have been found previously only in Francisella tularensis and Francisella novicida, suggesting a relationship between the "Philomiragia" bacterium and Francisella species. Of the 14 human "Philomiragia"-like isolates, 9 were from blood, 3 were from lung biopsies or pleural fluid, and one each was from peritoneal fluid and cerebrospinal fluid. DNA relatedness studies (hydroxyapatite method, 50 and 65 degrees C) showed that these 14 strains were a single group that was the same species as the "Philomiragia" bacterium. Two other human strains were oxidase negative and H2S negative. They formed a single DNA relatedness group that was indistinguishable from the type strains of both F. tularensis and F. novicida. DNA relatedness of "Philomiragia" bacterium type and other strains to strains of F. novicida and F. tularensis, including the type strains, was 35 to 46%. One of the two F. novicida- and F. tularensis-like strains was isolated from blood, and the other was isolated from a cervical lymph node. On the basis of these

  9. 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

  10. Electrochemiluminescence (ECL) immunosensor for detection of Francisella tularensis on screen-printed gold electrode array.

    PubMed

    Spehar-Délèze, Anna-Maria; Julich, Sandra; Gransee, Rainer; Tomaso, Herbert; Dulay, Samuel B; O'Sullivan, Ciara K

    2016-10-01

    An electrochemiluminescence (ECL) immunosensor for the rapid detection of the Francisella tularensis pathogen using whole antibodies or antibody fragments as capture biomolecule is described. A sandwich immunoassay was used with either lipopolysaccharide (LPS) or the whole inactivated bacterial cell (LVS) as a target, while Ru(bpy)3 (2+)-encapsulated silicate nanoparticles were linked to the secondary antibody and used as ECL labels. The assay was performed in a fluidic chip housed in a custom-built black box incorporating electronics, optics and fluidics. The obtained limit of detection for LPS was 0.4 ng/mL, while for the LVS it was 70 and 45 bacteria/mL when the capturing molecule was the whole antibody and the antibody F(ab) fragment, respectively.

  11. Large Scale Structural Rearrangement of a Serine Hydrolase from Francisella tularensis Facilitates Catalysis*

    PubMed Central

    Filippova, Ekaterina V.; Weston, Leigh A.; Kuhn, Misty L.; Geissler, Brett; Gehring, Alexandra M.; Armoush, Nicola; Adkins, Chinessa T.; Minasov, George; Dubrovska, Ievgeniia; Shuvalova, Ludmilla; Winsor, James R.; Lavis, Luke D.; Satchell, Karla J. F.; Becker, Daniel P.; Anderson, Wayne F.; Johnson, R. Jeremy

    2013-01-01

    Tularemia is a deadly, febrile disease caused by infection by the Gram-negative bacterium, Francisella tularensis. Members of the ubiquitous serine hydrolase protein family are among current targets to treat diverse bacterial infections. Herein we present a structural and functional study of a novel bacterial carboxylesterase (FTT258) from F. tularensis, a homologue of human acyl protein thioesterase (hAPT1). The structure of FTT258 has been determined in multiple forms, and unexpectedly large conformational changes of a peripheral flexible loop occur in the presence of a mechanistic cyclobutanone ligand. The concomitant changes in this hydrophobic loop and the newly exposed hydrophobic substrate binding pocket suggest that the observed structural changes are essential to the biological function and catalytic activity of FTT258. Using diverse substrate libraries, site-directed mutagenesis, and liposome binding assays, we determined the importance of these structural changes to the catalytic activity and membrane binding activity of FTT258. Residues within the newly exposed hydrophobic binding pocket and within the peripheral flexible loop proved essential to the hydrolytic activity of FTT258, indicating that structural rearrangement is required for catalytic activity. Both FTT258 and hAPT1 also showed significant association with liposomes designed to mimic bacterial or human membranes, respectively, even though similar structural rearrangements for hAPT1 have not been reported. The necessity for acyl protein thioesterases to have maximal catalytic activity near the membrane surface suggests that these conformational changes in the protein may dually regulate catalytic activity and membrane association in bacterial and human homologues. PMID:23430251

  12. Detection of Francisella tularensis within infected mouse tissues by using a hand-held PCR thermocycler.

    PubMed

    Emanuel, Peter A; Bell, Ryan; Dang, Jessica L; McClanahan, Rebecca; David, John C; Burgess, Robert J; Thompson, Joseph; Collins, Lisa; Hadfield, Ted

    2003-02-01

    The diagnosis of human cases of tularemia often relies upon the demonstration of an antibody response to Francisella tularensis or the direct culturing of the bacteria from the patient. Antibody response is not detectable until 2 weeks or more after infection, and culturing requires special media and suspicion of tularemia. In addition, handling live Francisella poses a risk to laboratory personnel due to the highly infectious nature of this pathogen. In an effort to develop a rapid diagnostic assay for tularemia, we investigated the use of TaqMan 5' hydrolysis fluorogenic PCR to detect the organism in tissues of infected mice. Mice were infected to produce respiratory tularemia. The fopA and tul4 genes of F. tularensis were amplified from infected spleen, lung, liver, and kidney tissues sampled over a 5-day period. The samples were analyzed using the laboratory-based Applied Biosystems International 7900 and the Smiths Detection-Edgewood BioSeeq, a hand-held portable fluorescence thermocycler designed for use in the field. A comparison of culturing and PCR for detection of bacteria in infected tissues shows that culturing was more sensitive than PCR. However, the results for culture take 72 h, whereas PCR results were available within 4 h. PCR was able to detect infection in all the tissues tested. Lung tissue showed the earliest response at 2 days when tested with the ABI 7900 and in 3 days when tested with the BioSeeq. The results were in agreement between the ABI 7900 and the BioSeeq when presented with the same sample. Template preparation may account for the loss of sensitivity compared to culturing techniques. The hand-held BioSeeq thermocycler shows promise as an expedient means of forward diagnosis of infection in the field.

  13. Why should we investigate the morphological disparity of plant clades?

    PubMed Central

    Oyston, Jack W.; Hughes, Martin; Gerber, Sylvain; Wills, Matthew A.

    2016-01-01

    Background Disparity refers to the morphological variation in a sample of taxa, and is distinct from diversity or taxonomic richness. Diversity and disparity are fundamentally decoupled; many groups attain high levels of disparity early in their evolution, while diversity is still comparatively low. Diversity may subsequently increase even in the face of static or declining disparity by increasingly fine sub-division of morphological ‘design’ space (morphospace). Many animal clades reached high levels of disparity early in their evolution, but there have been few comparable studies of plant clades, despite their profound ecological and evolutionary importance. This study offers a prospective and some preliminary macroevolutionary analyses. Methods Classical morphometric methods are most suitable when there is reasonable conservation of form, but lose traction where morphological differences become greater (e.g. in comparisons across higher taxa). Discrete character matrices offer one means to compare a greater diversity of forms. This study explores morphospaces derived from eight discrete data sets for major plant clades, and discusses their macroevolutionary implications. Key Results Most of the plant clades in this study show initial, high levels of disparity that approach or attain the maximum levels reached subsequently. These plant clades are characterized by an initial phase of evolution during which most regions of their empirical morphospaces are colonized. Angiosperms, palms, pines and ferns show remarkably little variation in disparity through time. Conifers furnish the most marked exception, appearing at relatively low disparity in the latest Carboniferous, before expanding incrementally with the radiation of successive, tightly clustered constituent sub-clades. Conclusions Many cladistic data sets can be repurposed for investigating the morphological disparity of plant clades through time, and offer insights that are complementary to more focused

  14. Clades reach highest morphological disparity early in their evolution

    PubMed Central

    Hughes, Martin; Gerber, Sylvain; Wills, Matthew Albion

    2013-01-01

    There are few putative macroevolutionary trends or rules that withstand scrutiny. Here, we test and verify the purported tendency for animal clades to reach their maximum morphological variety relatively early in their evolutionary histories (early high disparity). We present a meta-analysis of 98 metazoan clades radiating throughout the Phanerozoic. The disparity profiles of groups through time are summarized in terms of their center of gravity (CG), with values above and below 0.50 indicating top- and bottom-heaviness, respectively. Clades that terminate at one of the “big five” mass extinction events tend to have truncated trajectories, with a significantly top-heavy CG distribution overall. The remaining 63 clades show the opposite tendency, with a significantly bottom-heavy mean CG (relatively early high disparity). Resampling tests are used to identify groups with a CG significantly above or below 0.50; clades not terminating at a mass extinction are three times more likely to be significantly bottom-heavy than top-heavy. Overall, there is no clear temporal trend in disparity profile shapes from the Cambrian to the Recent, and early high disparity is the predominant pattern throughout the Phanerozoic. Our results do not allow us to distinguish between ecological and developmental explanations for this phenomenon. To the extent that ecology has a role, however, the paucity of bottom-heavy clades radiating in the immediate wake of mass extinctions suggests that early high disparity more probably results from the evolution of key apomorphies at the base of clades rather than from physical drivers or catastrophic ecospace clearing. PMID:23884651

  15. Clades reach highest morphological disparity early in their evolution

    NASA Astrophysics Data System (ADS)

    Hughes, Martin; Gerber, Sylvain; Albion Wills, Matthew

    2013-08-01

    There are few putative macroevolutionary trends or rules that withstand scrutiny. Here, we test and verify the purported tendency for animal clades to reach their maximum morphological variety relatively early in their evolutionary histories (early high disparity). We present a meta-analysis of 98 metazoan clades radiating throughout the Phanerozoic. The disparity profiles of groups through time are summarized in terms of their center of gravity (CG), with values above and below 0.50 indicating top- and bottom-heaviness, respectively. Clades that terminate at one of the "big five" mass extinction events tend to have truncated trajectories, with a significantly top-heavy CG distribution overall. The remaining 63 clades show the opposite tendency, with a significantly bottom-heavy mean CG (relatively early high disparity). Resampling tests are used to identify groups with a CG significantly above or below 0.50; clades not terminating at a mass extinction are three times more likely to be significantly bottom-heavy than top-heavy. Overall, there is no clear temporal trend in disparity profile shapes from the Cambrian to the Recent, and early high disparity is the predominant pattern throughout the Phanerozoic. Our results do not allow us to distinguish between ecological and developmental explanations for this phenomenon. To the extent that ecology has a role, however, the paucity of bottom-heavy clades radiating in the immediate wake of mass extinctions suggests that early high disparity more probably results from the evolution of key apomorphies at the base of clades rather than from physical drivers or catastrophic ecospace clearing.

  16. Genome sequencing identifies Listeria fleischmannii subsp. coloradonensis subsp. nov., isolated from a ranch.

    PubMed

    den Bakker, Henk C; Manuel, Clyde S; Fortes, Esther D; Wiedmann, Martin; Nightingale, Kendra K

    2013-09-01

    Twenty Listeria-like isolates were obtained from environmental samples collected on a cattle ranch in northern Colorado; all of these isolates were found to share an identical partial sigB sequence, suggesting close relatedness. The isolates were similar to members of the genus Listeria in that they were Gram-stain-positive, short rods, oxidase-negative and catalase-positive; the isolates were similar to Listeria fleischmannii because they were non-motile at 25 °C. 16S rRNA gene sequencing for representative isolates and whole genome sequencing for one isolate was performed. The genome of the type strain of Listeria fleischmannii (strain LU2006-1(T)) was also sequenced. The draft genomes were very similar in size and the average MUMmer nucleotide identity across 91% of the genomes was 95.16%. Genome sequence data were used to design primers for a six-gene multi-locus sequence analysis (MLSA) scheme. Phylogenies based on (i) the near-complete 16S rRNA gene, (ii) 31 core genes and (iii) six housekeeping genes illustrated the close relationship of these Listeria-like isolates to Listeria fleischmannii LU2006-1(T). Sufficient genetic divergence of the Listeria-like isolates from the type strain of Listeria fleischmannii and differing phenotypic characteristics warrant these isolates to be classified as members of a distinct infraspecific taxon, for which the name Listeria fleischmannii subsp. coloradonensis subsp. nov. is proposed. The type strain is TTU M1-001(T) ( =BAA-2414(T) =DSM 25391(T)). The isolates of Listeria fleischmannii subsp. coloradonensis subsp. nov. differ from the nominate subspecies by the inability to utilize melezitose, turanose and sucrose, and the ability to utilize inositol. The results also demonstrate the utility of whole genome sequencing to facilitate identification of novel taxa within a well-described genus. The genomes of both subspecies of Listeria fleischmannii contained putative enhancin genes; the Listeria fleischmannii subsp

  17. Adaptation of Francisella tularensis to the mammalian environment is governed by cues which can be mimicked in vitro.

    PubMed

    Hazlett, Karsten R O; Caldon, Seth D; McArthur, Debbie G; Cirillo, Kerry A; Kirimanjeswara, Girish S; Magguilli, Micheal L; Malik, Meenakshi; Shah, Aaloki; Broderick, Scott; Golovliov, Igor; Metzger, Dennis W; Rajan, Krishna; Sellati, Timothy J; Loegering, Daniel J

    2008-10-01

    The intracellular bacterium Francisella tularensis survives in mammals, arthropods, and freshwater amoeba. It was previously established that the conventional media used for in vitro propagation of this microbe do not yield bacteria that mimic those harvested from infected mammals; whether these in vitro-cultivated bacteria resemble arthropod- or amoeba-adapted Francisella is unknown. As a foundation for our goal of identifying F. tularensis outer membrane proteins which are expressed during mammalian infection, we first sought to identify in vitro cultivation conditions that induce the bacterium's infection-derived phenotype. We compared Francisella LVS grown in brain heart infusion broth (BHI; a standard microbiological medium rarely used in Francisella research) to that grown in Mueller-Hinton broth (MHB; the most widely used F. tularensis medium, used here as a negative control) and macrophages (a natural host cell, used here as a positive control). BHI- and macrophage-grown F. tularensis cells showed similar expression of MglA-dependent and MglA-independent proteins; expression of the MglA-dependent proteins was repressed by the supraphysiological levels of free amino acids present in MHB. We observed that during macrophage infection, protein expression by intracellular bacteria differed from that by extracellular bacteria; BHI-grown bacteria mirrored the latter, while MHB-grown bacteria resembled neither. Naïve macrophages responding to BHI- and macrophage-grown bacteria produced markedly lower levels of proinflammatory mediators than those in cells exposed to MHB-grown bacteria. In contrast to MHB-grown bacteria, BHI-grown bacteria showed minimal delay during intracellular replication. Cumulatively, our findings provide compelling evidence that growth in BHI yields bacteria which recapitulate the phenotype of Francisella organisms that have emerged from macrophages.

  18. Comparison of Five Commercial DNA Extraction Kits for the Recovery of Francisella Tularensis DNA from Spiked Soil Samples

    DTIC Science & Technology

    2007-01-01

    protozoa. The fact that F. tularensis has been shown to survive in amoebae in the laboratory [8], and has been isolated from natural water sources [9,10...play a role in the maintenance of this bacterium in the environment. Francisella strains are extremely difficult to culture from environmental...sources [12]; however, recent molecular surveys have identified Francisella-like bacteria in soil samples from Houston, TX, Denver, Co [13], and from

  19. Needle-Free Delivery of Acetalated Dextran-Encapsulated AR-12 Protects Mice from Francisella tularensis Lethal Challenge.

    PubMed

    Hoang, Ky V; Curry, Heather; Collier, Michael A; Borteh, Hassan; Bachelder, Eric M; Schlesinger, Larry S; Gunn, John S; Ainslie, Kristy M

    2016-04-01

    Francisella tularensiscauses tularemia and is a potential biothreat. Given the limited antibiotics for treating tularemia and the possible use of antibiotic-resistant strains as a biowarfare agent, new antibacterial agents are needed. AR-12 is an FDA-approved investigational new drug (IND) compound that induces autophagy and has shown host-directed, broad-spectrum activityin vitroagainstSalmonella entericaserovar Typhimurium andF. tularensis We have shown that AR-12 encapsulated within acetalated dextran (Ace-DEX) microparticles (AR-12/MPs) significantly reduces host cell cytotoxicity compared to that with free AR-12, while retaining the ability to controlS.Typhimurium within infected human macrophages. In the present study, the toxicity and efficacy of AR-12/MPs in controlling virulent type AF. tularensisSchuS4 infection were examinedin vitroandin vivo No significant toxicity of blank MPs or AR-12/MPs was observed in lung histology sections when the formulations were given intranasally to uninfected mice. In histology sections from the lungs of intranasally infected mice treated with the formulations, increased macrophage infiltration was observed for AR-12/MPs, with or without suboptimal gentamicin treatment, but not for blank MPs, soluble AR-12, or suboptimal gentamicin alone. AR-12/MPs dramatically reduced the burden ofF. tularensisin infected human macrophages, in a manner similar to that of free AR-12. However,in vivo, AR-12/MPs significantly enhanced the survival ofF. tularensisSchuS4-infected mice compared to that seen with free AR-12. In combination with suboptimal gentamicin treatment, AR-12/MPs further improved the survival ofF. tularensisSchuS4-infected mice. These studies provide support for Ace-DEX-encapsulated AR-12 as a promising new therapeutic agent for tularemia.

  20. Serogrouping of Bacteroides fragilis subsp. fragilis by the agglutination test.

    PubMed Central

    Lambe, D W; Moroz, D A

    1976-01-01

    The agglutination technique was used to establish a serological classification scheme for 98 strains of Bacteroides fragilis subsp. fragilis isolated from clinical specimens and normal human feces. Absorbed antisera were prepared to seven strains of B. fragilis subsp. fragilis. These seven absorbed antisera were species as well as subspecies specific and provided the basis of the serological classification scheme. This scheme was composed of 21 serogroups; seven of these serogroups contained only one group component. There was a total of 45 serological patterns. This serological scheme may be used for the serological classification of strains of B. fragilis subsp. fragilis and to study the epidemiology of this organism. PMID:950378

  1. Lack of OxyR and KatG Results in Extreme Susceptibility of Francisella tularensis LVS to Oxidative Stress and Marked Attenuation In vivo

    PubMed Central

    Honn, Marie; Lindgren, Helena; Bharath, Gurram K.; Sjöstedt, Anders

    2017-01-01

    Francisella tularensis is an intracellular bacterium and as such is expected to encounter a continuous attack by reactive oxygen species (ROS) in its intracellular habitat and efficiently coping with oxidative stress is therefore essential for its survival. The oxidative stress response system of F. tularensis is complex and includes multiple antioxidant enzymes and pathways, including the transcriptional regulator OxyR and the H2O2-decomposing enzyme catalase, encoded by katG. The latter is regulated by OxyR. A deletion of either of these genes, however, does not severely compromise the virulence of F. tularensis and we hypothesized that if the bacterium would be deficient of both catalase and OxyR, then the oxidative defense and virulence of F. tularensis would become severely hampered. To test this hypothesis, we generated a double deletion mutant, ΔoxyR/ΔkatG, of F. tularensis LVS and compared its phenotype to the parental LVS strain and the corresponding single deletion mutants. In accordance with the hypothesis, ΔoxyR/ΔkatG was distinctly more susceptible than ΔoxyR and ΔkatG to H2O2, ONOO−, and O2-, moreover, it hardly grew in mouse-derived BMDM or in mice, whereas ΔkatG and ΔoxyR grew as well as F. tularensis LVS in BMDM and exhibited only slight attenuation in mice. Altogether, the results demonstrate the importance of catalase and OxyR for a robust oxidative stress defense system and that they act cooperatively. The lack of both functions render F. tularensis severely crippled to handle oxidative stress and also much attenuated for intracellular growth and virulence. PMID:28174696

  2. Data supporting phylogenetic reconstructions of the Neotropical clade Gymnotiformes

    PubMed Central

    Tagliacollo, Victor A.; Bernt, Maxwell J.; Craig, Jack M.; Oliveira, Claudio; Albert, James S.

    2016-01-01

    Data is presented in support of model-based total evidence (MBTE) phylogenetic reconstructions of the Neotropical clade of Gymnotiformes “Model-based total evidence phylogeny of Neotropical electric knifefishes (Teleostei, Gymnotiformes)” (Tagliacollo et al., 2016) [1]). The MBTE phylogenies were inferred using a comprehensive dataset comprised of six genes (5277 bp) and 223 morphological characters for an ingroup taxon sample of 120 of 218 valid species and 33 of the 34 extant genera. The data in this article include primer sequences for gene amplification and sequencing, voucher information and GenBank accession numbers, descriptions of morphological characters, morphological synapomorphies for the recognized clades of Gymnotiformes, a supermatrix comprised of concatenated molecular and morphological data, and computer scripts to replicate MBTE inferences. We also included here Maximum-likelihood and Bayesian topologies, which support two main gymnotiform clades: Gymnotidae and Sternopygoidei, the latter comprised of Rhamphichthyoidea (Rhamphichthyidae+Hypopomidae) and Sinusoidea (Sternopygidae+Apteronotidae). PMID:26955648

  3. Monitoring biothreat agents (Francisella tularensis, Bacillus anthracis and Yersinia pestis) with a portable real-time PCR instrument.

    PubMed

    Mölsä, Markos; Hemmilä, Heidi; Katz, Anna; Niemimaa, Jukka; Forbes, Kristian M; Huitu, Otso; Stuart, Peter; Henttonen, Heikki; Nikkari, Simo

    2015-08-01

    In the event of suspected releases or natural outbreaks of contagious pathogens, rapid identification of the infectious agent is essential for appropriate medical intervention and disease containment. The purpose of this study was to compare the performance of a novel portable real-time PCR thermocycler, PikoReal™, to the standard real-time PCR thermocycler, Applied Biosystems® 7300 (ABI 7300), for the detection of three high-risk biothreat bacterial pathogens: Francisella tularensis, Bacillus anthracis and Yersinia pestis. In addition, a novel confirmatory real-time PCR assay for the detection of F. tularensis is presented and validated. The results show that sensitivity of the assays, based on a dilution series, for the three infectious agents ranged from 1 to 100 fg of target DNA with both instruments. No cross-reactivity was revealed in specificity testing. Duration of the assays with the PikoReal and ABI 7300 systems were 50 and 100 min, respectively. In field testing for F. tularensis, results were obtained with the PikoReal system in 95 min, as the pre-PCR preparation, including DNA extraction, required an additional 45 min. We conclude that the PikoReal system enables highly sensitive and rapid on-site detection of biothreat agents under field conditions, and may be a more efficient alternative to conventional diagnostic methods.

  4. Genetic engineering of Francisella tularensis LVS for use as a novel live vaccine platform against Pseudomonas aeruginosa infections.

    PubMed

    Robinson, Cory M; Kobe, Brianna N; Schmitt, Deanna M; Phair, Brian; Gilson, Tricia; Jung, Joo-Yong; Roberts, Lawton; Liao, Jialin; Camerlengo, Chelsea; Chang, Brandon; Davis, Mackenzie; Figurski, Leah; Sindeldecker, Devin; Horzempa, Joseph

    2015-01-01

    Francisella tularensis LVS (Live Vaccine Strain) is an attenuated bacterium that has been used as a live vaccine. Patients immunized with this organism show a very long-term memory response (over 30 years post vaccination) evidenced by the presence of indicators of robust cell-mediated immunity. Because F. tularensis LVS is such a potent vaccine, we hypothesized that this organism would be an effective vaccine platform. First, we sought to determine if we could genetically modify this strain to produce protective antigens of a heterologous pathogen. Currently, there is not a licensed vaccine against the important opportunistic bacterial pathogen, Pseudomonas aeruginosa. Because many P. aeruginosa strains are also drug resistant, the need for effective vaccines is magnified. Here, F. tularensis LVS was genetically modified to express surface proteins PilAPa, OprFPa, and FliCPa of P. aeruginosa. Immunization of mice with LVS expressing the recombinant FliCPa led to a significant production of antibodies specific for P. aeruginosa. However, mice that had been immunized with LVS expressing PilAPa or OprFPa did not produce high levels of antibodies specific for P. aerugionsa. Therefore, the recombinant LVS strain engineered to produce FliCPa may be able to provide immune protection against a P. aeruginosa challenge. However for future use of this vaccine platform, selection of the appropriate recombinant antigen is critical as not all recombinant antigens expressed in this strain were immunogenic.

  5. Francisella tularensis modulates a distinct subset of regulatory factors and sustains mitochondrial integrity to impair human neutrophil apoptosis

    PubMed Central

    McCracken, Jenna M.; Kinkead, Lauren C.; McCaffrey, Ramona L.; Allen, Lee-Ann H.

    2016-01-01

    Tularemia is a disease characterized by profound neutrophil accumulation and tissue destruction. The causative organism, Francisella tularensis, is a facultative intracellular bacterium that replicates in neutrophil cytosol, inhibits caspase activation, and profoundly prolongs cell lifespan. Herein we identify unique features of this infection and provide fundamental insight into the mechanisms of apoptosis inhibition. Mitochondria are critical regulators of neutrophil apoptosis. We demonstrate that F. tularensis significantly inhibits Bax translocation and Bid processing through 24–48 h of infection, and in this manner sustains mitochondrial integrity. Downstream of mitochondria, XIAP and PCNA inhibit caspase-9 and caspase-3 by direct binding. Notably, we find that PCNA disappeared rapidly and selectively from infected cells, thereby demonstrating that it is not essential for neutrophil survival, whereas upregulation of calpastatin correlated with diminished calpain activity and reduced XIAP degradation. In addition, R-roscovitine is a cyclin-dependent kinase inhibitor developed for treatment of cancer that also induces neutrophil apoptosis and can promote resolution of several infectious and inflammatory disorders. Herein we confirm the ability of R-roscovitine to induce neutrophil apoptosis, yet also demonstrate that its efficacy is significantly impaired by F. tularensis. Collectively, our findings advance understanding of neutrophil apoptosis and its capacity to be manipulated by pathogenic bacteria. PMID:26906922

  6. Prior Inoculation with Type B Strains of Francisella tularensis Provides Partial Protection against Virulent Type A Strains in Cottontail Rabbits

    PubMed Central

    Brown, Vienna R.; Adney, Danielle R.; Olea-Popelka, Francisco; Bowen, Richard A.

    2015-01-01

    Francisella tularensis is a highly virulent bacterium that is capable of causing severe disease (tularemia) in a wide range of species. This organism is characterized into two distinct subspecies: tularensis (type A) and holarctica (type B) which vary in several crucial ways, with some type A strains having been found to be considerably more virulent in humans and laboratory animals. Cottontail rabbits have been widely implicated as a reservoir species for this subspecies; however, experimental inoculation in our laboratory revealed type A organisms to be highly virulent, resulting in 100% mortality following challenge with 50–100 organisms. Inoculation of cottontail rabbits with the same number of organisms from type B strains of bacteria was found to be rarely lethal and to result in a robust humoral immune response. The objective of this study was to characterize the protection afforded by a prior challenge with type B strains against a later inoculation with a type A strain in North American cottontail rabbits (Sylvilagus spp). Previous infection with a type B strain of organism was found to lengthen survival time and in some cases prevent death following inoculation with a type A2 strain of F. tularensis. In contrast, inoculation of a type A1b strain was uniformly lethal in cottontail rabbits irrespective of a prior type B inoculation. These findings provide important insight about the role cottontail rabbits may play in environmental maintenance and transmission of this organism. PMID:26474413

  7. Genetic engineering of Francisella tularensis LVS for use as a novel live vaccine platform against Pseudomonas aeruginosa infections

    PubMed Central

    Robinson, Cory M; Kobe, Brianna N; Schmitt, Deanna M; Phair, Brian; Gilson, Tricia; Jung, Joo-Yong; Roberts, Lawton; Liao, Jialin; Camerlengo, Chelsea; Chang, Brandon; Davis, Mackenzie; Figurski, Leah; Sindeldecker, Devin; Horzempa, Joseph

    2015-01-01

    Francisella tularensis LVS (Live Vaccine Strain) is an attenuated bacterium that has been used as a live vaccine. Patients immunized with this organism show a very long-term memory response (over 30 years post vaccination) evidenced by the presence of indicators of robust cell-mediated immunity. Because F. tularensis LVS is such a potent vaccine, we hypothesized that this organism would be an effective vaccine platform. First, we sought to determine if we could genetically modify this strain to produce protective antigens of a heterologous pathogen. Currently, there is not a licensed vaccine against the important opportunistic bacterial pathogen, Pseudomonas aeruginosa. Because many P. aeruginosa strains are also drug resistant, the need for effective vaccines is magnified. Here, F. tularensis LVS was genetically modified to express surface proteins PilAPa, OprFPa, and FliCPa of P. aeruginosa. Immunization of mice with LVS expressing the recombinant FliCPa led to a significant production of antibodies specific for P. aeruginosa. However, mice that had been immunized with LVS expressing PilAPa or OprFPa did not produce high levels of antibodies specific for P. aerugionsa. Therefore, the recombinant LVS strain engineered to produce FliCPa may be able to provide immune protection against a P. aeruginosa challenge. However for future use of this vaccine platform, selection of the appropriate recombinant antigen is critical as not all recombinant antigens expressed in this strain were immunogenic. PMID:25617059

  8. Expanding the World of Marine Bacterial and Archaeal Clades

    PubMed Central

    Yilmaz, Pelin; Yarza, Pablo; Rapp, Josephine Z.; Glöckner, Frank O.

    2016-01-01

    Determining which microbial taxa are out there, where they live, and what they are doing is a driving approach in marine microbial ecology. The importance of these questions is underlined by concerted, large-scale, and global ocean sampling initiatives, for example the International Census of Marine Microbes, Ocean Sampling Day, or Tara Oceans. Given decades of effort, we know that the large majority of marine Bacteria and Archaea belong to about a dozen phyla. In addition to the classically culturable Bacteria and Archaea, at least 50 “clades,” at different taxonomic depths, exist. These account for the majority of marine microbial diversity, but there is still an underexplored and less abundant portion remaining. We refer to these hitherto unrecognized clades as unknown, as their boundaries, names, and classifications are not available. In this work, we were able to characterize up to 92 of these unknown clades found within the bacterial and archaeal phylogenetic diversity currently reported for marine water column environments. We mined the SILVA 16S rRNA gene datasets for sequences originating from the marine water column. Instead of the usual subjective taxa delineation and nomenclature methods, we applied the candidate taxonomic unit (CTU) circumscription system, along with a standardized nomenclature to the sequences in newly constructed phylogenetic trees. With this new phylogenetic and taxonomic framework, we performed an analysis of ICoMM rRNA gene amplicon datasets to gain insights into the global distribution of the new marine clades, their ecology, biogeography, and interaction with oceanographic variables. Most of the new clades we identified were interspersed by known taxa with cultivated members, whose genome sequences are available. This result encouraged us to perform metabolic predictions for the novel marine clades using the PICRUSt approach. Our work also provides an update on the taxonomy of several phyla and widely known marine clades as

  9. Draft Genome Sequences of 11 Lactococcus lactis subsp. cremoris Strains

    PubMed Central

    Backus, Lennart; Boekhorst, Jos; Dijkstra, Annereinou; Beerthuyzen, Marke; Siezen, Roland J.; Bachmann, Herwig; van Hijum, Sacha A. F. T.

    2017-01-01

    ABSTRACT The lactic acid bacterium Lactococcus lactis is widely used for the fermentation of dairy products. Here, we present the draft genome sequences of 11 L. lactis subsp. cremoris strains isolated from different environments. PMID:28302789

  10. Fatal pneumonia due to Serratia proteamaculans subsp. quinovora.

    PubMed Central

    Bollet, C; Grimont, P; Gainnier, M; Geissler, A; Sainty, J M; De Micco, P

    1993-01-01

    Serratia proteamaculans subsp. quinovora was isolated from several samples (blood cultures, tracheal aspirates, pleural effusion) from a patient with pneumonia. This is the first clinical isolate and the first documented human infection caused by this organism. PMID:8432835

  11. Mycobacterium avium subsp. paratuberculosis in Veterinary Medicine

    PubMed Central

    Harris, N. Beth; Barletta, Raúl G.

    2001-01-01

    Mycobacterium avium subsp. paratuberculosis (basonym M. paratuberculosis) is the etiologic agent of a severe gastroenteritis in ruminants known as Johne's disease. Economic losses to the cattle industry in the United States are staggering, reaching $1.5 billion annually. A potential pathogenic role in humans in the etiology of Crohn's disease is under investigation. In this article, we review the epidemiology, pathogenesis, diagnostics, and disease control measures of this important veterinary pathogen. We emphasize molecular genetic aspects including the description of markers used for strain identification, diagnostics, and phylogenetic analysis. Recent important advances in the development of animal models and genetic systems to study M. paratuberculosis virulence determinants are also discussed. We conclude with proposals for the applications of these models and recombinant technology to the development of diagnostic, control, and therapeutic measures. PMID:11432810

  12. Sulfitobacter pontiacus subsp. fungiae subsp. nov., Isolated from Coral Fungia seychellensis from Andaman Sea, and Description of Sulfitobacter pontiacus subsp. pontiacus subsp. nov.

    PubMed

    Zachariah, Sherin; Kumari, Prabla; Das, Subrata K

    2017-03-01

    Two closely related aerobic, Gram reaction-negative rod-shaped bacteria (S7-75(T) and S7-80) were isolated from mucus of coral Fungia seychellensis from Andaman Sea, India. Heterotrophic growth on marine agar was observed at 4-35 °C and pH 6.5-10.5; optimum growth occurred at 25-30 °C and pH 7-8. 16 S rRNA sequence analysis confirmed the strains belonged to the genus Sulfitobacter and the two isolates shared more than 99.28% pairwise sequence similarity. DNA-DNA similarity between two isolates S7-75(T) and S7-80 was above 96%. Strain S7-75(T) showed maximum 16S rRNA similarity of 99.64% with Sulfitobacter pontiacus LMG 19752(T). However, DNA-DNA relatedness between strain S7-75(T) and S. pontiacus LMG 19752(T) confirmed the placement of strain S7-75(T) as subspecies under the species S. pontiacus. Further, pulsed-field gel electrophoresis (PFGE), REP-PCR, ERIC-PCR fingerprint patterns and lipid profiles also differentiated strain S7-75(T) from the reference strain of S. pontiacus LMG 19752(T). The DNA G+C content was 59.8 mol%. Q10 was the major respiratory quinone. Based on polyphasic analysis, the isolate S7-75(T) represents a subspecies of S. pontiacus for which the name S. pontiacus subsp. fungiae subsp. nov. is proposed with S7-75(T) (=JCM 31094(T) = LMG 29158(T)) as type strain.

  13. Description of Klebsiella quasipneumoniae sp. nov., isolated from human infections, with two subspecies, Klebsiella quasipneumoniae subsp. quasipneumoniae subsp. nov. and Klebsiella quasipneumoniae subsp. similipneumoniae subsp. nov., and demonstration that Klebsiella singaporensis is a junior heterotypic synonym of Klebsiella variicola.

    PubMed

    Brisse, Sylvain; Passet, Virginie; Grimont, Patrick A D

    2014-09-01

    Strains previously classified as members of Klebsiella pneumoniae phylogroups KpI, KpII-A, KpII-B and KpIII were characterized by 16S rRNA (rrs) gene sequencing, multilocus sequence analysis based on rpoB, fusA, gapA, gyrA and leuS genes, average nucleotide identity and biochemical characteristics. Phylogenetic analysis demonstrated that KpI and KpIII corresponded to K. pneumoniae and Klebsiella variicola, respectively, whereas KpII-A and KpII-B formed two well-demarcated sequence clusters distinct from other members of the genus Klebsiella. Average nucleotide identity between KpII-A and KpII-B was 96.4 %, whereas values lower than 94 % were obtained for both groups when compared with K. pneumoniae and K. variicola. Biochemical properties differentiated KpII-A, KpII-B, K. pneumoniae and K. variicola, with acid production from adonitol and l-sorbose and ability to use 3-phenylproprionate, 5-keto-d-gluconate and tricarballylic acid as sole carbon sources being particularly useful. Based on their genetic and phenotypic characteristics, we propose the names Klebsiella quasipneumoniae subsp. quasipneumoniae subsp. nov. and K. quasipneumoniae subsp. similipneumoniae subsp. nov. for strains of KpII-A and KpII-B, respectively. The type strain of K. quasipneumoniae sp. nov. and of K. quasipneumoniae subsp. quasipneumoniae subsp. nov. is 01A030(T) ( = SB11(T) = CIP 110771(T) = DSM 28211(T)). The type strain of K. quasipneumoniae subsp. similipneumoniae subsp. nov. is 07A044(T) ( = SB30(T) = CIP 110770(T) = DSM 28212(T)). Both strains were isolated from human blood cultures. This work also showed that Klebsiella singaporensis is a junior heterotypic synonym of K. variicola.

  14. Description of two new plasmids isolated from Francisella philomiragia strains and construction of shuttle vectors for the study of Francisella tularensis.

    PubMed

    Le Pihive, E; Blaha, D; Chenavas, S; Thibault, F; Vidal, D; Valade, E

    2009-11-01

    Francisella tularensis is the causative agent of tularemia, a zoonotic disease often transmitted to humans by infected animals. The lack of useful specific genetic tools has long hampered the study of F. tularensis subspecies. We identified and characterized two new plasmids, pF242 and pF243, isolated from Francisella philomiragia strains ATCC 25016 and ATCC 25017, respectively. Sequence analysis revealed that pF242 and pF243 are closely related to pC194 and pFNL10 plasmids, respectively. Two generations of pF242- and pF243-based shuttle vectors, harboring several antibiotic resistance markers, were developed. We used the first generation to compare transformation efficiencies in two virulent F. tularensis subspecies. We found that electroporation was more efficient than cryotransformation: almost all vectors tested were successfully introduced by electroporation into Francisella strains with a high level of efficiency. The second generation of shuttle vectors, containing a multiple cloning site and/or gfp gene downstream of Francisella groES promotor, was used for GFP production in F. tularensis. The development of new shuttle vectors offers new perspectives in the genetic manipulation of F. tularensis, helping to elucidate the mechanisms underlying its virulence.

  15. Use of a capture-based pathogen transcript enrichment strategy for RNA-Seq analysis of the Francisella tularensis LVS transcriptome during infection of murine macrophages.

    PubMed

    Bent, Zachary W; Brazel, David M; Tran-Gyamfi, Mary B; Hamblin, Rachelle Y; VanderNoot, Victoria A; Branda, Steven S

    2013-01-01

    Francisella tularensis is a zoonotic intracellular pathogen that is capable of causing potentially fatal human infections. Like all successful bacterial pathogens, F. tularensis rapidly responds to changes in its environment during infection of host cells, and upon encountering different microenvironments within those cells. This ability to appropriately respond to the challenges of infection requires rapid and global shifts in gene expression patterns. In this study, we use a novel pathogen transcript enrichment strategy and whole transcriptome sequencing (RNA-Seq) to perform a detailed characterization of the rapid and global shifts in F. tularensis LVS gene expression during infection of murine macrophages. We performed differential gene expression analysis on all bacterial genes at two key stages of infection: phagosomal escape, and cytosolic replication. By comparing the F. tularensis transcriptome at these two stages of infection to that of the bacteria grown in culture, we were able to identify sets of genes that are differentially expressed over the course of infection. This analysis revealed the temporally dynamic expression of a number of known and putative transcriptional regulators and virulence factors, providing insight into their role during infection. In addition, we identified several F. tularensis genes that are significantly up-regulated during infection but had not been previously identified as virulence factors. These unknown genes may make attractive therapeutic or vaccine targets.

  16. Phenotypic characterization of the marine pathogen Photobacterium damselae subsp. piscicida.

    PubMed

    Thyssen, A; Grisez, L; van Houdt, R; Ollevier, F

    1998-10-01

    The taxonomic position of Photobacterium damselae subsp. piscicida, the causative agent of fish pasteurellosis, is controversial as this organism has also been described as 'Pasteurella piscicida'. To clarify the taxonomic position of the pathogen, a total of 113 P. damselae subsp. piscicida strains and 20 P. damselae subsp. damselae strains, isolated from different geographical areas and from the main affected fish species, were analysed using 129 morphological and biochemical tests, including the commercial API 20E and API CH50 test systems. For comparison, the type strains of other Photobacterium species (i.e. Photobacterium leiognathi and Photobacterium angustum) were included in the analyses. The results were statistically analysed by unweighted pair group average clustering and the distance between the different clusters was expressed as the percentage disagreement. The analyses showed that, based on morphological and biochemical identification tests, P. damselae subsp. piscicida is related to other Photobacterium species. However, it is clearly distinguishable from P. damselae subsp. damselae and no phenotypic evidence was found to include P. damselae subsp. piscicida as a subspecies in the species P. damselae.

  17. Structural differences in chromosomes distinguish species in the tomato clade.

    PubMed

    Anderson, L K; Covey, P A; Larsen, L R; Bedinger, P; Stack, S M

    2010-07-01

    The tomato clade of Solanaceae is composed of 12 species that are all diploid with the same chromosome number and morphology. Species in the tomato clade are considered to have evolved primarily by genic changes rather than large-scale chromosomal rearrangements because pachytene chromosomes in F(1) hybrids synapse normally along their lengths and linkage maps of intra- and inter-specific hybrids are co-linear. However, small inversions have been reported between tomato and some of its wild relatives. Therefore, we reevaluated 5 F(1) hybrids using high-resolution, electron microscopic examination of pachytene chromosome (= synaptonemal complex) spreads to determine whether any minor structural changes had occurred among species in the tomato clade, which were not easily visible using light microscopic analysis of conventional chromosome squashes. Our study revealed a number of unexpected synaptic configurations such as mismatched kinetochores, inversion loops and reciprocal translocations. Most of these structural differences were in or close to heterochromatin that has comparatively few genes and little recombination, so they would be expected to have little effect on the evident colinearity of linkage maps, especially in euchromatin. However, these results demonstrate that substantial changes in chromosome structure have occurred among species within the tomato clade.

  18. Photosynthetic pigments of oceanic Chlorophyta belonging to prasinophytes clade VII.

    PubMed

    Lopes Dos Santos, Adriana; Gourvil, Priscillia; Rodríguez, Francisco; Garrido, José Luis; Vaulot, Daniel

    2016-02-01

    The ecological importance and diversity of pico/nanoplanktonic algae remains poorly studied in marine waters, in part because many are tiny and without distinctive morphological features. Amongst green algae, Mamiellophyceae such as Micromonas or Bathycoccus are dominant in coastal waters while prasinophytes clade VII, yet not formerly described, appear to be major players in open oceanic waters. The pigment composition of 14 strains representative of different subclades of clade VII was analyzed using a method that improves the separation of loroxanthin and neoxanthin. All the prasinophytes clade VII analyzed here showed a pigment composition similar to that previously reported for RCC287 corresponding to pigment group prasino-2A. However, we detected in addition astaxanthin for which it is the first report in prasinophytes. Among the strains analyzed, the pigment signature is qualitatively similar within subclades A and B. By contrast, RCC3402 from subclade C (Picocystis) lacks loroxanthin, astaxanthin, and antheraxanthin but contains alloxanthin, diatoxanthin, and monadoxanthin that are usually found in diatoms or cryptophytes. For subclades A and B, loroxanthin was lowest at highest light irradiance suggesting a light-harvesting role of this pigment in clade VII as in Tetraselmis.

  19. Phylogeny and biogeography of an uncultured clade of snow chytrids.

    PubMed

    Naff, C S; Darcy, J L; Schmidt, S K

    2013-10-01

    Numerous studies have shown that snow can contain a diverse array of algae known as 'snow algae'. Some reports also indicate that parasites of algae (e.g. chytrids) are also found in snow, but efforts to phylogenetically identify 'snow chytrids' have not been successful. We used culture-independent molecular approaches to phylogenetically identify chytrids that are common in long-lived snowpacks of Colorado and Europe. The most remarkable finding of the present study was the discovery of a new clade of chytrids that has representatives in snowpacks of Colorado and Switzerland and cold sites in Nepal and France, but no representatives from warmer ecosystems. This new clade ('Snow Clade 1' or SC1) is as deeply divergent as its sister clade, the Lobulomycetales, and phylotypes of SC1 show significant (P < 0.003) genetic-isolation by geographic distance patterns, perhaps indicating a long evolutionary history in the cryosphere. In addition to SC1, other snow chytrids were phylogenetically shown to be in the order Rhizophydiales, a group with known algal parasites and saprotrophs. We suggest that these newly discovered snow chytrids are important components of snow ecosystems where they contribute to snow food-web dynamics and the release of nutrients due to their parasitic and saprotrophic activities.

  20. Pasteurella multocida subsp. multocida and P. multocida subsp. septica Differentiation by PCR Fingerprinting and α-Glucosidase Activity

    PubMed Central

    Hunt Gerardo, Sharon; Citron, Diane M.; Claros, Marina C.; Fernandez, Helen T.; Goldstein, Ellie J. C.

    2001-01-01

    Pasteurella multocida is composed of three subspecies that are often differentiated by fermentation of sorbitol and dulcitol. We studied 35 dulcitol-negative P. multocida isolates from infected dog and cat bite wounds, 16 of which yielded weak and/or conflicting fermentation reactions in Andrades sorbitol, thus making it difficult to distinguish between the two dulcitol-negative subspecies of P. multocida, i.e., P. multocida subsp. multocida and P. multocida subsp. septica. All isolates and two control strains were further analyzed using a PCR fingerprinting technique with a single primer (M13 core) and assessed for α-glucosidase (α-Glu) activity. Although the PCR fingerprint patterns and α-Glu activity did not correlate well with the sorbitol fermentation reactions, they did correlate well with each other. All strains identified as P. multocida subsp. septica were positive for α-Glu activity and exhibited the group I PCR fingerprint profile. All strains categorized as P. multocida subsp. multocida displayed either the group II or group III PCR fingerprint profile; 9 of 11 of these isolates were α-Glu negative. These data suggest that both PCR fingerprinting and α-Glu activity provide reliable means for differentiating P. multocida subsp. multocida from P. multocida subsp. septica, particularly in strains that produce weak and/or discrepant sorbitol fermentation reactions. PMID:11427568

  1. Bioavailability and efficacy of levofloxacin against Francisella tularensis in the common marmoset (Callithrix jacchus).

    PubMed

    Nelson, Michelle; Lever, Mark S; Dean, Rachel E; Pearce, Peter C; Stevens, Daniel J; Simpson, Andrew J H

    2010-09-01

    Pharmacokinetic and efficacy studies with levofloxacin were performed in the common marmoset (Callithrix jacchus) model of inhalational tularemia. Plasma levofloxacin pharmacokinetics were determined in six animals in separate single-dose and multidose studies. Plasma drug concentrations were analyzed using liquid chromatography-tandem mass spectrometry-electrospray ionization. On day 7 of a twice-daily dosing regimen of 40 mg/kg, the levofloxacin half-life, maximum concentration, and area under the curve in marmoset plasma were 2.3 h, 20.9 microg/ml, and 81.4 microg/liter/h, respectively. An efficacy study was undertaken using eight treated and two untreated control animals. Marmosets were challenged with a mean of 1.5 x 10(2) CFU of Francisella tularensis by the airborne route. Treated animals were administered 16.5 mg/kg levofloxacin by mouth twice daily, based on the pharmacokinetic parameters, beginning 24 h after challenge. Control animals had a raised core body temperature by 57 h postchallenge and died from infection by day 5. All of the other animals survived, remained afebrile, and lacked overt clinical signs. No bacteria were recovered from the organs of these animals at postmortem after culling at day 24 postchallenge. In conclusion, postexposure prophylaxis with orally administered levofloxacin was efficacious against acute inhalational tularemia in the common marmoset. The marmoset appears to be an appropriate animal model for the evaluation of postexposure therapies.

  2. Characterization of Monoclonal Antibodies to Terminal and Internal O-Antigen Epitopes of Francisella tularensis Lipopolysaccharide

    PubMed Central

    Roche, Marly I.; Lu, Zhaohua; Hui, Julia H.

    2011-01-01

    The lipopolysaccharide (LPS) of Francisella tularensis (Ft), the Gram negative bacterium that causes tularemia, has been shown to be a main protective antigen in mice and humans; we have previously demonstrated that murine anti-Ft LPS IgG2a monoclonal antibodies (MAbs) can protect mice against otherwise lethal intranasal infection with the Ft live vaccine strain (LVS). Here we show that four IgG2a anti-LPS MAbs are specific for the O-polysaccharide (O-antigen [OAg]) of Ft LPS. But whereas three of the MAbs bind to immunodominant repeating internal epitopes, one binds to a unique terminal epitope of Ft OAg. This was deduced from its even binding to both long and short chains of the LPS ladder in Western blots, its rapid decrease in ELISA binding to decreasing solid-phase LPS concentrations, its inability to compete for LPS binding with a representative of the other three MAbs, and its inability to immunoprecipitate OAg despite its superior agglutination titer. Biacore analysis showed the end-binding MAb to have higher bivalent avidity for Ft OAg than the internal-binding MAbs and provided an immunogenicity explanation for the predominance of internal-binding anti-Ft OAg MAbs. These findings demonstrate that non-overlapping epitopes can be targeted by antibodies to Ft OAg, which may inform the design of vaccines and immunotherapies against tularemia. PMID:21466282

  3. Exposure of laboratory workers to Francisella tularensis despite a bioterrorism procedure.

    PubMed

    Shapiro, Daniel S; Schwartz, Donald R

    2002-06-01

    A rapidly fatal case of pulmonary tularemia in a 43-year-old man who was transferred to a tertiary care facility is presented. The microbiology laboratory and autopsy services were not notified of the clinical suspicion of tularemia by the service caring for the patient. Despite having a laboratory bioterrorism procedure in place and adhering to established laboratory protocol, 12 microbiology laboratory employees were exposed to Francisella tularensis and the identification of the organism was delayed due to lack of notification of the laboratory of the clinical suspicion of tularemia. A total of 11 microbiology employees and two persons involved in performing the patient's autopsy received prophylactic doxycycline due to concerns of transmission. None of them developed signs or symptoms of tularemia. One microbiology laboratory employee was pregnant and declined prophylactic antibiotics. As a result of this event, the microbiology laboratory has incorporated flow charts directly into the bench procedures for several highly infectious agents that may be agents of bioterrorism. This should permit more rapid recognition of an isolate for referral to a Level B laboratory for definitive identification and should improve laboratory safety.

  4. Electrochemical detection of Francisella tularensis genomic DNA using solid-phase recombinase polymerase amplification.

    PubMed

    del Río, Jonathan Sabaté; Yehia Adly, Nouran; Acero-Sánchez, Josep Lluis; Henry, Olivier Y F; O'Sullivan, Ciara K

    2014-04-15

    Solid-phase isothermal DNA amplification was performed exploiting the homology protein recombinase A (recA). The system was primarily tested on maleimide activated microtitre plates as a proof-of-concept and later translated to an electrochemical platform. In both cases, forward primer for Francisella tularensis holarctica genomic DNA was surface immobilised via a thiol or an amino moiety and then elongated during the recA mediated amplification, carried out in the presence of specific target sequence and reverse primers. The formation of the subsequent surface tethered amplicons was either colorimetrically or electrochemically monitored using a horseradish peroxidase (HRP)-labelled DNA secondary probe complementary to the elongated strand. The amplification time was optimised to amplify even low amounts of DNA copies in less than an hour at a constant temperature of 37°C, achieving a limit of detection of 1.3×10(-13) M (4×10(6) copies in 50 μL) for the colorimetric assay and 3.3×10(-14) M (2×10(5) copies in 10 μL) for the chronoamperometric assay. The system was demonstrated to be highly specific with negligible cross-reactivity with non-complementary targets or primers.

  5. Immunization with heat-killed Francisella tularensis LVS elicits protective antibody-mediated immunity.

    PubMed

    Lavine, Christy L; Clinton, Shawn R; Angelova-Fischer, Irena; Marion, Tony N; Bina, Xiaowen R; Bina, James E; Whitt, Michael A; Miller, Mark A

    2007-11-01

    Francisella tularensis (FT) has been classified by the CDC as a category A pathogen because of its high virulence and the high mortality rate associated with infection via the aerosol route. Because there is no licensed vaccine available for FT, development of prophylactic and therapeutic regimens for the prevention/treatment of infection is a high priority. In this report, heat-killed FT live vaccine strain (HKLVS) was employed as a vaccine immunogen, either alone or in combination with an adjuvant, and was found to elicit protective immunity against high-dose FT live vaccine strain (FTLVS) challenge. FT-specific antibodies produced in response to immunization with HKLVS alone were subsequently found to completely protect naive mice against high-dose FT challenge in both infection-interference and passive immunization experiments. Additional passive immunization trials employing serum collected from mice immunized with a heat-killed preparation of an O-antigen-deficient transposon mutant of FTLVS (HKLVS-OAg(neg)) yielded similar results. These findings demonstrated that FT-specific antibodies alone can confer immunity against high-dose FTLVS challenge, and they reveal that antibody-mediated protection is not dependent upon production of LPS-specific antibodies.

  6. Francisella Tularensis Blue–Gray Phase Variation Involves Structural Modifications of Lipopolysaccharide O-Antigen, Core and Lipid A and Affects Intramacrophage Survival and Vaccine Efficacy

    PubMed Central

    Soni, Shilpa; Ernst, Robert K.; Muszyński, Artur; Mohapatra, Nrusingh P.; Perry, Malcolm B.; Vinogradov, Evgeny; Carlson, Russell W.; Gunn, John S.

    2010-01-01

    Francisella tularensis is a CDC Category A biological agent and a potential bioterrorist threat. There is no licensed vaccine against tularemia in the United States. A long-standing issue with potential Francisella vaccines is strain phase variation to a gray form that lacks protective capability in animal models. Comparisons of the parental strain (LVS) and a gray variant (LVSG) have identified lipopolysaccharide (LPS) alterations as a primary change. The LPS of the F. tularensis variant strain gains reactivity to F. novicida anti-LPS antibodies, suggesting structural alterations to the O-antigen. However, biochemical and structural analysis of the F. tularensis LVSG and LVS LPS demonstrated that LVSG has less O-antigen but no major O-antigen structural alterations. Additionally, LVSG possesses structural differences in both the core and lipid A regions, the latter being decreased galactosamine modification. Recent work has identified two genes important in adding galactosamine (flmF2 and flmK) to the lipid A. Quantitative real-time PCR showed reduced transcripts of both of these genes in the gray variant when compared to LVS. Loss of flmF2 or flmK caused less frequent phase conversion but did not alter intramacrophage survival or colony morphology. The LVSG strain demonstrated an intramacrophage survival defect in human and rat but not mouse macrophages. Consistent with this result, the LVSG variant demonstrated little change in LD50 in the mouse model of infection. Furthermore, the LVSG strain lacks the protective capacity of F. tularensis LVS against virulent Type A challenge. These data suggest that the LPS of the F. tularensis LVSG phase variant is dramatically altered. Understanding the mechanism of blue to gray phase variation may lead to a way to inhibit this variation, thus making future F. tularensis vaccines more stable and efficacious. PMID:21687776

  7. Comparative evaluation of automated and manual commercial DNA extraction methods for detection of Francisella tularensis DNA from suspensions and spiked swabs by real-time polymerase chain reaction.

    PubMed

    Dauphin, Leslie A; Walker, Roblena E; Petersen, Jeannine M; Bowen, Michael D

    2011-07-01

    This study evaluated commercial automated and manual DNA extraction methods for the isolation of Francisella tularensis DNA suitable for real-time polymerase chain reaction (PCR) analysis from cell suspensions and spiked cotton, foam, and polyester swabs. Two automated methods, the MagNA Pure Compact and the QIAcube, were compared to 4 manual methods, the IT 1-2-3 DNA sample purification kit, the MasterPure Complete DNA and RNA purification kit, the QIAamp DNA blood mini kit, and the UltraClean Microbial DNA isolation kit. The methods were compared using 6 F. tularensis strains representing the 2 subspecies which cause the majority of reported cases of tularemia in humans. Cell viability testing of the DNA extracts showed that all 6 extraction methods efficiently inactivated F. tularensis at concentrations of ≤10⁶ CFU/mL. Real-time PCR analysis using a multitarget 5' nuclease assay for F. tularensis revealed that the PCR sensitivity was equivalent using DNA extracted by the 2 automated methods and the manual MasterPure and QIAamp methods. These 4 methods resulted in significantly better levels of detection from bacterial suspensions and performed equivalently for spiked swab samples than the remaining 2. This study identifies optimal DNA extraction methods for processing swab specimens for the subsequent detection of F. tularensis DNA using real-time PCR assays. Furthermore, the results provide diagnostic laboratories with the option to select from 2 automated DNA extraction methods as suitable alternatives to manual methods for the isolation of DNA from F. tularensis.

  8. An improved Francisella tularensis Live Vaccine Strain (LVS) is well tolerated and highly immunogenic when administered to rabbits in escalating doses using various immunization routes

    PubMed Central

    Pasetti, Marcela F.; Cuberos, Lilian; Horn, Thomas L.; Shearer, Jeffry D.; Matthews, Stephen J.; House, Robert V.; Sztein, Marcelo B.

    2008-01-01

    Tularemia is a severe disease for which there is no licensed vaccine. An attenuated F. tularensis live vaccine strain (LVS) was protective when administered to humans but safety concerns precluded its licensure and use in large scale immunization. An improved F. tularensis LVS preparation was produced under current Good Manufacturing Practice (cGMP) guidelines for evaluation in clinical trials. Preclinical safety, tolerability and immunogenicity were investigated in rabbits that received LVS in escalating doses (1x105 to 1x109 CFU) by the intradermal, subcutaneous or percutaneous (scarification) route. This improved LVS formulation was well tolerated at all doses; no death or adverse clinical signs were observed and necropsies showed no signs of pathology. No live organisms were detected in liver or spleen. Transient local reactogenicity was observed after scarification injection. Erythema and edema developed after intradermal injection in the highest dose cohorts. High levels of F. tularensis-specific IgM, IgG and IgA developed early after immunization, in a dose-dependent fashion. Scarification elicited higher levels of IgA. Antibodies elicited by LVS also recognized F. tularensis Schu-S4 antigens and there was a significant correlation between antibody titers measured against both LVS and Schu-S4. The ELISA titers also correlated closely with those measured by microagglutination. This is the first report describing comprehensive toxicological and immunological studies of F. tularensis LVS in rabbits. This animal model, which closely resembles human disease, proved adequate to assess safety and immunogenicity of F. tularensis vaccine candidates. This new LVS vaccine preparation is being evaluated in human clinical studies. PMID:18308432

  9. Genome-Wide Diversity and Phylogeography of Mycobacterium avium subsp. paratuberculosis in Canadian Dairy Cattle.

    PubMed

    Ahlstrom, Christina; Barkema, Herman W; Stevenson, Karen; Zadoks, Ruth N; Biek, Roman; Kao, Rowland; Trewby, Hannah; Haupstein, Deb; Kelton, David F; Fecteau, Gilles; Labrecque, Olivia; Keefe, Greg P; McKenna, Shawn L B; Tahlan, Kapil; De Buck, Jeroen

    2016-01-01

    Mycobacterium avium subsp. paratuberculosis (MAP) is the causative bacterium of Johne's disease (JD) in ruminants. The control of JD in the dairy industry is challenging, but can be improved with a better understanding of the diversity and distribution of MAP subtypes. Previously established molecular typing techniques used to differentiate MAP have not been sufficiently discriminatory and/or reliable to accurately assess the population structure. In this study, the genetic diversity of 182 MAP isolates representing all Canadian provinces was compared to the known global diversity, using single nucleotide polymorphisms identified through whole genome sequencing. MAP isolates from Canada represented a subset of the known global diversity, as there were global isolates intermingled with Canadian isolates, as well as multiple global subtypes that were not found in Canada. One Type III and six "Bison type" isolates were found in Canada as well as one Type II subtype that represented 86% of all Canadian isolates. Rarefaction estimated larger subtype richness in Québec than in other Canadian provinces using a strict definition of MAP subtypes and lower subtype richness in the Atlantic region using a relaxed definition. Significant phylogeographic clustering was observed at the inter-provincial but not at the intra-provincial level, although most major clades were found in all provinces. The large number of shared subtypes among provinces suggests that cattle movement is a major driver of MAP transmission at the herd level, which is further supported by the lack of spatial clustering on an intra-provincial scale.

  10. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of macrophage growth locus A (MglA) protein from Francisella tularensis

    SciTech Connect

    Subburaman, P.; Austin, B.P.; Shaw, G.X.; Waugh, D.S.; Ji, X.

    2010-11-03

    Francisella tularensis, a potential bioweapon, causes a rare infectious disease called tularemia in humans and animals. The macrophage growth locus A (MglA) protein from F. tularensis associates with RNA polymerase to positively regulate the expression of multiple virulence factors that are required for its survival and replication within macrophages. The MglA protein was overproduced in Escherichia coli, purified and crystallized. The crystals diffracted to 7.5 {angstrom} resolution at the Advanced Photon Source, Argonne National Laboratory and belonged to the hexagonal space group P6{sub 1} or P6{sub 5}, with unit-cell parameters a = b = 125, c = 54 {angstrom}.

  11. Neonatal Mortality in Puppies Due to Bacteremia by Streptococcus dysgalactiae subsp. dysgalactiae

    PubMed Central

    Vela, Ana I.; Falsen, Enevold; Simarro, Isabel; Rollan, Eduardo; Collins, Matthew D.; Domínguez, Lucas; Fernandez-Garayzabal, Jose F.

    2006-01-01

    We report a case of bacteremia in puppies caused by Streptococcus dysgalactiae subsp. dysgalactiae. Identification was achieved by phenotypic and molecular genetic methods. This is the first report of the recovery of S. dysgalactiae subsp. dysgalactiae from dogs. PMID:16455943

  12. Complete Genome Sequence of Type Strain Campylobacter fetus subsp. fetus ATCC 27374

    PubMed Central

    Oliveira, Luciana M.; Resende, Daniela M.; Dorneles, Elaine M. S.; Horácio, Elvira C. A.; Alves, Fernanda L.; Gonçalves, Leilane O.; Tavares, Grace S.; Stynen, Ana Paula R.; Lage, Andrey P.

    2016-01-01

    Campylobacter fetus subsp. fetus is a zoonotic bacterium important for animal and public health. The complete sequencing and annotation of the genome of the type strain C. fetus subsp. fetus ATCC 27374 are reported here. PMID:27979934

  13. Complete Genome Sequence of Type Strain Campylobacter fetus subsp. fetus ATCC 27374.

    PubMed

    Oliveira, Luciana M; Resende, Daniela M; Dorneles, Elaine M S; Horácio, Elvira C A; Alves, Fernanda L; Gonçalves, Leilane O; Tavares, Grace S; Stynen, Ana Paula R; Lage, Andrey P; Ruiz, Jeronimo C

    2016-12-15

    Campylobacter fetus subsp. fetus is a zoonotic bacterium important for animal and public health. The complete sequencing and annotation of the genome of the type strain C. fetus subsp. fetus ATCC 27374 are reported here.

  14. Molecular characterization of virulence genes of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus in equines

    PubMed Central

    Javed, R.; Taku, A. K.; Gangil, Rakhi; Sharma, R. K.

    2016-01-01

    Aim: The aim was to determine the occurrence of streptococci in equines in Jammu (R. S. Pura, Katra), characterization of Streptococci equi subsp. equi and Streptococcus equi subsp. zooepidemicus with respect to their virulence traits and to determine antibiotic sensitivity pattern of virulent Streptococcus isolates. Materials and Methods: A total of 96 samples were collected from both clinically affected animals (exhibiting signs of respiratory tract disease) and apparently healthy animals and were sent to laboratory. The organisms were isolated on Columbia nalidixic acid agar containing 5% sheep blood as well as on sheep blood agar and confirmed by cultural characteristics and biochemical tests. Molecular detection of Streptococcus was done directly from cultures using sodA and seM gene-based polymerase chain reaction (PCR). Antibiogram was performed against five antibiotics such as amoxicillin, penicillin G, streptomycin, rifampicin, and methicillin. Results: During this study, a total 40 streptococcal isolates were obtained out of which 2 isolates were of S. equi subsp. equi, 12 isolates were from S. equi subsp. zooepidemicus. In the PCR-based detection, we revealed amplicons of 235 bp and 679 bp for confirmation of sodA and seM gene, respectively. In antibiogram, two isolates of S. equi subsp. equi were found resistant to penicillin G, and all other isolates were found sensitive to amoxicillin and streptomycin. Conclusion: The majority of streptococcal infections was due to S. equi subsp. Zooepidemicus, and thus was recognized as a potential pathogen of diseases of equines besides S. equi subsp. equi. PMID:27651677

  15. The automation and evaluation of nested clade phylogeographic analysis.

    PubMed

    Panchal, Mahesh; Beaumont, Mark A

    2007-06-01

    Nested clade phylogeographic analysis (NCPA) is a popular method for reconstructing the demographic history of spatially distributed populations from genetic data. Although some parts of the analysis are automated, there is no unique and widely followed algorithm for doing this in its entirety, beginning with the data, and ending with the inferences drawn from the data. This article describes a method that automates NCPA, thereby providing a framework for replicating analyses in an objective way. To do so, a number of decisions need to be made so that the automated implementation is representative of previous analyses. We review how the NCPA procedure has evolved since its inception and conclude that there is scope for some variability in the manual application of NCPA. We apply the automated software to three published datasets previously analyzed manually and replicate many details of the manual analyses, suggesting that the current algorithm is representative of how a typical user will perform NCPA. We simulate a large number of replicate datasets for geographically distributed, but entirely random-mating, populations. These are then analyzed using the automated NCPA algorithm. Results indicate that NCPA tends to give a high frequency of false positives. In our simulations we observe that 14% of the clades give a conclusive inference that a demographic event has occurred, and that 75% of the datasets have at least one clade that gives such an inference. This is mainly due to the generation of multiple statistics per clade, of which only one is required to be significant to apply the inference key. We survey the inferences that have been made in recent publications and show that the most commonly inferred processes (restricted gene flow with isolation by distance and contiguous range expansion) are those that are commonly inferred in our simulations. However, published datasets typically yield a richer set of inferences with NCPA than obtained in our random

  16. Revision of the Maddenia clade of Prunus (Rosaceae)

    PubMed Central

    Wen, Jun; Shi, Wenting

    2012-01-01

    Abstract The Maddenia clade of Prunus L. is monographed based on herbarium and field studies. Four species are currently accepted in this group: Prunus himalayana J.Wen, Prunus hypoleuca (Koehne) J.Wen, Prunus hypoxantha (Koehne) J.Wen, and Prunus gongshanensis J.Wen, with the last described herein as a new species. Maddenia fujianensis Y.T.Chang and Maddenia incisoserrata T.T.Yü & T.C.Ku are treated as synonyms of Prunus hypoleuca. PMID:22577333

  17. Biofilm formation of Francisella noatunensis subsp. orientalis

    USGS Publications Warehouse

    Soto, Esteban; Halliday-Wimmonds, Iona; Francis , Stewart; Kearney, Michael T; Hansen, John D.

    2015-01-01

    Francisella noatunensis subsp. orientalis (Fno) is an emergent fish pathogen in both marine and fresh water environments. The bacterium is suspected to persist in the environment even without the presence of a suitable fish host. In the present study, the influence of different abiotic factors such as salinity and temperature were used to study the biofilm formation of different isolates of Fno including intracellular growth loci C (iglC)and pathogenicity determinant protein A (pdpA) knockout strains. Finally, we compared the susceptibility of planktonic and biofilm to three disinfectants used in the aquaculture and ornamental fish industry, namely Virkon®, bleach and hydrogen peroxide. The data indicates that Fno is capable of producing biofilms within 24 h where both salinity as well as temperature plays a role in the growth and biofilm formation of Fno. Mutations in theiglC or pdpA, both known virulence factors, do not appear to affect the capacity of Fno to produce biofilms, and the minimum inhibitory concentration, and minimum biocidal concentration for the three disinfectants were lower than the minimum biofilm eradication concentration values. This information needs to be taken into account if trying to eradicate the pathogen from aquaculture facilities or aquariums.

  18. Disparate host immunity to Mycobacterium avium subsp. paratuberculosis antigens in calves inoculated with M. avium subsp. paratuberculosis, M. avium subsp. avium, M. kansasii and M. bovis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cross-reactivity of mycobacterial antigens in immune-based diagnostic assays has been a major concern and criticism of current tests for the detection of paratuberculosis. In the present study, host immune responses to antigen preparations of Mycobacterium avium subsp. paratuberculosis (MAP), consis...

  19. Live Attenuated Mutants of Francisella tularensis Protect Rabbits against Aerosol Challenge with a Virulent Type A Strain

    PubMed Central

    Smith, Le'Kneitah P.; Cole, Kelly Stefano; Santiago, Araceli E.; Mann, Barbara J.; Barry, Eileen M.

    2014-01-01

    Francisella tularensis, a Gram-negative bacterium, is the causative agent of tularemia. No licensed vaccine is currently available for protection against tularemia, although an attenuated strain, dubbed the live vaccine strain (LVS), is given to at-risk laboratory personnel as an investigational new drug (IND). In an effort to develop a vaccine that offers better protection, recombinant attenuated derivatives of a virulent type A strain, SCHU S4, were evaluated in New Zealand White (NZW) rabbits. Rabbits vaccinated via scarification with the three attenuated derivatives (SCHU S4 ΔguaBA, ΔaroD, and ΔfipB strains) or with LVS developed a mild fever, but no weight loss was detected. Twenty-one days after vaccination, all vaccinated rabbits were seropositive for IgG to F. tularensis lipopolysaccharide (LPS). Thirty days after vaccination, all rabbits were challenged with aerosolized SCHU S4 at doses ranging from 50 to 500 50% lethal doses (LD50). All rabbits developed fevers and weight loss after challenge, but the severity was greater for mock-vaccinated rabbits. The ΔguaBA and ΔaroD SCHU S4 derivatives provided partial protection against death (27 to 36%) and a prolonged time to death compared to results for the mock-vaccinated group. In contrast, LVS and the ΔfipB strain both prolonged the time to death, but there were no survivors from the challenge. This is the first demonstration of vaccine efficacy against aerosol challenge with virulent type A F. tularensis in a species other than a rodent since the original work with LVS in the 1960s. The ΔguaBA and ΔaroD SCHU S4 derivatives warrant further evaluation and consideration as potential vaccines for tularemia and for identification of immunological correlates of protection. PMID:24614653

  20. The historical biogeography of groupers: Clade diversification patterns and processes.

    PubMed

    Ma, Ka Yan; Craig, Matthew Thomas; Choat, John Howard; van Herwerden, Lynne

    2016-07-01

    Groupers (family Epinephelidae) are a clade of species-rich, biologically diverse reef fishes. Given their ecological variability and widespread distribution across ocean basins, it is important to scrutinize their evolutionary history that underlies present day distributions. This study investigated the patterns and processes by which grouper biodiversity has been generated and what factors have influenced their present day distributions. We reconstructed a robust, time-calibrated molecular phylogeny of Epinephelidae with comprehensive (∼87%) species sampling, whereby diversification rates were estimated and ancestral ranges were reconstructed. Our results indicate that groupers originated in what is now the East Atlantic during the mid-Eocene and diverged successively to form six strongly supported main clades. These clades differ in age (late Oligocene to mid-Miocene), geographic origin (West Atlantic to West Indo-Pacific) and temporal-spatial diversification pattern, ranging from constant rates of diversification to episodes of rapid radiation. Overall, divergence within certain biogeographic regions was most prevalent in groupers, while vicariant divergences were more common in Tropical Atlantic and East Pacific groupers. Our findings reveal that both biological and geographical factors have driven grouper diversification. They also underscore the importance of scrutinizing group-specific patterns to better understand reef fish evolution.

  1. Genome-scale evidence of the nematode-arthropod clade

    PubMed Central

    Dopazo, Hernán; Dopazo, Joaquín

    2005-01-01

    Background The issue of whether coelomates form a single clade, the Coelomata, or whether all animals that moult an exoskeleton (such as the coelomate arthropods and the pseudocoelomate nematodes) form a distinct clade, the Ecdysozoa, is the most puzzling issue in animal systematics and a major open-ended subject in evolutionary biology. Previous single-gene and genome-scale analyses designed to resolve the issue have produced contradictory results. Here we present the first genome-scale phylogenetic evidence that strongly supports the Ecdysozoa hypothesis. Results Through the most extensive phylogenetic analysis carried out to date, the complete genomes of 11 eukaryotic species have been analyzed in order to find homologous sequences derived from 18 human chromosomes. Phylogenetic analysis of datasets showing an increased adjustment to equal evolutionary rates between nematode and arthropod sequences produced a gradual change from support for Coelomata to support for Ecdysozoa. Transition between topologies occurred when fast-evolving sequences of Caenorhabditis elegans were removed. When chordate, nematode and arthropod sequences were constrained to fit equal evolutionary rates, the Ecdysozoa topology was statistically accepted whereas Coelomata was rejected. Conclusions The reliability of a monophyletic group clustering arthropods and nematodes was unequivocally accepted in datasets where traces of the long-branch attraction effect were removed. This is the first phylogenomic evidence to strongly support the 'moulting clade' hypothesis. PMID:15892869

  2. Experimentally induced bovine abortion with Mycoplasma agalactiae subsp bovis.

    PubMed

    Stalheim, O H; Proctor, S J

    1976-08-01

    Two pregnant cows aborted 11 and 18 days after Mycoplasma agalactiae subsp bovis was inoculated into the amniotic fluids. The placentas were retained. The fetuses (approx 100 and 150 days of age) were decomposed; M agalactiae subsp bovis was recovered from several tissues of the fetuses, the placentas, and fetal fluids. The same organism was given by intraperitoneal injection to 2 other pregnant (130 and 180 days, respectively) cows. At necropsy of the latter 36 days later, placentitis was severe; M agalactiae subsp bovis was recovered from the placentas of both cows and from the fetus of 1 cow. Control cows given sterile mycoplasma cultural medium by intraamnion or intraperitoneal injection did not abort and were not infected. When first recovered from the bovine placenta and fetus, M agalactiae subsp bovis grew slowly in liquid medium and assumed bizarre colonial morphology on solidified medium. Colonies were small (0.1 to 0.5 mm) and dark and lacked halos, but they reacted specifically in the direct fluorescent antibody test with equine M agalactiae subsp bovis antiserum. After 1 or 2 subcultures, the isolates grew at a normal rate and displayed their usual colonial morphology.

  3. Potential Transmission Pathways of Streptococcus gallolyticus subsp. gallolyticus

    PubMed Central

    Dumke, Jessika; Hinse, Dennis; Vollmer, Tanja; Schulz, Jochen; Knabbe, Cornelius; Dreier, Jens

    2015-01-01

    Streptococcus gallolyticus subsp. gallolyticus (S. gallolyticus subsp. gallolyticus), a member of group D streptococci, is an inhabitant of the animal and human gastrointestinal tract. Furthermore, it is a facultative pathogen which causes e.g. endocarditis, septicemia and mastitis. S. gallolyticus subsp. gallolyticus may be transmitted either directly or indirectly between animals and humans. However, the transmission routes are an unsolved issue. In this study, we present systematic analyses of an S. gallolyticus subsp. gallolyticus isolate of an infective endocarditis patient in relation to isolates of his laying hen flock. Isolates from pooled droppings of laying hens, pooled dust samples and human blood culture were characterized by using multilocus sequence typing (MLST) and DNA fingerprinting. MLST revealed the same allelic profile of isolates from the human blood culture and from the droppings of laying hens. In addition, these isolates showed clonal identity regarding a similar DNA fingerprinting pattern. For the first time, we received a hint that transmission of S. gallolyticus subsp. gallolyticus between poultry and humans may occur. This raises the question about the zoonotic potential of isolates from poultry and should be considered in future studies. PMID:25978355

  4. Anoxybacillus kamchatkensis subsp. asaccharedens subsp. nov., a thermophilic bacterium isolated from a hot spring in Batman.

    PubMed

    Gul-Guven, Reyhan; Guven, Kemal; Poli, Annarita; Nicolaus, Barbara

    2008-12-01

    A new thermophilic spore-forming strain KG8(T) was isolated from the mud of Taslidere hot spring in Batman. Strain KG8(T) was aerobe, Gram-positive, rod-shaped, motile, occurring in pairs or filamentous. Growth was observed from 35-65 degrees C (optimum 55 degrees C) and at pH 5.5-9.5 (optimum pH 7.5). It was capable of utilizing starch, growth was observed until 3% NaCl (w/v) and it was positive for nitrate reduction. On the basis of 16S rRNA gene sequence similarity, strain KG8(T) was shown to be related most closely to Anoxybacillus species. Chemotaxonomic data (major isoprenoid quinone-menaquinone-7; major fatty acid-iso-C15:0 and iso-C17:0) supported the affiliation of strain KG8(T) to the genus Anoxybacillus. The results of DNA-DNA hybridization, physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain KG8(T). Based on these results we propose assigning a novel subspecies of Anoxybacillus kamchatkensis, to be named Anoxybacillus kamchatkensis subsp. asaccharedens subsp. nov. with the type strain KG8(T) (DSM 18475(T)=CIP 109280(T)).

  5. Tulipa cinnabarina subsp. toprakii (Liliaceae), a new subspecies from southwestern Anatolia

    PubMed Central

    Eker, İsmail; Yıldırım, Hasan; Altıoğlu, Yusuf

    2016-01-01

    Abstract A new subpecies, Tulipa cinnabarina subsp. toprakii subsp. nov. (Liliaceae) from Turkey is described. Diagnostic characters, descriptions, detailed illustrations, geographical distribution, conservation status and ecological observations on the new taxon are provided. It is also compared with the closely related Tulipa cinnabarina subsp. cinnabarina. PMID:27698585

  6. Bartonella vinsonii subsp. berkhoffii in free-ranging white-tailed deer (Odocoileus virginianus).

    PubMed

    Chitwood, M Colter; Maggi, Ricardo G; Kennedy-Stoskopf, Suzanne; Toliver, Marcée; DePerno, Christopher S

    2013-04-01

    Bartonella vinsonii subsp. berkhoffii has not been detected previously in white-tailed deer (Odocoileus virginianus). We tested whole blood from 60 white-tailed deer for Bartonella spp. DNA; three (5%) were positive for Bartonella vinsonii subsp. berkhoffii. This is the first detection of Bartonella vinsonii subsp. berkhoffii in white-tailed deer.

  7. Tulipa cinnabarina subsp. toprakii (Liliaceae), a new subspecies from southwestern Anatolia.

    PubMed

    Eker, İsmail; Yıldırım, Hasan; Altıoğlu, Yusuf

    2016-01-01

    A new subpecies, Tulipa cinnabarina subsp. toprakii subsp. nov. (Liliaceae) from Turkey is described. Diagnostic characters, descriptions, detailed illustrations, geographical distribution, conservation status and ecological observations on the new taxon are provided. It is also compared with the closely related Tulipa cinnabarina subsp. cinnabarina.

  8. [Investigation of the presence of Francisella tularensis by culture, serology and molecular methods in mice of Thrace Region, Turkey].

    PubMed

    Unal Yilmaz, Gülizar; Gurcan, Saban; Ozkan, Beytullah; Karadenizli, Aynur

    2014-04-01

    Tularemia is a disease that has been reported in Turkey since 1936. Although mice are considered to have a role in the transmission of Francisella tularensis to man, this has not been exactly confirmed yet. The aim of this study was to investigate the presence of F. tularensis in mice by using culture, serology and molecular methods. For this purpose, four villages (Edirne-Demirkoy, Kirklareli-Kaynarca, Tekirdag-Muzruplu, Tekirdag-Sinanli) were selected in Thrace Region of Turkey where tularemia cases had been reported previously. A total of 126 live-catch mouse traps were established in warehouses, barns, areas near wells, water tanks and creeks in the villages in December 2012. Traps were kept overnight and the next day the animals collected were identified at species-level. The live-captured mice were anesthetized and their heart blood samples were obtained. Subsequently, liver and spleen tissues were removed from every mouse under aseptic conditions in the class-2 safety cabinet. These tissues were cultivated in Francis medium containing 5% sheep blood, 0.1% cystein, 1% glucose and incubated for seven days in both normal atmosphere and 5% carbondioxide incubator at 37°C. Tularemia microagglutination test was performed by using the sera which were obtained from live-captured mice. Finally, DNAs were isolated from both liver and spleen tissues of mice, and real-time polymerase chain reaction (Tularemia RT-PCR; Public Health Agency of Turkey, Ankara) were performed. In our study, a total of 19 mice were captured and of these 11 were alive. Ten mice were identified as Apodemus flavicollis, seven were Mus macedonicus and two were Mus musculus. There were no Francisella tularensis isolation in the cultures of mice liver and spleen tissues. Serological tests yielded negative results for 10 mice whose serum samples could be obtained. In RT-PCR, positivity were detected in spleen tissues of two mice which were captured from Kaynarca where first tularemia cases in

  9. Host-Adaptation of Francisella tularensis Alters the Bacterium's Surface-Carbohydrates to Hinder Effectors of Innate and Adaptive Immunity

    PubMed Central

    Zarrella, Tiffany M.; Singh, Anju; Bitsaktsis, Constantine; Rahman, Tabassum; Sahay, Bikash; Feustel, Paul J.; Gosselin, Edmund J.; Sellati, Timothy J.; Hazlett, Karsten R. O.

    2011-01-01

    Background The gram-negative bacterium Francisella tularensis survives in arthropods, fresh water amoeba, and mammals with both intracellular and extracellular phases and could reasonably be expected to express distinct phenotypes in these environments. The presence of a capsule on this bacterium has been controversial with some groups finding such a structure while other groups report that no capsule could be identified. Previously we reported in vitro culture conditions for this bacterium which, in contrast to typical methods, yielded a bacterial phenotype that mimics that of the bacterium's mammalian, extracellular phase. Methods/Findings SDS-PAGE and carbohydrate analysis of differentially-cultivated F. tularensis LVS revealed that bacteria displaying the host-adapted phenotype produce both longer polymers of LPS O-antigen (OAg) and additional HMW carbohydrates/glycoproteins that are reduced/absent in non-host-adapted bacteria. Analysis of wildtype and OAg-mutant bacteria indicated that the induced changes in surface carbohydrates involved both OAg and non-OAg species. To assess the impact of these HMW carbohydrates on the access of outer membrane constituents to antibody we used differentially-cultivated bacteria in vitro to immunoprecipitate antibodies directed against outer membrane moieties. We observed that the surface-carbohydrates induced during host–adaptation shield many outer membrane antigens from binding by antibody. Similar assays with normal mouse serum indicate that the induced HMW carbohydrates also impede complement deposition. Using an in vitro macrophage infection assay, we find that the bacterial HMW carbohydrate impedes TLR2-dependent, pro-inflammatory cytokine production by macrophages. Lastly we show that upon host-adaptation, the human-virulent strain, F. tularensis SchuS4 also induces capsule production with the effect of reducing macrophage-activation and accelerating tularemia pathogenesis in mice. Conclusion F. tularensis undergoes

  10. Staphylococcus equorum subsp. linens, subsp. nov., a starter culture component for surface ripened semi-hard cheeses.

    PubMed

    Place, Raymond B; Hiestand, Daniel; Gallmann, Hans Rudolf; Teuber, Michael

    2003-03-01

    Two staphylococcal strains, RP29T and RP33, were isolated from the main microflora of a surface ripened Swiss mountain cheese made from raw milk. These two strains were differentiated from the most closely related species Staphylococcus equorum on the basis of DNA-DNA hybridisation and phenotypic characteristics and are proposed as Staphylococcus equorum subsp. linens subsp. nov. They could be distinguished phenotypically from S. equorum by their sensitivity to all 14 tested antibiotics, especially to novobiocin, their incapability to ferment alpha-D-lactose, maltose, sucrose, D-trehalose, D-xylose, L-arabinose, salicin, D-ribose, D-raffinose, D-mannitol, and D-alanine. The GenBank accession numbers for the reference sequences of the 16S rDNA and the hsp60 gene used in this study are AF527483 and AF527484, respectively. 30 tons of a semi-hard Swiss cheese were produced with Staphylococcus equorum subsp. linens DSM 15097T as starter culture component in addition to Debaryomyces hansenii, Geotrichum candidum, Brevibacterium linens, Corynebacterium casei for surface ripened cheeses. The products were sensorically and hygienically perfect. Therefore, Staphylococcus equorum subsp. linens DSM 15097T can be proposed as starter culture component for surface ripened cheeses without any detected antibiotic resistances. The type strain of Staphylococcus equorum subsp. linens is DSM 15097T (CIP 107656T).

  11. The Trichoderma brevicompactum clade: a new lineage with new species, new peptaibiotics and mycotoxins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A new lineage is recognized in Trichoderma/Hypocrea, the Brevicompactum clade. This clade includes T. brevicompactum and the new species T. arundinaceum, T. turrialbense, T. protrudens and Hypocrea rodmanii. With the exception of H. rodmanii, all members of this clade produce trichothecenes harzian...

  12. Cross-clade neutralization patterns among HIV-1 strains from the six major clades of the pandemic evaluated and compared in two different models.

    PubMed

    Brown, Bruce K; Wieczorek, Lindsay; Sanders-Buell, Eric; Rosa Borges, Andrew; Robb, Merlin L; Birx, Deborah L; Michael, Nelson L; McCutchan, Francine E; Polonis, Victoria R

    2008-06-05

    A panel of paired primary virus isolates and envelope pseudoviruses from sixty strains representing six HIV-1 clades was tested for neutralization using pooled, clade-specific plasma in two prominently utilized neutralization platforms: a primary isolate assay using peripheral blood mononuclear cells (PBMC) and a pseudovirus assay using a reporter epithelial cell line. Using the PMBC assay, pairing of the antibody pool against homologous clade viruses generated the highest geometric mean neutralizing antibody titer in 4 out of 6 clades tested, and neutralization patterns showed numerous examples of reciprocal cross-recognition between antibody and viruses of specific clade pairs. In the pseudovirus assay, cross-clade neutralization was more limited, with fewer distinct cross-clade relationships evident. The clade C antibody pool was broadly cross-reactive, neutralizing the greatest number of viruses in both assays. These data highlight the importance of the neutralization assay format employed and suggest that clade C envelopes merit further evaluation for the elicitation of broadly neutralizing antibodies.

  13. Genome characterisation of the genus Francisella reveals insight into similar evolutionary paths in pathogens of mammals and fish

    PubMed Central

    2012-01-01

    Background Prior to this study, relatively few strains of Francisella had been genome-sequenced. Previously published Francisella genome sequences were largely restricted to the zoonotic agent F. tularensis. Only limited data were available for other members of the Francisella genus, including F. philomiragia, an opportunistic pathogen of humans, F. noatunensis, a serious pathogen of farmed fish, and other less well described endosymbiotic species. Results We determined the phylogenetic relationships of all known Francisella species, including some for which the phylogenetic positions were previously uncertain. The genus Francisella could be divided into two main genetic clades: one included F. tularensis, F. novicida, F. hispaniensis and Wolbachia persica, and another included F. philomiragia and F. noatunensis. Some Francisella species were found to have significant recombination frequencies. However, the fish pathogen F. noatunensis subsp. noatunensis was an exception due to it exhibiting a highly clonal population structure similar to the human pathogen F. tularensis. Conclusions The genus Francisella can be divided into two main genetic clades occupying both terrestrial and marine habitats. However, our analyses suggest that the ancestral Francisella species originated in a marine habitat. The observed genome to genome variation in gene content and IS elements of different species supports the view that similar evolutionary paths of host adaptation developed independently in F. tularensis (infecting mammals) and F. noatunensis subsp. noatunensis (infecting fish). PMID:22727144

  14. IL-12Rβ2 is critical for survival of primary Francisella tularensis LVS infection

    PubMed Central

    Melillo, Amanda A.; Foreman, Oded; Elkins, Karen L.

    2013-01-01

    Using a panel of vaccines that provided different degrees of protection, we previously identified the IL-12 receptor subunit β2 as a mediator, whose relative expression correlated with strength of protection against secondary lethal challenge of vaccinated mice with an intracellular bacterium, the LVS of Francisella tularensis. The present study therefore tested the hypothesis that IL-12Rβ2 is an important mediator in resistance to LVS by directly examining its role during infections. IL-12Rβ2 KO mice were highly susceptible to LVS primary infection, administered i.d. or i.n. The LD50 of LVS infection of KO mice were 2 logs lower than those of WT mice, regardless of route. Five days after infection with LVS, bacterial organ burdens were significantly higher in IL-12Rβ2 KO mice. IL-12Rβ2 KO mice infected with lethal doses of LVS had more severe liver pathology, including significant increases in the liver enzymes ALT and AST. Despite decreased levels of IFN-γ, LVS-vaccinated IL-12Rβ2 KO mice survived large lethal LVS secondary challenge. Consistent with in vivo protection, in vitro intramacrophage LVS growth was well-controlled in cocultures containing WT or IL-12Rβ2 KO LVS-immune splenocytes. Thus, survival of secondary LVS challenge was not strictly dependent on IL-12Rβ2. However, IL-12Rβ2 is important in parenteral and mucosal host resistance to primary LVS infection and in the ability of WT mice to clear LVS infection and serves to restrict liver damage. PMID:23440500

  15. Cytosolic clearance of replication-deficient mutants reveals Francisella tularensis interactions with the autophagic pathway.

    PubMed

    Chong, Audrey; Wehrly, Tara D; Child, Robert; Hansen, Bryan; Hwang, Seungmin; Virgin, Herbert W; Celli, Jean

    2012-09-01

    Cytosolic bacterial pathogens must evade intracellular innate immune recognition and clearance systems such as autophagy to ensure their survival and proliferation. The intracellular cycle of the bacterium Francisella tularensis is characterized by rapid phagosomal escape followed by extensive proliferation in the macrophage cytoplasm. Cytosolic replication, but not phagosomal escape, requires the locus FTT0369c, which encodes the dipA gene (deficient in intracellular replication A). Here, we show that a replication-deficient, ∆dipA mutant of the prototypical SchuS4 strain is eventually captured from the cytosol of murine and human macrophages into double-membrane vacuoles displaying the late endosomal marker, LAMP1, and the autophagy-associated protein, LC3, coinciding with a reduction in viable intracellular bacteria. Capture of SchuS4ΔdipA was not dipA-specific as other replication-deficient bacteria, such as chloramphenicol-treated SchuS4 and a purine auxotroph mutant SchuS4ΔpurMCD, were similarly targeted to autophagic vacuoles. Vacuoles containing replication-deficient bacteria were labeled with ubiquitin and the autophagy receptors SQSTM1/p62 and NBR1, and their formation was decreased in macrophages from either ATG5-, LC3B- or SQSTM1-deficient mice, indicating recognition by the ubiquitin-SQSTM1-LC3 pathway. While a fraction of both the wild-type and the replication-impaired strains were ubiquitinated and recruited SQSTM1, only the replication-defective strains progressed to autophagic capture, suggesting that wild-type Francisella interferes with the autophagic cascade. Survival of replication-deficient strains was not restored in autophagy-deficient macrophages, as these bacteria died in the cytosol prior to autophagic capture. Collectively, our results demonstrate that replication-impaired strains of Francisella are cleared by autophagy, while replication-competent bacteria seem to interfere with autophagic recognition, therefore ensuring survival

  16. Characterization of the O-antigen polymerase (Wzy) of Francisella tularensis.

    PubMed

    Kim, Tae-Hyun; Sebastian, Shite; Pinkham, Jessica T; Ross, Robin A; Blalock, LeeAnn T; Kasper, Dennis L

    2010-09-03

    The O-antigen polymerase of gram-negative bacteria has been difficult to characterize. Herein we report the biochemical and functional characterization of the protein product (Wzy) of the gene annotated as the putative O-antigen polymerase, which is located in the O-antigen biosynthetic locus of Francisella tularensis. In silico analysis (homology searching, hydropathy plotting, and codon usage assessment) strongly suggested that Wzy is an O-antigen polymerase whose function is to catalyze the addition of newly synthesized O-antigen repeating units to a glycolipid consisting of lipid A, inner core polysaccharide, and one repeating unit of the O-polysaccharide (O-PS). To characterize the function of the Wzy protein, a non-polar deletion mutant of wzy was generated by allelic replacement, and the banding pattern of O-PS was observed by immunoblot analysis of whole-cell lysates obtained by SDS-PAGE and stained with an O-PS-specific monoclonal antibody. These immunoblot analyses showed that O-PS of the wzy mutant expresses only one repeating unit of O-antigen. Further biochemical characterization of the subcellular fractions of the wzy mutant demonstrated that (as is characteristic of O-antigen polymerase mutants) the low molecular weight O-antigen accumulates in the periplasm of the mutant. Site-directed mutagenesis based on protein homology and topology, which was carried out to locate a catalytic residue of the protein, showed that modification of specific residues (Gly(176), Asp(177), Gly(323), and Tyr(324)) leads to a loss of O-PS polymerization. Topology models indicate that these amino acids most likely lie in close proximity on the bacterial surface.

  17. B-Cell Epitopes in GroEL of Francisella tularensis

    PubMed Central

    Lu, Zhaohua; Rynkiewicz, Michael J.; Madico, Guillermo; Li, Sheng; Yang, Chiou-Ying; Perkins, Hillary M.; Sompuram, Seshi R.; Kodela, Vani; Liu, Tong; Morris, Timothy; Wang, Daphne; Roche, Marly I.; Seaton, Barbara A.; Sharon, Jacqueline

    2014-01-01

    The chaperonin protein GroEL, also known as heat shock protein 60 (Hsp60), is a prominent antigen in the human and mouse antibody response to the facultative intracellular bacterium Francisella tularensis (Ft), the causative agent of tularemia. In addition to its presumed cytoplasmic location, FtGroEL has been reported to be a potential component of the bacterial surface and to be released from the bacteria. In the current study, 13 IgG2a and one IgG3 mouse monoclonal antibodies (mAbs) specific for FtGroEL were classified into eleven unique groups based on shared VH-VL germline genes, and seven crossblocking profiles revealing at least three non-overlapping epitope areas in competition ELISA. In a mouse model of respiratory tularemia with the highly pathogenic Ft type A strain SchuS4, the Ab64 and N200 IgG2a mAbs, which block each other’s binding to and are sensitive to the same two point mutations in FtGroEL, reduced bacterial burden indicating that they target protective GroEL B-cell epitopes. The Ab64 and N200 epitopes, as well as those of three other mAbs with different crossblocking profiles, Ab53, N3, and N30, were mapped by hydrogen/deuterium exchange–mass spectrometry (DXMS) and visualized on a homology model of FtGroEL. This model was further supported by its experimentally-validated computational docking to the X-ray crystal structures of Ab64 and Ab53 Fabs. The structural analysis and DXMS profiles of the Ab64 and N200 mAbs suggest that their protective effects may be due to induction or stabilization of a conformational change in FtGroEL. PMID:24968190

  18. Francisella tularensis novicida proteomic and transcriptomic data integration and annotation based on semantic web technologies

    PubMed Central

    Anwar, Nadia; Hunt, Ela

    2009-01-01

    Background This paper summarises the lessons and experiences gained from a case study of the application of semantic web technologies to the integration of data from the bacterial species Francisella tularensis novicida (Fn). Fn data sources are disparate and heterogeneous, as multiple laboratories across the world, using multiple technologies, perform experiments to understand the mechanism of virulence. It is hard to integrate these data sources in a flexible manner that allows new experimental data to be added and compared when required. Results Public domain data sources were combined in RDF. Using this connected graph of database cross references, we extended the annotations of an experimental data set by superimposing onto it the annotation graph. Identifiers used in the experimental data automatically resolved and the data acquired annotations in the rest of the RDF graph. This happened without the expensive manual annotation that would normally be required to produce these links. This graph of resolved identifiers was then used to combine two experimental data sets, a proteomics experiment and a transcriptomic experiment studying the mechanism of virulence through the comparison of wildtype Fn with an avirulent mutant strain. Conclusion We produced a graph of Fn cross references which enabled the combination of two experimental datasets. Through combination of these data we are able to perform queries that compare the results of the two experiments. We found that data are easily combined in RDF and that experimental results are easily compared when the data are integrated. We conclude that semantic data integration offers a convenient, simple and flexible solution to the integration of published and unpublished experimental data. PMID:19796400

  19. Characterization of the O-antigen Polymerase (Wzy) of Francisella tularensis*

    PubMed Central

    Kim, Tae-Hyun; Sebastian, Shite; Pinkham, Jessica T.; Ross, Robin A.; Blalock, LeeAnn T.; Kasper, Dennis L.

    2010-01-01

    The O-antigen polymerase of Gram-negative bacteria has been difficult to characterize. Herein we report the biochemical and functional characterization of the protein product (Wzy) of the gene annotated as the putative O-antigen polymerase, which is located in the O-antigen biosynthetic locus of Francisella tularensis. In silico analysis (homology searching, hydropathy plotting, and codon usage assessment) strongly suggested that Wzy is an O-antigen polymerase whose function is to catalyze the addition of newly synthesized O-antigen repeating units to a glycolipid consisting of lipid A, inner core polysaccharide, and one repeating unit of the O-polysaccharide (O-PS). To characterize the function of the Wzy protein, a non-polar deletion mutant of wzy was generated by allelic replacement, and the banding pattern of O-PS was observed by immunoblot analysis of whole-cell lysates obtained by SDS-PAGE and stained with an O-PS-specific monoclonal antibody. These immunoblot analyses showed that O-PS of the wzy mutant expresses only one repeating unit of O-antigen. Further biochemical characterization of the subcellular fractions of the wzy mutant demonstrated that (as is characteristic of O-antigen polymerase mutants) the low molecular weight O-antigen accumulates in the periplasm of the mutant. Site-directed mutagenesis based on protein homology and topology, which was carried out to locate a catalytic residue of the protein, showed that modification of specific residues (Gly176, Asp177, Gly323, and Tyr324) leads to a loss of O-PS polymerization. Topology models indicate that these amino acids most likely lie in close proximity on the bacterial surface. PMID:20605777

  20. Structural Analysis of a Protective Epitope of the Francisella tularensis O-Polysaccharide†

    PubMed Central

    Rynkiewicz, Michael J.; Lu, Zhaohua; Hui, Julia H.; Sharon, Jacqueline; Seaton, Barbara A.

    2012-01-01

    Francisella tularensis (Ft), the Gram negative facultative intracellular bacterium that causes tularemia, is considered a biothreat due to its high infectivity and the high mortality rate of respiratory disease. The Ft lipopolysaccharide (Ft LPS) is thought to be a main protective antigen in mice and humans, and we have previously demonstrated the protective effect of the Ft LPS-specific monoclonal antibody Ab52 in a mouse model of respiratory tularemia. Immunochemical characterization has shown that the epitope recognized by Ab52 is contained within two internal repeat units of the O-polysaccharide [O-antigen (OAg)] of Ft LPS. To further localize the Ab52 epitope and understand the molecular interactions between the antibody and the saccharide, we now solved the X-ray crystal structure of the Fab fragment of Ab52 and derived an antibody-antigen complex using molecular docking. The docked complex, refined through energy minimization, reveals an antigen binding site in the shape of a large canyon with a central pocket that accommodates a V-shaped epitope consisting of six sugar residues, α-D-GalpNAcAN(1→4)-α-D-GalpNAcAN(1→3)-β-D-QuipNAc(1→2)-β-D-Quip4NFm(1→4)-α-D-GalpNAcAN(1→4)-α-D-GalpNAcAN. These results inform the development of vaccines and immunotherapeutic/immunoprophylactic antibodies against Ft by suggesting a desired topology for antibody binding to internal epitopes of Ft LPS. This is the first report of an X-ray crystal structure of a monoclonal antibody that targets a protective Ft B cell epitope. PMID:22747335

  1. Utilization of an unstable plasmid and the I-SceI endonuclease to generate routine markerless deletion mutants in Francisella tularensis

    PubMed Central

    Horzempa, Joseph; Shanks, Robert M.Q.; Brown, Matthew J.; Russo, Brian C.; O’Dee, Dawn M.; Nau, Gerard J.

    2011-01-01

    We engineered an efficient system to make Francisella tularensis deletion mutations using an unstable, poorly maintained plasmid to enhance the likelihood of homologous recombination. For counterselection, we adapted a strategy using I-SceI, which causes a double-stranded break in the integrated suicide vector, forcing a second recombination to mediate allelic replacement. PMID:19879904

  2. Persistence of cell-mediated immunity three decades after vaccination with the live vaccine strain of Francisella tularensis

    PubMed Central

    Eneslätt, Kjell; Rietz, Cecilia; Rydén, Patrik; Stöven, Svenja; House, Robert V.; Wolfraim, Lawrence A.; Tärnvik, Arne; Sjöstedt, Anders

    2012-01-01

    Summary The efficacy of many vaccines against intracellular bacteria depends on the generation of cell-mediated immunity, but studies to determine the duration of immunity are usually confounded by re-exposure. The causative agent of tularemia, Francisella tularensis, is rare in most areas and, therefore, tularemia vaccination is an interesting model for studies of the longevity of vaccine-induced cell-mediated immunity. Here lymphocyte proliferation and cytokine production in response to F. tularensis were assayed in two groups of 16 individuals, vaccinated 1-3 or 27-34 years previously. As compared to naïve individuals, vaccinees of both groups showed higher proliferative responses and, out of 17 cytokines assayed, higher levels of MIP-1β, IFN-γ, IL-10, and IL-5 in response to recall stimulation. The responses were very similar in the two groups of vaccinees. A statistical model was developed to predict the immune status of the individuals and by use of two parameters, proliferative responses and levels of IFN-γ, 91.1% of the individuals were correctly classified. Using flow cytometry analysis, we demonstrated that during recall stimulation, expression of IFN-γ by CD4+CCR7+, CD4+CD62L+, CD8+CCR7+, and CD8+CD62L+ cells significantly increased in samples from vaccinated donors. In conclusion, cell-mediated immunity was found to persist three decades after tularemia vaccination without evidence of decline. PMID:21442618

  3. Cat-bite-induced Francisella tularensis infection with a false-positive serological reaction for Bartonella quintana

    PubMed Central

    Petersson, Evelina

    2017-01-01

    Introduction. Tularaemia is caused by infection with Francisella tularensistransmitted via direct contact with an infected hare carcass or indirectly through the bites of vectors, but may be cat-bite-associated as well. Medical history and reliable diagnostic analysis are important in order to differentiate it from other cat-associated infections, e.g. Bartonella spp. Case presentation. A healthy 56-year-old man was examined because of a cat-bite-associated ulceroglandular wound on his right thumb. Nineteen days after the cat bite occurred, a serology test was positive for anti-Bartonella quintana, but negative for anti-F. tularensis. Since Bartonella infections are rare in Sweden, another serology test was analysed 2 weeks later with a positive result for anti-F. tularensis. The patient was treated with doxycycline for 14 days and recovered. The patient was re-sampled after 18 months to obtain a convalescent sample. The acute and the convalescent samples were both analysed at a reference centre, with negative results for anti-Bartonella spp. this time. Conclusion. This case is enlightening about the importance of extending the medical history and re-sampling the patient for antibody detection when the clinical suspicion of cat-bite-associated tularaemia is high. The false-positive result for anti-B. quintana antibodies may have been due to technical issues with the assay, cross-reactivity or both. PMID:28348802

  4. Contribution of FcɛRI-associated vesicles to mast cell-macrophage communication following Francisella tularensis infection.

    PubMed

    Rodriguez, Annette R; Yu, Jieh-Juen; Navara, Christopher; Chambers, James P; Guentzel, M Neal; Arulanandam, Bernard P

    2016-10-01

    Understanding innate immune intercellular communication following microbial infection remains a key biological issue. Using live cell imaging, we demonstrate that mast cells actively extend cellular projections to sample the macrophage periphery during Francisella tularensis LVS infection. Mast cell MHCII(hi) expression was elevated from less than 1% to 13% during LVS infection. Direct contact during co-culture with macrophages further increased mast cell MHCII(hi) expression to approximately 87%. Confocal analyses of the cellular perimeter revealed mast cell caspase-1 was localized in close proximity with FcɛRI in uninfected mast cells, and repositioned to clustered regions upon LVS infection. Importantly, mast cell FcɛRI-encompassed vesicles are transferred to macrophages by trogocytosis, and macrophage caspase-1 expression is further up-regulated upon direct contact with mast cells. Our study reveals direct cellular interactions between innate cells that may impact the function of caspase-1, a known sensor of microbial danger and requirement for innate defense against many pathogenic microbes including F. tularensis.

  5. Lonsdalea quercina subsp. populi subsp. nov., isolated from bark canker of poplar trees.

    PubMed

    Tóth, Tímea; Lakatos, Tamás; Koltay, András

    2013-06-01

    Seven Gram-negative bacterial strains were isolated from oozing bark canker of poplar (Populus × euramericana) trees in Hungary. They showed high (>98.3%) 16S rRNA gene sequence similarity to Lonsdalea quercina; however, they differed from this species in several phenotypic characteristics. Multilocus sequence analysis based on three housekeeping genes (gyrB, atpD and infB) revealed, and DNA-DNA hybridization analysis confirmed, that this group of bacterial strains forms a distinct lineage within the species Lonsdalea quercina. A detailed study of phenotypic and physiological characteristics confirmed the separation of isolates from poplars from other subspecies of L. quercina; therefore, a novel subspecies, Lonsdalea quercina subsp. populi, type strain NY060(T) (=DSM 25466(T)=NCAIM B 02483(T)), is proposed.

  6. Juvenile skeletogenesis in anciently diverged sea urchin clades.

    PubMed

    Gao, Feng; Thompson, Jeffrey R; Petsios, Elizabeth; Erkenbrack, Eric; Moats, Rex A; Bottjer, David J; Davidson, Eric H

    2015-04-01

    Mechanistic understanding of evolutionary divergence in animal body plans devolves from analysis of those developmental processes that, in forms descendant from a common ancestor, are responsible for their morphological differences. The last common ancestor of the two extant subclasses of sea urchins, i.e., euechinoids and cidaroids, existed well before the Permian/Triassic extinction (252 mya). Subsequent evolutionary divergence of these clades offers in principle a rare opportunity to solve the developmental regulatory events underlying a defined evolutionary divergence process. Thus (i) there is an excellent and fairly dense (if yet incompletely analyzed) fossil record; (ii) cladistically confined features of the skeletal structures of modern euechinoid and cidaroid sea urchins are preserved in fossils of ancestral forms; (iii) euechinoids and cidaroids are among current laboratory model systems in molecular developmental biology (here Strongylocentrotus purpuratus [Sp] and Eucidaris tribuloides [Et]); (iv) skeletogenic specification in sea urchins is uncommonly well understood at the causal level of interactions of regulatory genes with one another, and with known skeletogenic effector genes, providing a ready arsenal of available molecular tools. Here we focus on differences in test and perignathic girdle skeletal morphology that distinguish all modern euechinoid from all modern cidaroid sea urchins. We demonstrate distinct canonical test and girdle morphologies in juveniles of both species by use of SEM and X-ray microtomography. Among the sharply distinct morphological features of these clades are the internal skeletal structures of the perignathic girdle to which attach homologous muscles utilized for retraction and protraction of Aristotles׳ lantern and its teeth. We demonstrate that these structures develop de novo between one and four weeks after metamorphosis. In order to study the underlying developmental processes, a method of section whole mount in

  7. Origin and Dispersal History of Two Colonial Ascidian Clades in the Botryllus schlosseri Species Complex

    PubMed Central

    Nydam, Marie L.

    2017-01-01

    Human-induced global warming and species introductions are rapidly altering the composition and functioning of Earth’s marine ecosystems. Ascidians (Phylum Chordata, Subphylum Tunicata, Class Ascidiacea) are likely to play an increasingly greater role in marine communities. The colonial ascidian B. schlosseri is a cryptic species complex comprising five genetically divergent clades (A-E). Clade A is a global species, and Clade E has so far been identified in European waters only. Using the largest mitochondrial cytochrome oxidase I datasets yet assembled, we determine the origin and dispersal history of these species. Nucleotide diversity and Approximate Bayesian Computation analyses support a Pacific origin for Clade A, with two likely dispersal scenarios that both show the northwestern Atlantic populations establishing early in the history of the species. Both Discrete Phylogeographic Analysis and Approximate Bayesian Computation support an origin of Clade E on the French side of the English Channel. An unsampled lineage evolved from the French lineage, which reflects the conclusion from the median joining network that not all Clade E lineages have been sampled. This unsampled lineage gave rise to the haplotypes on the English side of the English Channel, which were the ancestors to the Mediterranean and Bay of Biscay populations. Clade E has a wider geographic range than previously thought, and shows evidence of recent range expansion. Both Clade A and Clade E should be considered widespread species: Clade A globally and Clade E within Europe. PMID:28107476

  8. Overexpressed Proteins in Hypervirulent Clade 8 and Clade 6 Strains of Escherichia coli O157:H7 Compared to E. coli O157:H7 EDL933 Clade 3 Strain

    PubMed Central

    Amigo, Natalia; Zhang, Qi; Amadio, Ariel; Zhang, Qunjie; Silva, Wanderson M.; Cui, Baiyuan; Chen, Zhongjian; Larzabal, Mariano; Bei, Jinlong; Cataldi, Angel

    2016-01-01

    Escherichia coli O157:H7 is responsible for severe diarrhea and hemolytic uremic syndrome (HUS), and predominantly affects children under 5 years. The major virulence traits are Shiga toxins, necessary to develop HUS and the Type III Secretion System (T3SS) through which bacteria translocate effector proteins directly into the host cell. By SNPs typing, E. coli O157:H7 was separated into nine different clades. Clade 8 and clade 6 strains were more frequently associated with severe disease and HUS. In this study, we aimed to identify differentially expressed proteins in two strains of E. coli O157:H7 (clade 8 and clade 6), obtained from cattle and compared them with the well characterized reference EDL933 strain (clade 3). Clade 8 and clade 6 strains show enhanced pathogenicity in a mouse model and virulence-related properties. Proteins were extracted and analyzed using the TMT-6plex labeling strategy associated with two dimensional liquid chromatography and mass spectrometry in tandem. We detected 2241 proteins in the cell extract and 1787 proteins in the culture supernatants. Attention was focused on the proteins related to virulence, overexpressed in clade 6 and 8 strains compared to EDL933 strain. The proteins relevant overexpressed in clade 8 strain were the curli protein CsgC, a transcriptional activator (PchE), phage proteins, Stx2, FlgM and FlgD, a dienelactone hydrolase, CheW and CheY, and the SPATE protease EspP. For clade 6 strain, a high overexpression of phage proteins was detected, mostly from Stx2 encoding phage, including Stx2, flagellin and the protease TagA, EDL933_p0016, dienelactone hydrolase, and Haemolysin A, amongst others with unknown function. Some of these proteins were analyzed by RT-qPCR to corroborate the proteomic data. Clade 6 and clade 8 strains showed enhanced transcription of 10 out of 12 genes compared to EDL933. These results may provide new insights in E. coli O157:H7 mechanisms of pathogenesis. PMID:27880834

  9. Description of Teunomyces gen. nov. for the Candida kruisii clade, Suhomyces gen. nov. for the Candida tanzawaensis clade and Suhomyces kilbournensis sp. nov.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DNA sequence analysis has shown that species of the Candida kruisii clade and species of the C. tanzawaensis clade represent phylogenetically circumscribed genera, which are described as Teunomyces gen. nov., type species T. kruisii, and Suhomyces gen. nov., type species S. tanzawaensis. Many of the...

  10. Complete Genome Sequence of Beijerinckia indica subsp. indica▿

    PubMed Central

    Tamas, Ivica; Dedysh, Svetlana N.; Liesack, Werner; Stott, Matthew B.; Alam, Maqsudul; Murrell, J. Colin; Dunfield, Peter F.

    2010-01-01

    Beijerinckia indica subsp. indica is an aerobic, acidophilic, exopolysaccharide-producing, N2-fixing soil bacterium. It is a generalist chemoorganotroph that is phylogenetically closely related to facultative and obligate methanotrophs of the genera Methylocella and Methylocapsa. Here we report the full genome sequence of this bacterium. PMID:20601475

  11. Streptococcus dysgalactiae subsp. equisimilis Bacteremia, Finland, 1995–2004

    PubMed Central

    Vähäkuopus, Susanna; Vuopio-Varkila, Jaana; Vuento, Risto; Syrjänen, Jaana

    2010-01-01

    We conducted a retrospective population-based study of 140 episodes of Streptococcus dysgalactiae subsp. equisimilis bacteremia occurring in Finland during 1995–2004. Rare emm types were associated with more severe disease and increased mortality rates. Skin and soft tissue infections were more frequent clinical signs among cases caused by common emm types. PMID:20409380

  12. Streptococcus dysgalactiae subsp. equisimilis Bacteremia, Finland, 1995-2004.

    PubMed

    Rantala, Sari; Vahakuopus, Susanna; Vuopio-Varkila, Jaana; Vuento, Risto; Syrjanen, Jaana

    2010-05-01

    We conducted a retrospective population-based study of 140 episodes of Streptococcus dysgalactiae subsp. equisimilis bacteremia occurring in Finland during 1995-2004. Rare emm types were associated with more severe disease and increased mortality rates. Skin and soft tissue infections were more frequent clinical signs among cases caused by common emm types.

  13. Bartonella vinsonii subsp. berkhoffii endocarditis in a dog from Saskatchewan

    PubMed Central

    Cockwill, Ken R.; Taylor, Susan M.; Philibert, Helene M.; Breitschwerdt, Edward B.; Maggi, Ricardo G.

    2007-01-01

    A dog referred for lameness was diagnosed with culture-negative endocarditis. Antibodies to Bartonella spp. were detected. Antibiotic treatment resulted in transient clinical improvement, but the dog developed cardiac failure and was euthanized. Bartonella vinsonii subsp. berkhoffii genotype IV was identified within the aortic heart valve lesions by PCR amplification and DNA sequencing. PMID:17824328

  14. Laminaria japonica Extract, an Inhibitor of Clavibater michiganense Subsp. Sepedonicum

    PubMed Central

    Cai, Jin; Feng, Jia; Xie, Shulian; Wang, Feipeng; Xu, Qiufeng

    2014-01-01

    Bacterial ring rot of potato is one of the most serious potato plant and tuber diseases. Laminaria japonica extract was investigated for its antimicrobial activity against Clavibater michiganense subsp. sepedonicum (Spieckermann & Kotthoff) Davis et al., the causative agent of bacterial ring rot of potato. The results showed that the optimum extraction conditions of antimicrobial substances from L. japonica were an extraction temperature of 80°C, an extraction time of 12 h, and a solid to liquid ratio of 1∶25. Active compounds of L. japonica were isolated by solvent partition, thin layer chromatography (TLC) and column chromatography. All nineteen fractionations had antimicrobial activities against C. michiganense subsp. sepedonicum, while Fractionation three (Fr.3) had the highest (P<0.05) antimicrobial activity. Chemical composition analysis identified a total of 26 components in Fr.3. The main constituents of Fr.3 were alkanes (80.97%), esters (5.24%), acids (4.87%) and alcohols (2.21%). Antimicrobial activity of Fr.3 against C. michiganense subsp. sepedonicum could be attributed to its ability to damage the cell wall and cell membrane, induce the production of reactive oxygen species (ROS), increase cytosolic Ca2+ concentration, inhibit the glycolytic pathway (EMP) and tricarboxylic acid (TCA) cycle, inhibit protein and nucleic acid synthesis, and disrupt the normal cycle of DNA replication. These findings indicate that L. japonica extracts have potential for inhibiting C. michiganense subsp. sepedonicum. PMID:24714388

  15. Characterization of Lactococcus lactis subsp. lactis isolated from surface waters.

    PubMed

    Svec, P; Sedlácek, I

    2008-01-01

    A group of nine presumptive enterococci was isolated on enterococcal selective media Slanetz-Bartley agar and/or kanamycin-esculin-azide agar during a screening of Enterococcus spp. in surface waters. All strains formed a homogeneous cluster separated from all enterococcal species using rep-PCR fingerprinting with the (GTG)5 primer but they matched fingerprints revealed by Lactococcus lactis subsp. lactis representatives. Further identification using extensive biotyping and automated ribotyping with EcoRI (RiboPrinter microbial characterization system) confirmed all strains as L. lactis subsp. lactis in full correspondence with the (GTG)5-PCR. We demonstrated that L. lactis subsp. lactis strains occur in different surface waters and can be confused with enterococci due to their positive growth on selective enterococcal media as well as positive results in tests commonly used for identification of the genus Enterococcus (esculin hydrolysis, acetoin and pyrrolidonyl arylamidase production, growth at 10 degrees C and in 6.5% NaCl). The (GTG)5-PCR fingerprinting was revealed as a reliable and fast method for the identification of L. lactis subsp lactis while automated ribotyping with EcoRI proved to be a good tool for intrasubspecies typing purposes.

  16. A new flavan-3-ol from Artocarpus nitidus subsp. lingnanensis.

    PubMed

    Ti, Hui-Hui; Lin, Li-Dong; Ding, Wen-Bing; Wei, Xiao-Yi

    2012-01-01

    Further investigation on the stems of Artocarpus nitidus subsp. lingnanensis led to the isolation and characterization of a new flavan-3-ol, named artoflavanocoumarin, along with three known compounds (+)-catechin, (+)-afzelechin 3-O-α-L-rhamnoside, and (+)-catechin 3-O-α-L-rhamnoside. Their structures were elucidated on the basis of spectroscopic data.

  17. Streptococcus equi subsp. zooepidemicus Infections Associated with Guinea Pigs

    PubMed Central

    Young, Andrea; Levine, Seth J.; Garvin, Joseph P.; Brown, Susan; Turner, Lauren; Fritzinger, Angela; Gertz, Robert E.; Murphy, Julia M.; Vogt, Marshall; Beall, Bernard

    2015-01-01

    Streptococcus equi subsp. zooepidemicus is a known zoonotic pathogen. In this public health investigation conducted in Virginia, USA, in 2013, we identified a probable family cluster of S. zooepidemicus cases linked epidemiologically and genetically to infected guinea pigs. S. zooepidemicus infections should be considered in patients who have severe clinical illness and report guinea pig exposure. PMID:25531424

  18. Description and history of Syringa oblata subsp. oblata 'Frank Meyer'

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An accession of Syringa oblata subsp. oblata (PI 23031) collected in China by Frank Meyer in 1908was given the name ‘Frank Meyer’ by Father Fiala in 1988. To be established according to the International Code of Nomenclature for Cultivated Plants, a new cultivar name must be accompanied by a descrip...

  19. A Proteomic Study of Clavibacter Michiganensis Subsp. Michiganensis Culture Supernatants

    PubMed Central

    Hiery, Eva; Poetsch, Ansgar; Moosbauer, Tanja; Amin, Bushra; Hofmann, Jörg; Burkovski, Andreas

    2015-01-01

    Clavibacter michiganensis, subsp. michiganensis is a Gram-positive plant pathogen infecting tomato (Solanum lycopersicum). Despite a considerable economic importance due to significant losses of infected plants and fruits, knowledge about virulence factors of C. michiganensis subsp. michiganensis and host-pathogen interactions on a molecular level are rather limited. In the study presented here, the proteome of culture supernatants from C. michiganensis subsp. michiganensis NCPPB382 was analyzed. In total, 1872 proteins were identified in M9 and 1766 proteins in xylem mimicking medium. Filtration of supernatants before protein precipitation reduced these to 1276 proteins in M9 and 976 proteins in the xylem mimicking medium culture filtrate. The results obtained indicate that C. michiganensis subsp. michiganensis reacts to a sucrose- and glucose-depleted medium similar to the xylem sap by utilizing amino acids and host cell polymers as well as their degradation products, mainly peptides, amino acids and various C5 and C6 sugars. Interestingly, the bacterium expresses the previously described virulence factors Pat-1 and CelA not exclusively after host cell contact in planta but already in M9 minimal and xylem mimicking medium. PMID:28248277

  20. Cellular Interactions in Mycobacterium avium subsp. paratuberculosis Infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The study of host immune responses to Mycobacterium avium subsp. paratuberculosis (MAP) is complicated by a number of factors, including the protracted nature of the disease and the stealthy nature of the pathogen. Noted as one of the more fastidious mycobacteria, infection with MAP is often chara...

  1. Mycobacterium avium subsp. paratuberculosis infection, immunology and pathology of livestock

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP) infection in ruminants leads to a chronic and progressive enteric disease (Johne’s disease) that results in loss of intestinal function, poor body condition, and eventual death. Transmission is primarily through a fecal-oral route in neonates but con...

  2. Complete genome sequence of Clavibacter michiganensis subsp. insidiosus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clavibacter michiganensis subsp. insidiosus (Cmi) causes bacterial wilt disease of alfalfa (Medicago sativa L.) and can also infect the model legume plant M. truncatula. The virulence mechanisms of Cmi are yet to be identified, hampered by the lack of efficient mutagenesis tools as well as by the la...

  3. Decoupled form and function in disparate herbivorous dinosaur clades

    NASA Astrophysics Data System (ADS)

    Lautenschlager, Stephan; Brassey, Charlotte A.; Button, David J.; Barrett, Paul M.

    2016-05-01

    Convergent evolution, the acquisition of morphologically similar traits in unrelated taxa due to similar functional demands or environmental factors, is a common phenomenon in the animal kingdom. Consequently, the occurrence of similar form is used routinely to address fundamental questions in morphofunctional research and to infer function in fossils. However, such qualitative assessments can be misleading and it is essential to test form/function relationships quantitatively. The parallel occurrence of a suite of morphologically convergent craniodental characteristics in three herbivorous, phylogenetically disparate dinosaur clades (Sauropodomorpha, Ornithischia, Theropoda) provides an ideal test case. A combination of computational biomechanical models (Finite Element Analysis, Multibody Dynamics Analysis) demonstrate that despite a high degree of morphological similarity between representative taxa (Plateosaurus engelhardti, Stegosaurus stenops, Erlikosaurus andrewsi) from these clades, their biomechanical behaviours are notably different and difficult to predict on the basis of form alone. These functional differences likely reflect dietary specialisations, demonstrating the value of quantitative biomechanical approaches when evaluating form/function relationships in extinct taxa.

  4. Decoupled form and function in disparate herbivorous dinosaur clades.

    PubMed

    Lautenschlager, Stephan; Brassey, Charlotte A; Button, David J; Barrett, Paul M

    2016-05-20

    Convergent evolution, the acquisition of morphologically similar traits in unrelated taxa due to similar functional demands or environmental factors, is a common phenomenon in the animal kingdom. Consequently, the occurrence of similar form is used routinely to address fundamental questions in morphofunctional research and to infer function in fossils. However, such qualitative assessments can be misleading and it is essential to test form/function relationships quantitatively. The parallel occurrence of a suite of morphologically convergent craniodental characteristics in three herbivorous, phylogenetically disparate dinosaur clades (Sauropodomorpha, Ornithischia, Theropoda) provides an ideal test case. A combination of computational biomechanical models (Finite Element Analysis, Multibody Dynamics Analysis) demonstrate that despite a high degree of morphological similarity between representative taxa (Plateosaurus engelhardti, Stegosaurus stenops, Erlikosaurus andrewsi) from these clades, their biomechanical behaviours are notably different and difficult to predict on the basis of form alone. These functional differences likely reflect dietary specialisations, demonstrating the value of quantitative biomechanical approaches when evaluating form/function relationships in extinct taxa.

  5. Direct evidence for the Homo-Pan clade.

    PubMed

    Wimmer, Rainer; Kirsch, Stefan; Rappold, Gudrun A; Schempp, Werner

    2002-01-01

    For a long time, the evolutionary relationship between human and African apes, the 'trichotomy problem', has been debated with strong differences in opinion and interpretation. Statistical analyses of different molecular DNA data sets have been carried out and have primarily supported a Homo-Pan clade. An alternative way to address this question is by the comparison of evolutionarily relevant chromosomal breakpoints. Here, we made use of a P1-derived artificial chromosome (PAC)/bacterial artificial chromosome (BAC) contig spanning approximately 2.8 Mb on the long arm of the human Y chromosome, to comparatively map individual PAC clones to chromosomes from great apes, gibbons, and two species of Old World monkeys by fluorescence in-situ hybridization. During our search for evolutionary breakpoints on the Y chromosome, it transpired that a transposition of an approximately 100-kb DNA fragment from chromosome 1 onto the Y chromosome must have occurred in a common ancestor of human, chimpanzee and bonobo. Only the Y chromosomes of these three species contain the chromosome-1-derived fragment; it could not be detected on the Y chromosomes of gorillas or the other primates examined. Thus, this shared derived (synapomorphic) trait provides clear evidence for a Homo-Pan clade independent of DNA sequence analysis.

  6. Deep phylogenetic incongruence in the angiosperm clade Rosidae.

    PubMed

    Sun, Miao; Soltis, Douglas E; Soltis, Pamela S; Zhu, Xinyu; Burleigh, J Gordon; Chen, Zhiduan

    2015-02-01

    Analysis of large data sets can help resolve difficult nodes in the tree of life and also reveal complex evolutionary histories. The placement of the Celastrales-Oxalidales-Malpighiales (COM) clade within Rosidae remains one of the most confounding phylogenetic questions in angiosperms, with previous analyses placing it with either Fabidae or Malvidae. To elucidate the position of COM, we assembled multi-gene matrices of chloroplast, mitochondrial, and nuclear sequences, as well as large single- and multi-copy nuclear gene data sets. Analyses of multi-gene data sets demonstrate conflict between the chloroplast and both nuclear and mitochondrial data sets, and the results are robust to various character-coding and data-exclusion treatments. Analyses of single- and multi-copy nuclear loci indicate that most loci support the placement of COM with Malvidae, fewer loci support COM with Fabidae, and almost no loci support COM outside a clade of Fabidae and Malvidae. Although incomplete lineage sorting and ancient introgressive hybridization remain as plausible explanations for the conflict among loci, more complete sampling is necessary to evaluate these hypotheses fully. Our results emphasize the importance of genomic data sets for revealing deep incongruence and complex patterns of evolution.

  7. Decoupled form and function in disparate herbivorous dinosaur clades

    PubMed Central

    Lautenschlager, Stephan; Brassey, Charlotte A.; Button, David J.; Barrett, Paul M.

    2016-01-01

    Convergent evolution, the acquisition of morphologically similar traits in unrelated taxa due to similar functional demands or environmental factors, is a common phenomenon in the animal kingdom. Consequently, the occurrence of similar form is used routinely to address fundamental questions in morphofunctional research and to infer function in fossils. However, such qualitative assessments can be misleading and it is essential to test form/function relationships quantitatively. The parallel occurrence of a suite of morphologically convergent craniodental characteristics in three herbivorous, phylogenetically disparate dinosaur clades (Sauropodomorpha, Ornithischia, Theropoda) provides an ideal test case. A combination of computational biomechanical models (Finite Element Analysis, Multibody Dynamics Analysis) demonstrate that despite a high degree of morphological similarity between representative taxa (Plateosaurus engelhardti, Stegosaurus stenops, Erlikosaurus andrewsi) from these clades, their biomechanical behaviours are notably different and difficult to predict on the basis of form alone. These functional differences likely reflect dietary specialisations, demonstrating the value of quantitative biomechanical approaches when evaluating form/function relationships in extinct taxa. PMID:27199098

  8. The genetic code of the fungal CTG clade.

    PubMed

    Santos, Manuel A S; Gomes, Ana C; Santos, Maria C; Carreto, Laura C; Moura, Gabriela R

    2011-01-01

    Genetic code alterations discovered over the last 40 years in bacteria and eukaryotes invalidate the hypothesis that the code is universal and frozen. Mitochondria of various yeast species translate the UGA stop codon as tryptophan (Trp) and leucine (Leu) CUN codons (N = any nucleotide) as threonine (Thr) and fungal CTG clade species reassigned Leu CUG codons to serine and translate them ambiguously in their cytoplasms. This unique sense-to-sense genetic code alteration is mediated by a Ser-tRNA containing a Leu 5'-CAG-3'anticodon (ser-tRNA(CAG)), which is recognized and charged with Ser (~97%) by the seryl-tRNA synthetase (SerRS) and with Leu (~3%) by the leucyl-tRNA synthetase (LeuRS). This unusual tRNA appeared 272 ± 25 million years ago and had a profound impact on the evolution of the CTG clade species. Here, we review the most recent results and concepts arising from the study of this codon reassignment and we highlight how its study is changing our views of the evolution of the genetic code.

  9. Five novel species in the Lodderomyces clade associated with insects.

    PubMed

    Liu, Xiao-Jing; Yi, Ze-Hao; Ren, Yong-Cheng; Li, Ying; Hui, Feng-Li

    2016-11-01

    During a survey of yeasts associated with insects in Central China's natural ecosystems, 116 yeast strains were isolated from the gut of adult insects in two families and from one beetle larva. Among the yeasts isolated in this study, 102 strains were identified as 20 known species in the class Saccharomycetes. The remaining 14 strains were identified as representing five novel species in the Lodderomyces clade based on the combined sequences of the D1/D2 domains of the LSU rRNA gene and the internal transcribed spacer (ITS) regions, as well as other taxonomic characteristics. Lodderomyces beijingensis sp. nov. (type strain CBS 14171T=CICC 33087T=NYNU 15764T) formed a clade with Lodderomyces elongisporus and Candida oxycetoniae. The other four novel species, namely Candida margitis sp. nov. (type strain CBS 14175T=CICC 33091T=NYNU 15857T), Candida xiaguanensis sp. nov. (type strain CBS 13923T=CICC 33056T=NYNU 1488T), Candida parachauliodis sp. nov. (type strain CBS 13928T=CICC 33058T=NYNU 14959T) and Candida coleopterorum sp. nov. (type strain CBS 14180T=CICC 33084T=NYNU 1582T), showed close relationships to the species near Candida parapsilosis, Candida sakaeoensis, Candida chauliodes and Candida corydalis. Descriptions of these novel yeast species are provided as well as discussions of their ecology in relation to their insect hosts.

  10. Novel Temperate Phages of Salmonella enterica subsp. salamae and subsp. diarizonae and Their Activity against Pathogenic S. enterica subsp. enterica Isolates.

    PubMed

    Mikalová, Lenka; Bosák, Juraj; Hříbková, Hana; Dědičová, Daniela; Benada, Oldřich; Šmarda, Jan; Šmajs, David

    2017-01-01

    Forty strains of Salmonella enterica (S. enterica) subspecies salamae (II), arizonae (IIIa), diarizonae (IIIb), and houtenae (IV) were isolated from human or environmental samples and tested for bacteriophage production. Production of bacteriophages was observed in 15 S. enterica strains (37.5%) belonging to either the subspecies salamae (8 strains) or diarizonae (7 strains). Activity of phages was tested against 52 pathogenic S. enterica subsp. enterica isolates and showed that phages produced by subsp. salamae had broader activity against pathogenic salmonellae compared to phages from the subsp. diarizonae. All 15 phages were analyzed using PCR amplification of phage-specific regions and 9 different amplification profiles were identified. Five phages (SEN1, SEN4, SEN5, SEN22, and SEN34) were completely sequenced and classified as temperate phages. Phages SEN4 and SEN5 were genetically identical, thus representing a single phage type (i.e. SEN4/5). SEN1 and SEN4/5 fit into the group of P2-like phages, while the SEN22 phage showed sequence relatedness to P22-like phages. Interestingly, while phage SEN34 was genetically distantly related to Lambda-like phages (Siphoviridae), it had the morphology of the Myoviridae family. Based on sequence analysis and electron microscopy, phages SEN1 and SEN4/5 were members of the Myoviridae family and phage SEN22 belonged to the Podoviridae family.

  11. Novel Temperate Phages of Salmonella enterica subsp. salamae and subsp. diarizonae and Their Activity against Pathogenic S. enterica subsp. enterica Isolates

    PubMed Central

    Hříbková, Hana; Dědičová, Daniela; Benada, Oldřich; Šmarda, Jan; Šmajs, David

    2017-01-01

    Forty strains of Salmonella enterica (S. enterica) subspecies salamae (II), arizonae (IIIa), diarizonae (IIIb), and houtenae (IV) were isolated from human or environmental samples and tested for bacteriophage production. Production of bacteriophages was observed in 15 S. enterica strains (37.5%) belonging to either the subspecies salamae (8 strains) or diarizonae (7 strains). Activity of phages was tested against 52 pathogenic S. enterica subsp. enterica isolates and showed that phages produced by subsp. salamae had broader activity against pathogenic salmonellae compared to phages from the subsp. diarizonae. All 15 phages were analyzed using PCR amplification of phage-specific regions and 9 different amplification profiles were identified. Five phages (SEN1, SEN4, SEN5, SEN22, and SEN34) were completely sequenced and classified as temperate phages. Phages SEN4 and SEN5 were genetically identical, thus representing a single phage type (i.e. SEN4/5). SEN1 and SEN4/5 fit into the group of P2-like phages, while the SEN22 phage showed sequence relatedness to P22-like phages. Interestingly, while phage SEN34 was genetically distantly related to Lambda-like phages (Siphoviridae), it had the morphology of the Myoviridae family. Based on sequence analysis and electron microscopy, phages SEN1 and SEN4/5 were members of the Myoviridae family and phage SEN22 belonged to the Podoviridae family. PMID:28118395

  12. Crystal Structures of the Histidine Acid Phosphatase from Francisella tularensis Provide Insight into Substrate Recognition

    SciTech Connect

    Singh, Harkewal; Felts, Richard L.; Schuermann, Jonathan P.; Reilly, Thomas J.; Tanner, John J.

    2009-12-01

    Histidine acid phosphatases catalyze the transfer of a phosphoryl group from phosphomonoesters to water at acidic pH using an active-site histidine. The histidine acid phosphatase from the category A pathogen Francisella tularensis (FtHAP) has been implicated in intramacrophage survival and virulence, motivating interest in understanding the structure and mechanism of this enzyme. Here, we report a structure-based study of ligand recognition by FtHAP. The 1.70-{angstrom}-resolution structure of FtHAP complexed with the competitive inhibitor L(+)-tartrate was solved using single-wavelength anomalous diffraction phasing. Structures of the ligand-free enzyme and the complex with inorganic phosphate were determined at resolutions of 1.85 and 1.70 {angstrom}, respectively. The structure of the Asp261Ala mutant enzyme complexed with the substrate 3'-AMP was determined at 1.50 {angstrom} resolution to gain insight into substrate recognition. FtHAP exhibits a two-domain fold similar to that of human prostatic acid phosphatase, consisting of an {alpha}/{beta} core domain and a smaller domain that caps the core domain. The structures show that the core domain supplies the phosphoryl binding site, catalytic histidine (His17), and an aspartic acid residue (Asp261) that protonates the leaving group, while the cap domain contributes residues that enforce substrate preference. FtHAP and human prostatic acid phosphatase differ in the orientation of the crucial first helix of the cap domain, implying differences in the substrate preferences of the two enzymes. 3'-AMP binds in one end of a 15-{angstrom}-long tunnel, with the adenine clamped between Phe23 and Tyr135, and the ribose 2'-hydroxyl interacting with Gln132. The importance of the clamp is confirmed with site-directed mutagenesis; mutation of Phe23 and Tyr135 individually to Ala increases K{sub m} by factors of 7 and 10, respectively. The structural data are consistent with a role for FtHAP in scavenging phosphate from small

  13. Whole genome sequences of three Clade 3 Clostridium difficile strains carrying binary toxin genes in China.

    PubMed

    Chen, Rong; Feng, Yu; Wang, Xiaohui; Yang, Jingyu; Zhang, Xiaoxia; Lü, Xiaoju; Zong, Zhiyong

    2017-03-06

    Clostridium difficile consists of six clades but studies on Clade 3 are limited. Here, we report genome sequences of three Clade 3 C. difficile strains carrying genes encoding toxin A and B and the binary toxin. Isolates 103 and 133 (both of ST5) and isolate 106 (ST285) were recovered from three ICU patients. Whole genome sequencing using HiSeq 2500 revealed 4.1-Mb genomes with 28-29% GC content. There were ≥1,104 SNP between the isolates, suggesting they were not of a single clone. The toxin A and B gene-carrying pathogenicity locus (PaLoc) of the three isolates were identical and had the insertion of the transposon Tn6218. The genetic components of PaLoc among Clade 3 strains were the same with only a few nucleotide mutations and deletions/insertions, suggesting that the Tn6218 insertion might have occurred before the divergence within Clade 3. The binary toxin-genes carrying CDT locus (CdtLoc) of the three isolates were identical and were highly similar to those of other Clade 3 strains, but were more divergent from those of other clades. In conclusion, Clade 3 has an unusual clade-specific PaLoc characteristic of a Tn6218 insertion which appears to be the main feature to distinguish Clade 3 from other C. difficile.

  14. Variable depth distribution of Trichodesmium clades in the North Pacific Ocean.

    PubMed

    Rouco, Mónica; Haley, Sheean T; Alexander, Harriet; Wilson, Samuel T; Karl, David M; Dyhrman, Sonya T

    2016-12-01

    Populations of nitrogen-fixing cyanobacteria in the genus Trichodesmium are critical to ocean ecosystems, yet predicting patterns of Trichodesmium distribution and their role in ocean biogeochemistry is an ongoing challenge. This may, in part, be due to differences in the physiological ecology of Trichodesmium species, which are not typically considered independently in field studies. In this study, the abundance of the two dominant Trichodesmium clades (Clade I and Clade III) was investigated during a survey at Station ALOHA in the North Pacific Subtropical Gyre (NPSG) using a clade-specific qPCR approach. While Clade I dominated the Trichodesmium community, Clade III abundance was >50% in some NPSG samples, in contrast to the western North Atlantic where Clade III abundance was always <10%. Clade I populations were distributed down to depths >80 m, while Clade III populations were only observed in the mixed layer and found to be significantly correlated with depth and temperature. These data suggest active niche partitioning of Trichodesmium species from different clades, as has been observed in other cyanobacteria. Tracking the distribution and physiology of Trichodesmium spp. would contribute to better predictions of the physiological ecology of this biogeochemically important genus in the present and future ocean.

  15. Whole genome sequences of three Clade 3 Clostridium difficile strains carrying binary toxin genes in China

    PubMed Central

    Chen, Rong; Feng, Yu; Wang, Xiaohui; Yang, Jingyu; Zhang, Xiaoxia; Lü, Xiaoju; Zong, Zhiyong

    2017-01-01

    Clostridium difficile consists of six clades but studies on Clade 3 are limited. Here, we report genome sequences of three Clade 3 C. difficile strains carrying genes encoding toxin A and B and the binary toxin. Isolates 103 and 133 (both of ST5) and isolate 106 (ST285) were recovered from three ICU patients. Whole genome sequencing using HiSeq 2500 revealed 4.1-Mb genomes with 28–29% GC content. There were ≥1,104 SNP between the isolates, suggesting they were not of a single clone. The toxin A and B gene-carrying pathogenicity locus (PaLoc) of the three isolates were identical and had the insertion of the transposon Tn6218. The genetic components of PaLoc among Clade 3 strains were the same with only a few nucleotide mutations and deletions/insertions, suggesting that the Tn6218 insertion might have occurred before the divergence within Clade 3. The binary toxin-genes carrying CDT locus (CdtLoc) of the three isolates were identical and were highly similar to those of other Clade 3 strains, but were more divergent from those of other clades. In conclusion, Clade 3 has an unusual clade-specific PaLoc characteristic of a Tn6218 insertion which appears to be the main feature to distinguish Clade 3 from other C. difficile. PMID:28262711

  16. [Evaluation of a newly-developed ready-to-use commercial PCR kit for the molecular diagnosis of Francisella tularensis].

    PubMed

    Celebi, Bekir; Kılıç, Selçuk; Yeşilyurt, Murat; Acar, Bülent

    2014-01-01

    Tularemia is a rare zoonotic infection, however, considerations of tularemia as a biological weapon and several recent major epidemics have caused renewed interest in this disease. Laboratory diagnosis of tularemia is done in the presence of appropriate epidemiological data, by the demonstration of specific antibodies in the serum samples obtained with 1-2 week intervals following the development of symptoms. It is an a posteriori analysis with limited use for prompt diagnosis of the patient during the early symptomatic phase and deliberate release of biological agents. Limitations in both culture and serology have led to substantial research in the development of new diagnostic techniques. Several PCR methods for tularemia have been developed, both for conventional and real-time polymerase chain reaction (rtPCR). However, PCR methods are hard to be deployed in remote endemic areas that lack sufficient infrastructure. Recently a "Toolbox" which includes all instruments, equipments and solutions [DNA4U® Bacteria Genomic DNA Isolation Kit, CubeCycler® (Personal Thermal Cycler), PCR4U® Bioterrorism Agents Detection Kit, electrophoresis tank, power supply, ready-agarose gel and electrophoresis buffer] necessary for conventional PCR, was developed for the identification of bioterrorism agents in the field. In this study we aimed to evaluate the efficacy of a ready-to-use commercial PCR kit (Nanobiz, Ankara, Turkey) targeting the tul4 gene, for the diagnosis of tularemia and to compare the results with an in-house conventional PCR and a rtPCR test. We applied the assay to a collection of four F.tularensis standard strains, 15 field isolates (from humans, animals, water), 13 non-Francisella strains which are phylogenetically related to F.tularensis and a total of 60 lymph node aspirates obtained from suspected tularemia cases. Compared to the in-house PCR method used in our laboratory, the sensitivity, specificity, positive and negative predictive values of Nanobiz PCR

  17. Genome-Wide Diversity and Phylogeography of Mycobacterium avium subsp. paratuberculosis in Canadian Dairy Cattle

    PubMed Central

    Ahlstrom, Christina; Barkema, Herman W.; Stevenson, Karen; Zadoks, Ruth N.; Biek, Roman; Kao, Rowland; Trewby, Hannah; Haupstein, Deb; Kelton, David F.; Fecteau, Gilles; Labrecque, Olivia; Keefe, Greg P.; McKenna, Shawn L. B.; Tahlan, Kapil; De Buck, Jeroen

    2016-01-01

    Mycobacterium avium subsp. paratuberculosis (MAP) is the causative bacterium of Johne’s disease (JD) in ruminants. The control of JD in the dairy industry is challenging, but can be improved with a better understanding of the diversity and distribution of MAP subtypes. Previously established molecular typing techniques used to differentiate MAP have not been sufficiently discriminatory and/or reliable to accurately assess the population structure. In this study, the genetic diversity of 182 MAP isolates representing all Canadian provinces was compared to the known global diversity, using single nucleotide polymorphisms identified through whole genome sequencing. MAP isolates from Canada represented a subset of the known global diversity, as there were global isolates intermingled with Canadian isolates, as well as multiple global subtypes that were not found in Canada. One Type III and six “Bison type” isolates were found in Canada as well as one Type II subtype that represented 86% of all Canadian isolates. Rarefaction estimated larger subtype richness in Québec than in other Canadian provinces using a strict definition of MAP subtypes and lower subtype richness in the Atlantic region using a relaxed definition. Significant phylogeographic clustering was observed at the inter-provincial but not at the intra-provincial level, although most major clades were found in all provinces. The large number of shared subtypes among provinces suggests that cattle movement is a major driver of MAP transmission at the herd level, which is further supported by the lack of spatial clustering on an intra-provincial scale. PMID:26871723

  18. Acyl-Homoserine Lactone Quorum Sensing in the Roseobacter Clade

    PubMed Central

    Zan, Jindong; Liu, Yue; Fuqua, Clay; Hill, Russell T.

    2014-01-01

    Members of the Roseobacter clade are ecologically important and numerically abundant in coastal environments and can associate with marine invertebrates and nutrient-rich marine snow or organic particles, on which quorum sensing (QS) may play an important role. In this review, we summarize current research progress on roseobacterial acyl-homoserine lactone-based QS, particularly focusing on three relatively well-studied representatives, Phaeobacter inhibens DSM17395, the marine sponge symbiont Ruegeria sp. KLH11 and the dinoflagellate symbiont Dinoroseobacter shibae. Bioinformatic survey of luxI homologues revealed that over 80% of available roseobacterial genomes encode at least one luxI homologue, reflecting the significance of QS controlled regulatory pathways in adapting to the relevant marine environments. We also discuss several areas that warrant further investigation, including studies on the ecological role of these diverse QS pathways in natural environments. PMID:24402124

  19. Differential niche dynamics among major marine invertebrate clades.

    PubMed

    Hopkins, Melanie J; Simpson, Carl; Kiessling, Wolfgang

    2014-03-01

    The degree to which organisms retain their environmental preferences is of utmost importance in predicting their fate in a world of rapid climate change. Notably, marine invertebrates frequently show strong affinities for either carbonate or terrigenous clastic environments. This affinity is due to characteristics of the sediments as well as correlated environmental factors. We assessed the conservatism of substrate affinities of marine invertebrates over geological timescales, and found that niche conservatism is prevalent in the oceans, and largely determined by the strength of initial habitat preference. There is substantial variation in niche conservatism among major clades with corals and sponges being among the most conservative. Time-series analysis suggests that niche conservatism is enhanced during times of elevated nutrient flux, whereas niche evolution tends to occur after mass extinctions. Niche evolution is not necessarily elevated in genera exhibiting higher turnover in species composition.

  20. Differential niche dynamics among major marine invertebrate clades

    PubMed Central

    Hopkins, Melanie J; Simpson, Carl; Kiessling, Wolfgang

    2014-01-01

    The degree to which organisms retain their environmental preferences is of utmost importance in predicting their fate in a world of rapid climate change. Notably, marine invertebrates frequently show strong affinities for either carbonate or terrigenous clastic environments. This affinity is due to characteristics of the sediments as well as correlated environmental factors. We assessed the conservatism of substrate affinities of marine invertebrates over geological timescales, and found that niche conservatism is prevalent in the oceans, and largely determined by the strength of initial habitat preference. There is substantial variation in niche conservatism among major clades with corals and sponges being among the most conservative. Time-series analysis suggests that niche conservatism is enhanced during times of elevated nutrient flux, whereas niche evolution tends to occur after mass extinctions. Niche evolution is not necessarily elevated in genera exhibiting higher turnover in species composition. PMID:24313951

  1. Evaluation of the FilmArray® system for detection of Bacillus anthracis, Francisella tularensis, and Yersinia pestis

    SciTech Connect

    Seiner, Derrick R.; Colburn, Heather A.; Baird, Cheryl L.; Bartholomew, Rachel A.; Straub, Tim M.; Victry, Kristin D.; Hutchison, Janine R.; Valentine, Nancy B.; Bruckner-Lea, Cindy J.

    2013-04-29

    To evaluate the sensitivity and specificity of the Idaho Technologies FilmArray® Biothreat Panel for the detection of Bacillus anthracis (Ba), Francisella tularensis (Ft), and Yersinia pestis (Yp) DNA, and demonstrate the detection of Ba spores. Methods and Results: DNA samples from Ba, Ft and Yp strains and near-neighbors, and live Ba spores were analyzed using the Biothreat Panel, a multiplexed PCR-based assay for 17 pathogens and toxins. Sensitivity studies with DNA suggest a limit of detection of 250 genome equivalents (GEs) per sample. Furthermore, the correct call of Ft, Yp or Bacillus species was made in 63 of 72 samples tested at 25 GE or less. With samples containing 25 Ba Sterne spores, at least one of the two possible Ba markers were identified in all samples tested. We observed no cross-reactivity with near-neighbor DNAs.

  2. The Role of the Francisella Tularensis Pathogenicity Island in Type VI Secretion, Intracellular Survival, and Modulation of Host Cell Signaling

    PubMed Central

    Bröms, Jeanette E.; Sjöstedt, Anders; Lavander, Moa

    2010-01-01

    Francisella tularensis is a highly virulent gram-negative intracellular bacterium that causes the zoonotic disease tularemia. Essential for its virulence is the ability to multiply within host cells, in particular monocytic cells. The bacterium has developed intricate means to subvert host immune mechanisms and thereby facilitate its intracellular survival by preventing phagolysosomal fusion followed by escape into the cytosol, where it multiplies. Moreover, it targets and manipulates numerous host cell signaling pathways, thereby ameliorating the otherwise bactericidal capacity. Many of the underlying molecular mechanisms still remain unknown but key elements, directly or indirectly responsible for many of the aforementioned mechanisms, rely on the expression of proteins encoded by the Francisella pathogenicity island (FPI), suggested to constitute a type VI secretion system. We here describe the current knowledge regarding the components of the FPI and the roles that have been ascribed to them. PMID:21687753

  3. A Molecular Survey for Francisella tularensis and Rickettsia spp. in Haemaphysalis leporispalustris (Acari: Ixodidae) in Northern California.

    PubMed

    Roth, Tara; Lane, Robert S; Foley, Janet

    2016-12-28

    Francisella tularensis and Rickettsia spp. have been cultured from Haemaphysalis leporispalustris Packard, but their prevalence in this tick has not been determined using modern molecular methods. We collected H. leporispalustris by flagging vegetation and leaf litter and from lagomorphs (Lepus californicus Gray and Sylvilagus bachmani (Waterhouse)) in northern California. Francisella tularensis DNA was not detected in any of 1,030 ticks tested by polymerase chain reaction (PCR), whereas 0.4% of larvae tested in pools, 0 of 117 individual nymphs, and 2.3% of 164 adult ticks were PCR-positive for Rickettsia spp. Positive sites were Laurel Canyon Trail in Tilden Regional Park in Alameda Contra Costa County, with a Rickettsia spp. prevalence of 0.6% in 2009, and Hopland Research and Extension Center in Mendocino County, with a prevalence of 4.2% in 1988. DNA sequencing revealed R. felis, the agent of cat-flea typhus, in two larval pools from shaded California bay and live oak leaf litter in Contra Costa County and one adult tick from a L. californicus in chaparral in Mendocino County. The R. felis in unfed, questing larvae demonstrates that H. leporispalustris can transmit this rickettsia transovarially. Although R. felis is increasingly found in diverse arthropods and geographical regions, prior literature suggests a typical epidemiological cycle involving mesocarnivores and the cat flea, Ctenocephalides felis. To our knowledge, this is the first report of R. felis in H. leporispalustris. Natural infection and transovarial transmission of this pathogen in the tick indicate the existence of a previously undocumented wild-lands transmission cycle that may intersect mesocarnivore-reservoired cycles and collectively affect human health risk.

  4. Rapid Focused Sequencing: A Multiplexed Assay for Simultaneous Detection and Strain Typing of Bacillus anthracis, Francisella tularensis, and Yersinia pestis

    PubMed Central

    Zolotova, Anna; Tan, Eugene; Selden, Richard F.

    2013-01-01

    Background The intentional release of Bacillus anthracis in the United States in 2001 has heightened concern about the use of pathogenic microorganisms in bioterrorism attacks. Many of the deadliest bacteria, including the Class A Select Agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis, are highly infectious via the pulmonary route when released in aerosolized form. Hence, rapid, sensitive, and reliable methods for detection of these biothreats and characterization of their potential impact on the exposed population are of critical importance to initiate and support rapid military, public health, and clinical responses. Methodology/Principal Findings We have developed microfluidic multiplexed PCR and sequencing assays based on the simultaneous interrogation of three pathogens per assay and ten loci per pathogen. Microfluidic separation of amplified fluorescently labeled fragments generated characteristic electrophoretic signatures for identification of each agent. The three sets of primers allowed significant strain typing and discrimination from non-pathogenic closely-related species and environmental background strains based on amplicon sizes alone. Furthermore, sequencing of the 10 amplicons per pathogen, termed “Rapid Focused Sequencing,” allowed an even greater degree of strain discrimination and, in some cases, can be used to determine virulence. Both amplification and sequencing assays were performed in microfluidic biochips developed for fast thermal cycling and requiring 7 µL per reaction. The 30-plex sequencing assay resulted in genotypic resolution of 84 representative strains belonging to each of the three biothreat species. Conclusions/Significance The microfluidic multiplexed assays allowed identification and strain differentiation of the biothreat agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis and clear discrimination from closely-related species and several environmental background strains. The

  5. Reproducible and Quantitative Model of Infection of Dermacentor variabilis with the Live Vaccine Strain of Francisella tularensis

    PubMed Central

    Coburn, Jenifer; Maier, Tamara; Casey, Monika; Padmore, Lavinia; Sato, Hiromi

    2014-01-01

    Pathogen life cycles in mammalian hosts have been studied extensively, but studies with arthropod vectors represent considerable challenges. In part this is due to the difficulty of delivering a reproducible dose of bacteria to follow arthropod-associated replication. We have established reproducible techniques to introduce known numbers of Francisella tularensis strain LVS from mice into Dermacentor variabilis nymphs. Using this model infection system, we performed dose-response infection experiments and followed bacterial replication through the molt to adults and at later time points. During development to adults, bacteria replicate to high numbers and can be found associated with the gut tissues, salivary glands, and hemolymph of adult ticks. Further, we can transmit a mutant of LVS (LVS ΔpurMCD) that cannot replicate in macrophages in vitro or in mice to nymphs. Our data show that the LVS ΔpurMCD mutant cannot be transstadially transmitted from nymphs to adult ticks. We then show that a plasmid-complemented strain of this mutant is recoverable in adult ticks and necessary for bacterial replication during the molt. In a mixed-infection assay (ΔpurMCD mutant versus ΔpurMCD complement), 98% of the recovered bacteria retained the plasmid marker. These data suggest that the ΔpurMCD mutation cannot be rescued by the presence a complemented strain in a mixed infection. Importantly, our infection model provides a platform to test specific mutants for their replication in ticks, perform competition studies, and use other genetic techniques to identify F. tularensis genes that are expressed or required in this unique environment. PMID:25362054

  6. Development of functional and molecular correlates of vaccine-induced protection for a model intracellular pathogen, F. tularensis LVS.

    PubMed

    De Pascalis, Roberto; Chou, Alicia Y; Bosio, Catharine M; Huang, Chiung-Yu; Follmann, Dean A; Elkins, Karen L

    2012-01-01

    In contrast with common human infections for which vaccine efficacy can be evaluated directly in field studies, alternative strategies are needed to evaluate efficacy for slowly developing or sporadic diseases like tularemia. For diseases such as these caused by intracellular bacteria, serological measures of antibodies are generally not predictive. Here, we used vaccines varying in efficacy to explore development of clinically useful correlates of protection for intracellular bacteria, using Francisella tularensis as an experimental model. F. tularensis is an intracellular bacterium classified as Category A bioterrorism agent which causes tularemia. The primary vaccine candidate in the U.S., called Live Vaccine Strain (LVS), has been the subject of ongoing clinical studies; however, safety and efficacy are not well established, and LVS is not licensed by the U.S. FDA. Using a mouse model, we compared the in vivo efficacy of a panel of qualitatively different Francisella vaccine candidates, the in vitro functional activity of immune lymphocytes derived from vaccinated mice, and relative gene expression in immune lymphocytes. Integrated analyses showed that the hierarchy of protection in vivo engendered by qualitatively different vaccines was reflected by the degree of lymphocytes' in vitro activity in controlling the intramacrophage growth of Francisella. Thus, this assay may be a functional correlate. Further, the strength of protection was significantly related to the degree of up-regulation of expression of a panel of genes in cells recovered from the assay. These included IFN-γ, IL-6, IL-12Rβ2, T-bet, SOCS-1, and IL-18bp. Taken together, the results indicate that an in vitro assay that detects control of bacterial growth, and/or a selected panel of mediators, may ultimately be developed to predict the outcome of vaccine efficacy and to complement clinical trials. The overall approach may be applicable to intracellular pathogens in general.

  7. Insights into the activity of maturation inhibitor PF-46396 on HIV-1 clade C

    PubMed Central

    Ghimire, Dibya; Timilsina, Uddhav; Srivastava, Tryambak Pratap; Gaur, Ritu

    2017-01-01

    HIV maturation inhibitors are an emerging class of anti-retroviral compounds that inhibit the viral protease-mediated cleavage of the Gag, CA-SP1 (capsid-spacer peptide 1) peptide to mature CA. The first-in-class maturation inhibitor bevirimat (BVM) displayed potent activity against HIV-1 clade B but was ineffective against other HIV-1 clades including clade C. Another pyridone-based maturation inhibitor, PF-46396 displayed potent activity against HIV-1 clade B. In this study, we aimed at determining the activity of PF-46396 against HIV-1 clade C. We employed various biochemical and virological assays to demonstrate that PF-46396 is effective against HIV-1 clade C. We observed a dose dependent accumulation of CA-SP1 intermediate in presence of the compound. We carried out mutagenesis in the CA- SP1 region of HIV-1 clade C Gag and observed that the mutations conferred resistance against the compound. Many mutations inhibited Gag processing thereby reducing virus release in the absence of the compound. However, presence of PF-46396 rescued these defects and enhanced virus release, replication capacity and infectivity of HIV-1 clade C. These results put together identify PF-46396 as a broadly active maturation inhibitor against HIV-1 clade B and C and help in rational designing of novel analogs with reduced toxicity and increased efficacy for its potential use in clinics. PMID:28252110

  8. Draft Genome Sequences for Canadian Isolates of Pectobacterium carotovorum subsp. brasiliense with Weak Virulence on Potato

    PubMed Central

    Yuan, Kat (Xiaoli); Cullis, Jeff; Lévesque, C. André; Chen, Wen; Lewis, Christopher T.; De Boer, Solke H.

    2015-01-01

    Pectobacterium carotovurum subsp. brasiliense causes soft rot and blackleg diseases on potato. Here, we report the draft genome sequences of three weakly virulent P. carotovurum subsp. brasiliense strains isolated in Canada. Analysis of these genome sequences will help to pinpoint differences in virulence among P. carotovurum subsp. brasiliense strains from tropical/subtropical and temperate regions, such as Canada and United States. A small number of key factors for adaptation to this bacterium's specific environmental niche were also evaluated. PMID:25858837

  9. Draft Genome Sequences of Salmonella enterica subsp. enterica Serovars Typhimurium and Nottingham Isolated from Food Products

    PubMed Central

    Zheng, Jie; Ayers, Sherry; Melka, David C.; Curry, Phillip E.; Payne, Justin S.; Laasri, Anna; Wang, Charles; Hammack, Thomas S.; Brown, Eric W.

    2016-01-01

    A quantitative real-time PCR (qPCR) designed to detect Salmonella enterica subsp. enterica serovar Enteritidis, targeting the sdf gene, generated positive results for S. enterica subsp. enterica serovar Typhimurium (CFSAN033950) and S. enterica subsp. enterica serovar Nottingham (CFSAN006803) isolated from food samples. Both strains show pulsed-field gel electrophoresis (PFGE) patterns distinct from those of S. Enteritidis. Here, we report the genome sequences of these two strains. PMID:27445384

  10. Genetic Basis of Tetracycline Resistance in Bifidobacterium animalis subsp. lactis▿

    PubMed Central

    Gueimonde, Miguel; Flórez, Ana Belén; van Hoek, Angela H. A. M.; Stuer-Lauridsen, Birgitte; Strøman, Per; de los Reyes-Gavilán, Clara G.; Margolles, Abelardo

    2010-01-01

    All strains of Bifidobacterium animalis subsp. lactis described to date show medium level resistance to tetracycline. Screening of 26 strains from a variety of sources revealed the presence of tet(W) in all isolates. A transposase gene upstream of tet(W) was found in all strains, and both genes were cotranscribed in strain IPLAIC4. Mutants with increased tetracycline resistance as well as tetracycline-sensitive mutants of IPLAIC4 were isolated and genetically characterized. The native tet(W) gene was able to restore the resistance phenotype to a mutant with an alteration in tet(W) by functional complementation, indicating that tet(W) is necessary and sufficient for the tetracycline resistance seen in B. animalis subsp. lactis. PMID:20348299

  11. Allelopathic activity of Nepeta nuda L. subsp. nuda water extracts

    NASA Astrophysics Data System (ADS)

    Dragoeva, Asya; Stoyanova, Zheni; Koleva, Vanya; Dragolova, Daniela

    2017-03-01

    Nepeta nuda subsp. nuda is a medicinal plant growing wild in Bulgaria. Different species of Nepeta genus have been reported to possess allelopathic potential. The present study was conducted to observe its phytotoxic effects on T. aestivum and C. sativus L. seeds in laboratory conditions. Nepeta water extracts (NWE) prepared from aerial parts of plants at concentrations 2, 4, 6, 8, 10, 12 and 14 g/l were tested. The rate of seed germination, the root and shoot length, fresh and dry weight of seedlings were observed after treatment with NWE. As a control served seeds treated with distilled water. Germination was not affected, but NWE showed deterioration in seedling growth. Roots were more affected than shoots. The fresh and dry weights were reduced upon treatment with the extracts tested. These negative effects were dose-dependent. The overall results indicate presence of water soluble allelochemicals in Nepeta nuda subsp. nuda.

  12. Thermal Inactivation of Mycobacterium avium subsp. paratuberculosis in Artificially Contaminated Milk by Direct Steam Injection

    PubMed Central

    Butot, Sophie; Jagadeesan, Balamurugan; Bakker, Douwe; Donaghy, John

    2016-01-01

    ABSTRACT The efficiency of direct steam injection (DSI) at 105°C for 3 s to inactivate Mycobacterium avium subsp. paratuberculosis in milk at a pilot-plant scale was investigated. Milk samples were artificially contaminated with M. avium subsp. paratuberculosis and also with cow fecal material naturally infected with M. avium subsp. paratuberculosis. We also tested milk artificially contaminated with Mycobacterium smegmatis as a candidate surrogate to compare thermal inactivation between M. smegmatis and M. avium subsp. paratuberculosis. Following the DSI process, no viable M. avium subsp. paratuberculosis or M. smegmatis was recovered using culture methods for both strains. For pure M. avium subsp. paratuberculosis cultures, a minimum reduction of 5.6 log10 was achieved with DSI, and a minimum reduction of 5.7 log10 was found with M. smegmatis. The minimum log10 reduction for wild-type M. avium subsp. paratuberculosis naturally present in feces was 3.3. In addition, 44 dairy and nondairy powdered infant formula (PIF) ingredients used during the manufacturing process of PIF were tested for an alternate source for M. avium subsp. paratuberculosis and were found to be negative by quantitative PCR (qPCR). In conclusion, the results obtained from this study indicate that a >7-fold-log10 reduction of M. avium subsp. paratuberculosis in milk can be achieved with the applied DSI process. IMPORTANCE M. avium subsp. paratuberculosis is widespread in dairy herds in many countries. M. avium subsp. paratuberculosis is the causative agent of Johne's disease in cattle, and infected animals can directly or indirectly (i.e., fecal contamination) contaminate milk. Despite much research and debate, there is no conclusive evidence that M. avium subsp. paratuberculosis is a zoonotic bacterium, i.e., one that causes disease in humans. The presence of M. avium subsp. paratuberculosis or its DNA has been reported in dairy products, including pasteurized milk, cheese, and infant formula

  13. Isolation by genomic subtraction of DNA probes specific for Erwinia carotovora subsp. atroseptica.

    PubMed Central

    Darrasse, A; Kotoujansky, A; Bertheau, Y

    1994-01-01

    Erwinia carotovora subsp. atroseptica is a pathogen of potatoes in Europe because of its ability to induce blackleg symptoms early in the growing season. However, E. carotovora subsp. carotovora is not able to produce such severe symptoms under the same conditions. On the basis of the technique described by Straus and Ausubel (Proc. Natl. Acad. Sci. USA 87:1889-1893, 1990), we isolated DNA sequences of E. carotovora subsp. atroseptica 86.20 that were absent from the genomic DNA of E. carotovora subsp. carotovora CH26. Six DNA fragments ranging from ca. 180 to 400 bp were isolated, cloned, and sequenced. Each fragment was further hybridized with 130 microorganisms including 87 E. carotovora strains. One probe was specific for typical E. carotovora subsp. atroseptica strains, two probes hybridized with all E. carotovora subsp. atroseptica strains and with a few E. carotovora subsp. carotovora strains, and two probes recognized only a subset of E. carotovora subsp. atroseptica strains. The last probe was absent from the genomic DNA of E. carotovora subsp. carotovora CH26 but was present in the genomes of many strains, including those of other species and genera. This probe is homologous to the putP gene of Escherichia coli, which encodes a proline carrier. Further use of the probes is discussed. Images PMID:8117082

  14. Characterization of the arginine deiminase of Streptococcus equi subsp. zooepidemicus.

    PubMed

    Hong, Kyongsu

    2006-09-01

    Streptococcus equi subsp. zooepidemicus is an important cause of infectious diseases in horses and rarely humans. Little is known about the virulence factors or protective antigens of S. equi subsp. zooepidemicus. In the present study, I designed original primers based on an alignment of the gene sagp(arcA) from Streptococcus pyogenes encoding streptococcal acid glycoprotein-arginine deiminase (SAGP/AD) to amplify the S. equi subsp. zooepidemicus counterpart sequence by polymerase chain reaction, and I analyzed the sagp(arcA) gene of the organism. Using chromosomal walking steps, I identified a contiguous eight-gene locus involved in SAGP/AD production. Their open reading frames were found to share significant homologies and to correspond closely in molecular mass to previously sequenced arc genes of S. pyogenes, thus they were designated ahrC.2 (arginine repressor), arcR (CRP/FNR transcription regulator), sagp(arcA) (streptococcal acid glycoprotein-arginine deiminase), putative acetyltransferase gene, arcB (ornithine carbamyl transferase), arcD (arginine-ornithine antiporter), arcT (Xaa-His peptidase), and arcC (carbamate kinase). The SAGP homologue of S. equi subsp. zooepidemicus (SzSAGP), encoded by arcA gene of the bacteria (arcA(SZ)), was successfully expressed in Escherichia coli and purified to homogeneity. When in vitro growth inhibitory activity of the recombinant SzSAGP was tested against MOLT-3 cells, it inhibited the growth of the cells during the 3 days of culture in a dose-dependent manner, accompanied by the induction of apoptotic cell death. The recombinant protein also possessed AD activity. By immunoblot analysis using both anti-SzSAGP-SfbI(H8) and anti-SfbI(H8) sera, I was able to demonstrate that the SzSAGP protein is expressed on the streptococcal surface.

  15. Nanolipoprotein Particles (NLPs) as Versatile Vaccine Platforms for Co-delivery of Multiple Adjuvants with Subunit Antigens from Burkholderia spp. and F. tularensis - Technical Report

    SciTech Connect

    Fischer, N. O.

    2015-01-13

    The goal of this proposal is to demonstrate that colocalization of protein subunit antigens and adjuvants on nanolipoprotein particles (NLPs) can increase the protective efficacy of subunit antigens from Burkholderia spp. and Francisella tularensis against an aerosol challenge. In the third quarter of the third year, F344 rats vaccinated with adjuvanted NLP formulations were challenged with F. tularensis SCHU S4 at Battelle. Preliminary data indicate that up to 65% of females vaccinated intranasally with an NLP-based formulation survived this challenge, compared to only 20% survival of naïve animals. In addition, NLPs were successfully formulated with Burkholderia protein antigens. IACUC approval for immunological assessments in BALB/c mice was received and we anticipate that these assessments will begin by March 2015, pending ACURO approval.

  16. Structural and biological evaluation of a novel series of benzimidazole inhibitors of Francisella tularensis enoyl-ACP reductase (FabI).

    PubMed

    Mehboob, Shahila; Song, Jinhua; Hevener, Kirk E; Su, Pin-Chih; Boci, Teuta; Brubaker, Libby; Truong, Lena; Mistry, Tina; Deng, Jiangping; Cook, James L; Santarsiero, Bernard D; Ghosh, Arun K; Johnson, Michael E

    2015-03-15

    Francisella tularensis, the causative agent of tularemia, presents a significant biological threat and is a Category A priority pathogen due to its potential for weaponization. The bacterial FASII pathway is a viable target for the development of novel antibacterial agents treating Gram-negative infections. Here we report the advancement of a promising series of benzimidazole FabI (enoyl-ACP reductase) inhibitors to a second-generation using a systematic, structure-guided lead optimization strategy, and the determination of several co-crystal structures that confirm the binding mode of designed inhibitors. These compounds display an improved low nanomolar enzymatic activity as well as promising low microgram/mL antibacterial activity against both F. tularensis and Staphylococcus aureus and its methicillin-resistant strain (MRSA). The improvements in activity accompanying structural modifications lead to a better understanding of the relationship between the chemical structure and biological activity that encompasses both enzymatic and whole-cell activity.

  17. Structural and Enzymatic Analyses Reveal the Binding Mode of a Novel Series of Francisella tularensis Enoyl Reductase (FabI) Inhibitors

    SciTech Connect

    Mehboob, Shahila; Hevener, Kirk E.; Truong, Kent; Boci, Teuta; Santarsiero, Bernard D.; Johnson, Michael E.

    2012-10-10

    Because of structural and mechanistic differences between eukaryotic and prokaryotic fatty acid synthesis enzymes, the bacterial pathway, FAS-II, is an attractive target for the design of antimicrobial agents. We have previously reported the identification of a novel series of benzimidazole compounds with particularly good antibacterial effect against Francisella tularensis, a Category A biowarfare pathogen. Herein we report the crystal structure of the F. tularensis FabI enzyme in complex with our most active benzimidazole compound bound with NADH. The structure reveals that the benzimidazole compounds bind to the substrate site in a unique conformation that is distinct from the binding motif of other known FabI inhibitors. Detailed inhibition kinetics have confirmed that the compounds possess a novel inhibitory mechanism that is unique among known FabI inhibitors. These studies could have a strong impact on future antimicrobial design efforts and may reveal new avenues for the design of FAS-II active antibacterial compounds.

  18. Serological investigation of wild boars (Sus scrofa) and red foxes (Vulpes vulpes) as indicator animals for circulation of Francisella tularensis in Germany.

    PubMed

    Otto, Peter; Chaignat, Valerie; Klimpel, Diana; Diller, Roland; Melzer, Falk; Müller, Wolfgang; Tomaso, Herbert

    2014-01-01

    Tularemia outbreaks in humans have recently been reported in many European countries, but data on the occurrence in the animal population are scarce. In North America, seroconversion of omnivores and carnivores was used as indicator for the presence of tularemia, for the European fauna, however, data are barely available. Therefore, the suitability of wild boars (Sus scrofa) and red foxes (Vulpes vulpes) as indicators for the circulation of F. tularensis in Germany was evaluated. Serum samples from 566 wild boars and 457 red foxes were collected between 1995 and 2012 in three federal states in Central Germany (Hesse, Saxony-Anhalt, and Thuringia). The overall rate of seropositive animals was 1.1% in wild boars and 7.4% in red foxes. In conclusion, serological examination of red foxes is recommended, because they can be reliably used as indicator animals for the presence of F. tularensis in the environment.

  19. The complexity and diversity of the Pathogenicity Locus in Clostridium difficile clade 5.

    PubMed

    Elliott, Briony; Dingle, Kate E; Didelot, Xavier; Crook, Derrick W; Riley, Thomas V

    2014-11-08

    The symptoms of Clostridium difficile infection are caused by two closely related toxins, TcdA and TcdB, which are encoded by the 19.6 kb Pathogenicity Locus (PaLoc). The PaLoc is variably present among strains, and in this respect it resembles a mobile genetic element. The C. difficile population structure consists mainly of five phylogenetic clades designated 1-5. Certain genotypes of clade 5 are associated with recently emergent highly pathogenic strains causing human disease and animal infections. The aim of this study was to explore the evolutionary history of the PaLoc in C. difficile clade 5. Phylogenetic analyses and annotation of clade 5 PaLoc variants and adjoining genomic regions were undertaken using a representative collection of toxigenic and nontoxigenic strains. Comparison of the core genome and PaLoc phylogenies obtained for clade 5 and representatives of the other clades identified two distinct PaLoc acquisition events, one involving a toxin A(+)B(+) PaLoc variant and the other an A(-)B(+) variant. Although the exact mechanism of each PaLoc acquisition is unclear, evidence of possible homologous recombination with other clades and between clade 5 lineages was found within the PaLoc and adjacent regions. The generation of nontoxigenic variants by PaLoc loss via homologous recombination with PaLoc-negative members of other clades was suggested by analysis of cdu2, although none is likely to have occurred recently. A variant of the putative holin gene present in the clade 5 A(-)B(+) PaLoc was likely acquired via allelic exchange with an unknown element. Fine-scale phylogenetic analysis of C. difficile clade 5 revealed the extent of its genetic diversity, consistent with ancient evolutionary origins and a complex evolutionary history for the PaLoc.

  20. Francisella tularensis Schu S4 lipopolysaccharide core sugar and O-antigen mutants are attenuated in a mouse model of tularemia.

    PubMed

    Rasmussen, Jed A; Post, Deborah M B; Gibson, Bradford W; Lindemann, Stephen R; Apicella, Michael A; Meyerholz, David K; Jones, Bradley D

    2014-04-01

    The virulence factors mediating Francisella pathogenesis are being investigated, with an emphasis on understanding how the organism evades innate immunity mechanisms. Francisella tularensis produces a lipopolysaccharide (LPS) that is essentially inert and a polysaccharide capsule that helps the organism to evade detection by components of innate immunity. Using an F. tularensis Schu S4 mutant library, we identified strains that are disrupted for capsule and O-antigen production. These serum-sensitive strains lack both capsule production and O-antigen laddering. Analysis of the predicted protein sequences for the disrupted genes (FTT1236 and FTT1238c) revealed similarity to those for waa (rfa) biosynthetic genes in other bacteria. Mass spectrometry further revealed that these proteins are involved in LPS core sugar biosynthesis and the ligation of O antigen to the LPS core sugars. The 50% lethal dose (LD50) values of these strains are increased 100- to 1,000-fold for mice. Histopathology revealed that the immune response to the F. tularensis mutant strains was significantly different from that observed with wild-type-infected mice. The lung tissue from mutant-infected mice had widespread necrotic debris, but the spleens lacked necrosis and displayed neutrophilia. In contrast, the lungs of wild-type-infected mice had nominal necrosis, but the spleens had widespread necrosis. These data indicate that murine death caused by wild-type strains occurs by a mechanism different from that by which the mutant strains kill mice. Mice immunized with these mutant strains displayed >10-fold protective effects against virulent type A F. tularensis challenge.

  1. Francisella tularensis Schu S4 Lipopolysaccharide Core Sugar and O-Antigen Mutants Are Attenuated in a Mouse Model of Tularemia

    PubMed Central

    Rasmussen, Jed A.; Post, Deborah M. B.; Gibson, Bradford W.; Lindemann, Stephen R.; Apicella, Michael A.; Meyerholz, David K.

    2014-01-01

    The virulence factors mediating Francisella pathogenesis are being investigated, with an emphasis on understanding how the organism evades innate immunity mechanisms. Francisella tularensis produces a lipopolysaccharide (LPS) that is essentially inert and a polysaccharide capsule that helps the organism to evade detection by components of innate immunity. Using an F. tularensis Schu S4 mutant library, we identified strains that are disrupted for capsule and O-antigen production. These serum-sensitive strains lack both capsule production and O-antigen laddering. Analysis of the predicted protein sequences for the disrupted genes (FTT1236 and FTT1238c) revealed similarity to those for waa (rfa) biosynthetic genes in other bacteria. Mass spectrometry further revealed that these proteins are involved in LPS core sugar biosynthesis and the ligation of O antigen to the LPS core sugars. The 50% lethal dose (LD50) values of these strains are increased 100- to 1,000-fold for mice. Histopathology revealed that the immune response to the F. tularensis mutant strains was significantly different from that observed with wild-type-infected mice. The lung tissue from mutant-infected mice had widespread necrotic debris, but the spleens lacked necrosis and displayed neutrophilia. In contrast, the lungs of wild-type-infected mice had nominal necrosis, but the spleens had widespread necrosis. These data indicate that murine death caused by wild-type strains occurs by a mechanism different from that by which the mutant strains kill mice. Mice immunized with these mutant strains displayed >10-fold protective effects against virulent type A F. tularensis challenge. PMID:24452684

  2. Proposal for 'Candidatus Mycoplasma haemomuris subsp. musculi' in mice, and 'Candidatus Mycoplasma haemomuris subsp. ratti' in rats.

    PubMed

    Harasawa, Ryô; Fujita, Hiromi; Kadosaka, Teruki; Ando, Shuji; Rikihisa, Yasuko

    2015-02-01

    Mycoplasma haemomuris is causative of infectious anaemia or splenomegaly in rodents. We examined the nucleotide sequences of the non-ribosomal genes, rnpB and dnaK, in strains of the species M. haemomuris detected in small field mice and black rats. rnpB nucleotide sequences in strains of the species M. haemomuris isolated from small field mice and black rats had only 89 % sequence similarity, suggesting their separation into two distinct subgroups. dnaK had a nucleotide sequence similarity of 84 % between the subgroups. These results support the classification of M. haemomuris into two genetically distinct subgroups. Here we propose the establishment of these subgroups as 'Candidatus Mycoplasma haemomuris subsp. musculi', detected in small field mice (Apodemus argenteus), and 'Candidatus Mycoplasma haemomuris subsp. ratti', detected in black rats (Rattus rattus).

  3. Staphylococcus schleiferi subsp. coagulans subsp. nov., isolated from the external auditory meatus of dogs with external ear otitis.

    PubMed

    Igimi, S; Takahashi, E; Mitsuoka, T

    1990-10-01

    A new subspecies, Staphylococcus schleiferi subsp. coagulans, was isolated from the external auditory meatus of dogs suffering from external ear otitis and is described on the basis of studies of 21 strains. Phenotypic studies showed that these strains are more closely related to Staphylococcus intermedius than to other staphylococci, but DNA hybridization studies indicated that they are closely related to Staphylococcus schleiferi N850274T. On the basis of biochemical distinctiveness (positive test tube coagulase test and different carbohydrate reactions) and the etiological importance (frequent isolation from otitis specimens from dogs) of these strains, we propose to classify them as a subspecies of S. schleiferi. The strains of this new subspecies are coagulase tube test, beta-hemolysin, and heat-stable nuclease positive but clumping factor negative. A simple scheme for the differentiation of S. schleiferi subsp. coagulans from the other coagulase-positive staphylococci is presented. The type strain is GA211 (= JCM 7470).

  4. Identification of Genome-Wide Mutations in Ciprofloxacin-Resistant F. tularensis LVS Using Whole Genome Tiling Arrays and Next Generation Sequencing

    DOE PAGES

    Jaing, Crystal J.; McLoughlin, Kevin S.; Thissen, James B.; ...

    2016-09-26

    Francisella tularensis is classified as a Class A bioterrorism agent by the U.S. government due to its high virulence and the ease with which it can be spread as an aerosol. It is a facultative intracellular pathogen and the causative agent of tularemia. Ciprofloxacin (Cipro) is a broad spectrum antibiotic effective against Gram-positive and Gram-negative bacteria. Increased Cipro resistance in pathogenic microbes is of serious concern when considering options for medical treatment of bacterial infections. Identification of genes and loci that are associated with Ciprofloxacin resistance will help advance the understanding of resistance mechanisms and may, in the future, providemore » better treatment options for patients. It may also provide information for development of assays that can rapidly identify Cipro-resistant isolates of this pathogen. In this study, we then selected a large number of F. tularensis live vaccine strain (LVS) isolates that survived in progressively higher Ciprofloxacin concentrations, screened the isolates using a whole genome F. tularensis LVS tiling microarray and Illumina sequencing, and identified both known and novel mutations associated with resistance. For genes containing mutations encode DNA gyrase subunit A, a hypothetical protein, an asparagine synthase, a sugar transamine/perosamine synthetase and others. Finally, structural modeling performed on these proteins provides insights into the potential function of these proteins and how they might contribute to Cipro resistance mechanisms.« less

  5. Nanolipoprotein Particles (NLPs) as Versatile Vaccine Platforms for Co-delivery of Multiple Adjuvants with Subunit Antigens from Burkholderia spp. and F. tularensis - Annual Technical Report

    SciTech Connect

    Fischer, N. O.

    2015-04-16

    The goal of this proposal is to demonstrate that co-localization of protein subunit antigens and adjuvants on nanolipoprotein particles (NLPs) can increase the protective efficacy of recombinant subunit antigens from Burkholderia spp. and Francisella tularensis against an aerosol challenge. NLPs are are biocompatible, high-density lipoprotein mimetics that are amenable to the incorporation of multiple, chemically-disparate adjuvant and antigen molecules. We hypothesize that the ability to co-localize optimized adjuvant formulations with subunit antigens within a single particle will enhance the stimulation and activation of key immune effector cells, increasing the protective efficacy of subunit antigen-based vaccines. While Burkholderia spp. and F. tularensis subunit antigens are the focus of this proposal, we anticipate that this approach is applicable to a wide range of DOD-relevant biothreat agents. The F344 rat aerosol challenge model for F. tularensis has been successfully established at Battelle under this contract, and Year 3 efficacy studies performed at Battelle demonstrated that an NLP vaccine formulation was able to enhance survival of female F344 rats relative to naïve animals. In addition, Year 3 focused on the incorporation of multiple Burkholderia antigens (both polysaccharides and proteins) onto adjuvanted NLPs, with immunological analysis poised to begin in the next quarter.

  6. Identification of Genome-Wide Mutations in Ciprofloxacin-Resistant F. tularensis LVS Using Whole Genome Tiling Arrays and Next Generation Sequencing

    SciTech Connect

    Jaing, Crystal J.; McLoughlin, Kevin S.; Thissen, James B.; Zemla, Adam; Gardner, Shea N.; Vergez, Lisa M.; Bourguet, Feliza; Mabery, Shalini; Fofanov, Viacheslav Y.; Koshinsky, Heather; Jackson, Paul J.; Wang, Junwen

    2016-09-26

    Francisella tularensis is classified as a Class A bioterrorism agent by the U.S. government due to its high virulence and the ease with which it can be spread as an aerosol. It is a facultative intracellular pathogen and the causative agent of tularemia. Ciprofloxacin (Cipro) is a broad spectrum antibiotic effective against Gram-positive and Gram-negative bacteria. Increased Cipro resistance in pathogenic microbes is of serious concern when considering options for medical treatment of bacterial infections. Identification of genes and loci that are associated with Ciprofloxacin resistance will help advance the understanding of resistance mechanisms and may, in the future, provide better treatment options for patients. It may also provide information for development of assays that can rapidly identify Cipro-resistant isolates of this pathogen. In this study, we then selected a large number of F. tularensis live vaccine strain (LVS) isolates that survived in progressively higher Ciprofloxacin concentrations, screened the isolates using a whole genome F. tularensis LVS tiling microarray and Illumina sequencing, and identified both known and novel mutations associated with resistance. For genes containing mutations encode DNA gyrase subunit A, a hypothetical protein, an asparagine synthase, a sugar transamine/perosamine synthetase and others. Finally, structural modeling performed on these proteins provides insights into the potential function of these proteins and how they might contribute to Cipro resistance mechanisms.

  7. Systems approach to investigating host-pathogen interactions in infections with the biothreat agent Francisella. Constraints-based model of Francisella tularensis

    PubMed Central

    2010-01-01

    Background Francisella tularensis is a prototypic example of a pathogen for which few experimental datasets exist, but for which copious high-throughout data are becoming available because of its re-emerging significance as biothreat agent. The virulence of Francisella tularensis depends on its growth capabilities within a defined environmental niche of the host cell. Results We reconstructed the metabolism of Francisella as a stoichiometric matrix. This systems biology approach demonstrated that changes in carbohydrate utilization and amino acid metabolism play a pivotal role in growth, acid resistance, and energy homeostasis during infection with Francisella. We also show how varying the expression of certain metabolic genes in different environments efficiently controls the metabolic capacity of F. tularensis. Selective gene-expression analysis showed modulation of sugar catabolism by switching from oxidative metabolism (TCA cycle) in the initial stages of infection to fatty acid oxidation and gluconeogenesis later on. Computational analysis with constraints derived from experimental data revealed a limited set of metabolic genes that are operational during infection. Conclusions This integrated systems approach provides an important tool to understand the pathogenesis of an ill-characterized biothreat agent and to identify potential novel drug targets when rapid target identification is required should such microbes be intentionally released or become epidemic. PMID:20731870

  8. Identification of Genome-Wide Mutations in Ciprofloxacin-Resistant F. tularensis LVS Using Whole Genome Tiling Arrays and Next Generation Sequencing

    PubMed Central

    Jaing, Crystal J.; McLoughlin, Kevin S.; Thissen, James B.; Zemla, Adam; Vergez, Lisa M.; Bourguet, Feliza; Mabery, Shalini; Fofanov, Viacheslav Y.; Koshinsky, Heather; Jackson, Paul J.

    2016-01-01

    Francisella tularensis is classified as a Class A bioterrorism agent by the U.S. government due to its high virulence and the ease with which it can be spread as an aerosol. It is a facultative intracellular pathogen and the causative agent of tularemia. Ciprofloxacin (Cipro) is a broad spectrum antibiotic effective against Gram-positive and Gram-negative bacteria. Increased Cipro resistance in pathogenic microbes is of serious concern when considering options for medical treatment of bacterial infections. Identification of genes and loci that are associated with Ciprofloxacin resistance will help advance the understanding of resistance mechanisms and may, in the future, provide better treatment options for patients. It may also provide information for development of assays that can rapidly identify Cipro-resistant isolates of this pathogen. In this study, we selected a large number of F. tularensis live vaccine strain (LVS) isolates that survived in progressively higher Ciprofloxacin concentrations, screened the isolates using a whole genome F. tularensis LVS tiling microarray and Illumina sequencing, and identified both known and novel mutations associated with resistance. Genes containing mutations encode DNA gyrase subunit A, a hypothetical protein, an asparagine synthase, a sugar transamine/perosamine synthetase and others. Structural modeling performed on these proteins provides insights into the potential function of these proteins and how they might contribute to Cipro resistance mechanisms. PMID:27668749

  9. Modulation of Human Airway Barrier Functions during Burkholderia thailandensis and Francisella tularensis Infection Running Title: Airway Barrier Functions during Bacterial Infections.

    PubMed

    Blume, Cornelia; David, Jonathan; Bell, Rachel E; Laver, Jay R; Read, Robert C; Clark, Graeme C; Davies, Donna E; Swindle, Emily J

    2016-08-03

    The bronchial epithelium provides protection against pathogens from the inhaled environment through the formation of a highly-regulated barrier. In order to understand the pulmonary diseases melioidosis and tularemia caused by Burkholderia thailandensis and Fransicella tularensis, respectively, the barrier function of the human bronchial epithelium were analysed. Polarised 16HBE14o- or differentiated primary human bronchial epithelial cells (BECs) were exposed to increasing multiplicities of infection (MOI) of B. thailandensis or F. tularensis Live Vaccine Strain and barrier responses monitored over 24-72 h. Challenge of polarized BECs with either bacterial species caused an MOI- and time-dependent increase in ionic permeability, disruption of tight junctions, and bacterial passage from the apical to the basolateral compartment. B. thailandensis was found to be more invasive than F. tularensis. Both bacterial species induced an MOI-dependent increase in TNF-α release. An increase in ionic permeability and TNF-α release was induced by B. thailandensis in differentiated BECs. Pretreatment of polarised BECs with the corticosteroid fluticasone propionate reduced bacterial-dependent increases in ionic permeability, bacterial passage, and TNF-α release. TNF blocking antibody Enbrel(®) reduced bacterial passage only. BEC barrier properties are disrupted during respiratory bacterial infections and targeting with corticosteroids or anti-TNF compounds may represent a therapeutic option.

  10. The Type III Secretion System Effector SeoC of Salmonella enterica subsp. salamae and S. enterica subsp. arizonae ADP-Ribosylates Src and Inhibits Opsonophagocytosis.

    PubMed

    Pollard, Dominic J; Young, Joanna C; Covarelli, Valentina; Herrera-León, Silvia; Connor, Thomas R; Fookes, Maria; Walker, Danielle; Echeita, Aurora; Thomson, Nicholas R; Berger, Cedric N; Frankel, Gad

    2016-12-01

    Salmonella species utilize type III secretion systems (T3SSs) to translocate effectors into the cytosol of mammalian host cells, subverting cell signaling and facilitating the onset of gastroenteritis. In this study, we compared a draft genome assembly of Salmonella enterica subsp. salamae strain 3588/07 against the genomes of S. enterica subsp. enterica serovar Typhimurium strain LT2 and Salmonella bongori strain 12419. S. enterica subsp. salamae encodes the Salmonella pathogenicity island 1 (SPI-1), SPI-2, and the locus of enterocyte effacement (LEE) T3SSs. Though several key S Typhimurium effector genes are missing (e.g., avrA, sopB, and sseL), S. enterica subsp. salamae invades HeLa cells and contains homologues of S. bongori sboK and sboC, which we named seoC SboC and SeoC are homologues of EspJ from enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively), which inhibit Src kinase-dependent phagocytosis by ADP-ribosylation. By screening 73 clinical and environmental Salmonella isolates, we identified EspJ homologues in S. bongori, S. enterica subsp. salamae, and Salmonella enterica subsp. arizonae The β-lactamase TEM-1 reporter system showed that SeoC is translocated by the SPI-1 T3SS. All the Salmonella SeoC/SboC homologues ADP-ribosylate Src E310 in vitro Ectopic expression of SeoC/SboC inhibited phagocytosis of IgG-opsonized beads into Cos-7 cells stably expressing green fluorescent protein (GFP)-FcγRIIa. Concurrently, S. enterica subsp. salamae infection of J774.A1 macrophages inhibited phagocytosis of beads, in a seoC-dependent manner. These results show that S. bongori, S. enterica subsp. salamae, and S. enterica subsp. arizonae share features of the infection strategy of extracellular pathogens EPEC and EHEC and shed light on the complexities of the T3SS effector repertoires of Enterobacteriaceae.

  11. Linkage maps for Arabidopsis lyrata subsp. lyrata and Arabidopsis lyrata subsp. petraea combining anonymous and Arabidopsis thaliana-derived markers.

    PubMed

    Beaulieu, Julien; Jean, Martine; Belzile, François

    2007-02-01

    Arabidopsis lyrata, a close relative of the model plant Arabidopsis thaliana, is 1 of a few plant species for which the genome is to be entirely sequenced, which promises to yield important insights into genome evolution. Only 2 sparse linkage maps have been published, and these were based solely on markers derived from the A. thaliana genome. Because the genome of A. lyrata is practically twice as large as that of A. thaliana, the extent of map coverage of the A. lyrata genome remains uncertain. In this study, a 2-way pseudo-testcross strategy was used to construct genetic linkage maps of A. lyrata subsp. petraea and A. lyrata subsp. lyrata, using simple sequence repeat (SSR) and cleaved amplified polymorphic sequence (CAPS) markers from the A. thaliana genome, and anonymous amplified fragment length polymorphism (AFLP) markers that could potentially uncover regions unique to the A. lyrata genome. The SSR and CAPS markers largely confirmed the relationships between linkage groups in A. lyrata and A. thaliana. AFLP markers slightly increased the coverage of the A. lyrata maps, but mostly increased marker density on the linkage groups. We noted a much lower level of polymorphism and a greater segregation distortion in A. lyrata subsp. lyrata markers. The implications of these findings for the sequencing of the A. lyrata genome are discussed.

  12. Plasmodium falciparum dolichol phosphate mannose synthase represents a novel clade

    SciTech Connect

    Shams-Eldin, Hosam Santos de Macedo, Cristiana; Niehus, Sebastian; Dorn, Caroline; Kimmel, Juergen; Azzouz, Nahid; Schwarz, Ralph T.

    2008-06-06

    Dolichol phosphate mannose synthase (DPM) catalyzes the reaction between dolichol phosphate (Dol-P) and guanosine diphosphate mannose (GDP-Man) to form dolichol-phosphate-mannose (Dol-P-Man). This molecule acts as mannose donor for N-glycosylation and glycosylphosphatidylinositol (GPI) biosynthesis. The Plasmodium falciparum DPM1 (Pfdpm1) possesses a single predicted transmembrane region near the N-, but not the C-terminus. Here we show that the cloned Pfdpm1 gene failed to complement a Saccharomyces cerevisiae mutant indicating that the parasite gene does not belong to the baker's yeast group, as was previously assumed. Furthermore, Pfdpm1 was unable to complement a mouse mutant deficient in DPM but efficiently complements the Schizosaccharomyces pombe fission yeast mutant, indicating a difference between fission yeast and mammalian DPM genes. Therefore, we reanalyzed the hydrophobicity scales of all known DPMs and consequently reclassify the DPM clade into six major novel subgroups. Furthermore, we show that Pfdpm1 represents a unique enzyme among these subgroups.

  13. Evolution of pollination niches in a generalist plant clade.

    PubMed

    Gómez, José María; Perfectti, Francisco; Abdelaziz, Mohamed; Lorite, Juan; Muñoz-Pajares, Antonio Jesús; Valverde, Javier

    2015-01-01

    It is widely assumed that floral diversification occurs by adaptive shifts between pollination niches. In contrast to specialized flowers, identifying pollination niches of generalist flowers is a challenge. Consequently, how generalist pollination niches evolve is largely unknown. We apply tools from network theory and comparative methods to investigate the evolution of pollination niches among generalist species belonging to the genus Erysimum. These species have similar flowers. We found that the studied species may be grouped in several multidimensional niches separated not by a shift of pollinators, but instead by quantitative variation in the relative abundance of pollinator functional groups. These pollination niches did not vary in generalization degree; we did not find any evolutionary trend toward specialization within the studied clade. Furthermore, the evolution of pollination niche fitted to a Brownian motion model without phylogenetic signal, and was characterized by frequent events of niche convergences and divergences. We presume that the evolution of Erysimum pollination niches has occurred mostly by recurrent shifts between slightly different generalized pollinator assemblages varying spatially as a mosaic and without any change in specialization degree. Most changes in pollination niches do not prompt floral divergence, a reason why adaptation to pollinators is uncommon in generalist plants.

  14. Genome size increases in recently diverged hornwort clades.

    PubMed

    Bainard, Jillian D; Villarreal, Juan Carlos

    2013-08-01

    As our knowledge of plant genome size estimates continues to grow, one group has continually been neglected: the hornworts. Hornworts (Anthocerotophyta) have been traditionally grouped with liverworts and mosses because they share a haploid dominant life cycle; however, recent molecular studies place hornworts as the sister lineage to extant tracheophytes. Given the scarcity of information regarding the DNA content of hornworts, our objective was to estimate the 1C-value for a range of hornwort species within a phylogenetic context. Using flow cytometry, we estimated genome size for 36 samples representing 24 species. This accounts for roughly 10% of known hornwort species. Haploid genome sizes (1C-value) ranged from 160 Mbp or 0.16 pg (Leiosporoceros dussii) to 719 Mbp or 0.73 pg (Nothoceros endiviifolius). The average 1C-value was 261 ± 104 Mbp (0.27 ± 0.11 pg). Ancestral reconstruction of genome size on a hornwort phylogeny suggests a small ancestral genome size and revealed increases in genome size in the most recently divergent clades. Much more work is needed to understand DNA content variation in this phylogenetically important group, but this work has significantly increased our knowledge of genome size variation in hornworts.

  15. Feline immunodeficiency virus clade C mucosal transmission and disease courses.

    PubMed

    Obert, L A; Hoover, E A

    2000-05-01

    The transmissibility and pathogenicity of a clade C feline immunodeficiency virus (FIV-C) was examined via the oral-nasal, vaginal, or rectal mucosa. FIV-C was transmissible by all three mucosal routes. Vaginal transmission was most efficient (100%), oral exposure resulted in a 80% infection rate, and rectal transmission was least effective (44%). In contrast to previous intravenous passage studies, a broader range of host-virus relationships was observed after mucosal exposure. Three categories of FIV-C infection were defined: (1) rapidly progressive infection marked by high virus burdens and rapid CD4+ cell depletion (43% of vaginally exposed animals); (2) conventional (typical) infection featuring slowly progressive CD4+ cell decline (61% of all exposed animals); and (3) regressive (transient) infection marked by low and then barely detectable virus burdens and no CD4+ cell alterations (22% of rectally inoculated cats). These disease courses appear to have parallels in mucosal HIV and SIV infections, emphasizing the importance of the virus-mucosa interface in lentiviral pathogenesis.

  16. Burkholderia pseudomallei sequencing identifies genomic clades with distinct recombination, accessory, and epigenetic profiles

    PubMed Central

    Nandi, Tannistha; Holden, Matthew T.G.; Didelot, Xavier; Mehershahi, Kurosh; Boddey, Justin A.; Beacham, Ifor; Peak, Ian; Harting, John; Baybayan, Primo; Guo, Yan; Wang, Susana; How, Lee Chee; Sim, Bernice; Essex-Lopresti, Angela; Sarkar-Tyson, Mitali; Nelson, Michelle; Smither, Sophie; Ong, Catherine; Aw, Lay Tin; Hoon, Chua Hui; Michell, Stephen; Studholme, David J.; Titball, Richard; Chen, Swaine L.; Parkhill, Julian

    2015-01-01

    Burkholderia pseudomallei (Bp) is the causative agent of the infectious disease melioidosis. To investigate population diversity, recombination, and horizontal gene transfer in closely related Bp isolates, we performed whole-genome sequencing (WGS) on 106 clinical, animal, and environmental strains from a restricted Asian locale. Whole-genome phylogenies resolved multiple genomic clades of Bp, largely congruent with multilocus sequence typing (MLST). We discovered widespread recombination in the Bp core genome, involving hundreds of regions associated with multiple haplotypes. Highly recombinant regions exhibited functional enrichments that may contribute to virulence. We observed clade-specific patterns of recombination and accessory gene exchange, and provide evidence that this is likely due to ongoing recombination between clade members. Reciprocally, interclade exchanges were rarely observed, suggesting mechanisms restricting gene flow between clades. Interrogation of accessory elements revealed that each clade harbored a distinct complement of restriction-modification (RM) systems, predicted to cause clade-specific patterns of DNA methylation. Using methylome sequencing, we confirmed that representative strains from separate clades indeed exhibit distinct methylation profiles. Finally, using an E. coli system, we demonstrate that Bp RM systems can inhibit uptake of non-self DNA. Our data suggest that RM systems borne on mobile elements, besides preventing foreign DNA invasion, may also contribute to limiting exchanges of genetic material between individuals of the same species. Genomic clades may thus represent functional units of genetic isolation in Bp, modulating intraspecies genetic diversity. PMID:25236617

  17. Burkholderia pseudomallei sequencing identifies genomic clades with distinct recombination, accessory, and epigenetic profiles.

    PubMed

    Nandi, Tannistha; Holden, Matthew T G; Holden, Mathew T G; Didelot, Xavier; Mehershahi, Kurosh; Boddey, Justin A; Beacham, Ifor; Peak, Ian; Harting, John; Baybayan, Primo; Guo, Yan; Wang, Susana; How, Lee Chee; Sim, Bernice; Essex-Lopresti, Angela; Sarkar-Tyson, Mitali; Nelson, Michelle; Smither, Sophie; Ong, Catherine; Aw, Lay Tin; Hoon, Chua Hui; Michell, Stephen; Studholme, David J; Titball, Richard; Chen, Swaine L; Parkhill, Julian; Tan, Patrick

    2015-01-01

    Burkholderia pseudomallei (Bp) is the causative agent of the infectious disease melioidosis. To investigate population diversity, recombination, and horizontal gene transfer in closely related Bp isolates, we performed whole-genome sequencing (WGS) on 106 clinical, animal, and environmental strains from a restricted Asian locale. Whole-genome phylogenies resolved multiple genomic clades of Bp, largely congruent with multilocus sequence typing (MLST). We discovered widespread recombination in the Bp core genome, involving hundreds of regions associated with multiple haplotypes. Highly recombinant regions exhibited functional enrichments that may contribute to virulence. We observed clade-specific patterns of recombination and accessory gene exchange, and provide evidence that this is likely due to ongoing recombination between clade members. Reciprocally, interclade exchanges were rarely observed, suggesting mechanisms restricting gene flow between clades. Interrogation of accessory elements revealed that each clade harbored a distinct complement of restriction-modification (RM) systems, predicted to cause clade-specific patterns of DNA methylation. Using methylome sequencing, we confirmed that representative strains from separate clades indeed exhibit distinct methylation profiles. Finally, using an E. coli system, we demonstrate that Bp RM systems can inhibit uptake of non-self DNA. Our data suggest that RM systems borne on mobile elements, besides preventing foreign DNA invasion, may also contribute to limiting exchanges of genetic material between individuals of the same species. Genomic clades may thus represent functional units of genetic isolation in Bp, modulating intraspecies genetic diversity.

  18. A functional genomic analysis of Arabidopsis thaliana PP2C clade D

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the reference dicot plant Arabidopsis thaliana, the PP2C family of P-protein phosphatases includes the products of 80 genes that have been separated into 10 multi-protein clades plus six singletons. Clade D includes the products of nine genes distributed among 3 chromosomes (PPD1, At3g12620; PPD2...

  19. Progress to extinction: increased specialisation causes the demise of animal clades

    PubMed Central

    Raia, P.; Carotenuto, F.; Mondanaro, A.; Castiglione, S.; Passaro, F.; Saggese, F.; Melchionna, M.; Serio, C.; Alessio, L.; Silvestro, D.; Fortelius, M.

    2016-01-01

    Animal clades tend to follow a predictable path of waxing and waning during their existence, regardless of their total species richness or geographic coverage. Clades begin small and undifferentiated, then expand to a peak in diversity and range, only to shift into a rarely broken decline towards extinction. While this trajectory is now well documented and broadly recognised, the reasons underlying it remain obscure. In particular, it is unknown why clade extinction is universal and occurs with such surprising regularity. Current explanations for paleontological extinctions call on the growing costs of biological interactions, geological accidents, evolutionary traps, and mass extinctions. While these are effective causes of extinction, they mainly apply to species, not clades. Although mass extinctions is the undeniable cause for the demise of a sizeable number of major taxa, we show here that clades escaping them go extinct because of the widespread tendency of evolution to produce increasingly specialised, sympatric, and geographically restricted species over time. PMID:27507121

  20. Progress to extinction: increased specialisation causes the demise of animal clades

    NASA Astrophysics Data System (ADS)

    Raia, P.; Carotenuto, F.; Mondanaro, A.; Castiglione, S.; Passaro, F.; Saggese, F.; Melchionna, M.; Serio, C.; Alessio, L.; Silvestro, D.; Fortelius, M.

    2016-08-01

    Animal clades tend to follow a predictable path of waxing and waning during their existence, regardless of their total species richness or geographic coverage. Clades begin small and undifferentiated, then expand to a peak in diversity and range, only to shift into a rarely broken decline towards extinction. While this trajectory is now well documented and broadly recognised, the reasons underlying it remain obscure. In particular, it is unknown why clade extinction is universal and occurs with such surprising regularity. Current explanations for paleontological extinctions call on the growing costs of biological interactions, geological accidents, evolutionary traps, and mass extinctions. While these are effective causes of extinction, they mainly apply to species, not clades. Although mass extinctions is the undeniable cause for the demise of a sizeable number of major taxa, we show here that clades escaping them go extinct because of the widespread tendency of evolution to produce increasingly specialised, sympatric, and geographically restricted species over time.

  1. New insights into the phylogeny and historical biogeography of the Lellingeria myosuroides clade (Polypodiaceae)

    PubMed Central

    Ranker, Tom A.; Sundue, Michael; Labiak, Paulo; Parris, Barbara; Rouhan, Germinal

    2010-01-01

    Grammitid ferns are a well-supported clade of ~900 primarily tropical epiphytic species. Recent phylogenetic studies have found support for a distinctive, geographically diverse group of 24 species referred to as the Lellingeria myosuroides clade and have provided evidence for a variety of phylogenetic relationships within the group, as well as hypotheses of historical processes that have produced current biogeographical patterns. We present new data and analyses that support the following primary conclusions: 1) the L. myosuroides clade is monophyletic and pantropical; 2) that clade is sister to a more species rich clade of entirely Neotropical species (Lellingeria s.s.); 3) we infer two independent dispersal events from the Neotropics to Pacific islands, five independent dispersal events from the Neotropics to the Paleotropics, and two separate dispersal events from mainland tropical America to the West Indies. PMID:21113337

  2. Clade 8 and Clade 6 Strains of Escherichia coli O157:H7 from Cattle in Argentina have Hypervirulent-Like Phenotypes

    PubMed Central

    Amigo, Natalia; Mercado, Elsa; Bentancor, Adriana; Singh, Pallavi; Vilte, Daniel; Gerhardt, Elisabeth; Zotta, Elsa; Ibarra, Cristina; Manning, Shannon D.; Larzábal, Mariano; Cataldi, Angel

    2015-01-01

    The hemolytic uremic syndrome (HUS) whose main causative agent is enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a disease that mainly affects children under 5 years of age. Argentina is the country with the highest incidence of HUS in the world. Cattle are a major reservoir and source of infection with E. coli O157:H7. To date, the epidemiological factors that contribute to its prevalence are poorly understood. Single nucleotide polymorphism (SNP) typing has helped to define nine E. coli O157:H7 clades and the clade 8 strains were associated with most of the cases of severe disease. In this study, eight randomly selected isolates of EHEC O157:H7 from cattle in Argentina were studied as well as two human isolates. Four of them were classified as clade 8 through the screening for 23 SNPs; the two human isolates grouped in this clade as well, while two strains were closely related to strains representing clade 6. To assess the pathogenicity of these strains, we assayed correlates of virulence. Shiga toxin production was determined by an ELISA kit. Four strains were high producers and one of these strains that belonged to a novel genotype showed high verocytotoxic activity in cultured cells. Also, these clade 8 and 6 strains showed high RBC lysis and adherence to epithelial cells. One of the clade 6 strains showed stronger inhibition of normal water absorption than E. coli O157:H7 EDL933 in human colonic explants. In addition, two of the strains showing high levels of Stx2 production and RBC lysis activity were associated with lethality and uremia in a mouse model. Consequently, circulation of such strains in cattle may partially contribute to the high incidence of HUS in Argentina. PMID:26030198

  3. Clade 8 and Clade 6 Strains of Escherichia coli O157:H7 from Cattle in Argentina have Hypervirulent-Like Phenotypes.

    PubMed

    Amigo, Natalia; Mercado, Elsa; Bentancor, Adriana; Singh, Pallavi; Vilte, Daniel; Gerhardt, Elisabeth; Zotta, Elsa; Ibarra, Cristina; Manning, Shannon D; Larzábal, Mariano; Cataldi, Angel

    2015-01-01

    The hemolytic uremic syndrome (HUS) whose main causative agent is enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a disease that mainly affects children under 5 years of age. Argentina is the country with the highest incidence of HUS in the world. Cattle are a major reservoir and source of infection with E. coli O157:H7. To date, the epidemiological factors that contribute to its prevalence are poorly understood. Single nucleotide polymorphism (SNP) typing has helped to define nine E. coli O157:H7 clades and the clade 8 strains were associated with most of the cases of severe disease. In this study, eight randomly selected isolates of EHEC O157:H7 from cattle in Argentina were studied as well as two human isolates. Four of them were classified as clade 8 through the screening for 23 SNPs; the two human isolates grouped in this clade as well, while two strains were closely related to strains representing clade 6. To assess the pathogenicity of these strains, we assayed correlates of virulence. Shiga toxin production was determined by an ELISA kit. Four strains were high producers and one of these strains that belonged to a novel genotype showed high verocytotoxic activity in cultured cells. Also, these clade 8 and 6 strains showed high RBC lysis and adherence to epithelial cells. One of the clade 6 strains showed stronger inhibition of normal water absorption than E. coli O157:H7 EDL933 in human colonic explants. In addition, two of the strains showing high levels of Stx2 production and RBC lysis activity were associated with lethality and uremia in a mouse model. Consequently, circulation of such strains in cattle may partially contribute to the high incidence of HUS in Argentina.

  4. Models derived from in vitro analyses of spleen, liver, and lung leukocyte functions predict vaccine efficacy against the Francisella tularensis Live Vaccine Strain (LVS).

    PubMed

    De Pascalis, Roberto; Chou, Alicia Y; Ryden, Patrik; Kennett, Nikki J; Sjöstedt, Anders; Elkins, Karen L

    2014-04-08

    Currently, there are no licensed vaccines and no correlates of protection against Francisella tularensis, which causes tularemia. We recently demonstrated that measuring in vitro control of intramacrophage bacterial growth by murine F. tularensis-immune splenocytes, as well as transcriptional analyses, discriminated Francisella vaccines of different efficacies. Further, we identified potential correlates of protection against systemic challenge. Here, we extended this approach by studying leukocytes derived from lungs and livers of mice immunized by parenteral and respiratory routes with F. tularensis vaccines. Liver and lung leukocytes derived from intradermally and intranasally vaccinated mice controlled in vitro Francisella Live Vaccine Strain (LVS) intramacrophage replication in patterns similar to those of splenocytes. Gene expression analyses of potential correlates also revealed similar patterns in liver cells and splenocytes. In some cases (e.g., tumor necrosis factor alpha [TNF-α], interleukin 22 [IL-22], and granulocyte-macrophage colony-stimulating factor [GM-CSF]), liver cells exhibited even higher relative gene expression, whereas fewer genes exhibited differential expression in lung cells. In contrast with their strong ability to control LVS replication, splenocytes from intranasally vaccinated mice expressed few genes with a hierarchy of expression similar to that of splenocytes from intradermally vaccinated mice. Thus, the relative levels of gene expression vary between cell types from different organs and by vaccination route. Most importantly, because studies comparing cell sources and routes of vaccination supported the predictive validity of this coculture and gene quantification approach, we combined in vitro LVS replication with gene expression data to develop analytical models that discriminated between vaccine groups and successfully predicted the degree of vaccine efficacy. Thus, this strategy remains a promising means of identifying and

  5. Preliminary investigation of a mice model of Klebsiella pneumoniae subsp. ozaenae induced pneumonia.

    PubMed

    Renois, Fanny; Jacques, Jérôme; Guillard, Thomas; Moret, Hélène; Pluot, Michel; Andreoletti, Laurent; de Champs, Christophe

    2011-11-01

    In the present study, we comparatively assessed the pathophysiological mechanisms developed during lung infection of BALB/C female mice infected by an original wild type Klebsiella pneumoniae subsp. ozaenae strain (CH137) or by a referent subspecies K. pneumoniae. subsp. pneumoniae strain (ATCC10031). The mice infected with 2.10⁶ CFU K. p. subsp. pneumoniae (n = 10) showed transient signs of infection and all of them recovered. All of those infected with 1.10⁶ CFU K. p. subsp. ozaenae (n = 10) developed pneumonia within 24 h and died between 48 and 72 h. Few macrophages, numerous polymorphonuclear cells and lymphocytes were observed in their lungs in opposite to K. p. subsp. pneumoniae. In bronchoalveolar lavage, a significant increase in MIP-2, IL-6, KC and MCP-1 levels was only observed in K. p. subsp. ozaenae infected mice whereas high levels of TNF-α were evidenced with the two subspecies. Our findings indicated a lethal effect of a wild type K. p. subsp. ozaenae strain by acute pneumonia reflecting an insufficient alveolar macrophage response. This model might be of a major interest to comparatively explore the pathogenicity of K. p. subsp ozaenae strains and to further explore the physiopathological mechanisms of gram-negative bacteria induced human pneumonia.

  6. Genome Sequence of Klebsiella quasipneumoniae subsp. similipneumoniae MB373, an Effective Bioremediator

    PubMed Central

    Aslam, Fozia; Thomas, Torsten

    2016-01-01

    Klebsiella quasipneumoniae subsp. similipneumoniae MB373 was isolated from effluent of the Hattar Industrial Estate, Haripur, Pakistan. K. quasipneumoniae subsp. similipneumoniae has few cultivated/characterized members so far. Whole-genome sequencing revealed its potential for metal and toxin resistance, which further elucidated various enzymatic processes for the degradation of xenobiotics, illuminating its bioremediation applications. PMID:27688323

  7. Complete Genome Sequence of the Endophytic Enterobacter cloacae subsp. cloacae Strain ENHKU01

    PubMed Central

    Liu, Wing-Yee; Chung, Karl Ming-Kar; Wong, Chi-Fat; Jiang, Jing-Wei; Hui, Raymond Kin-Hi

    2012-01-01

    Enterobacter cloacae subsp. cloacae strain ENHKU01 is a Gram-negative endophyte isolated from a diseased pepper (Capsicum annuum) plant in Hong Kong. This is the first complete genome sequence report of a plant-endophytic strain of E. cloacae subsp. cloacae. PMID:23045485

  8. A Gene Specific to Mycobacterium avium subsp. paratuberculosis, But Only at the Transcription-translation Level

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There is no known antibody that detects M. avium subsp paratuberculosis and does not cross react with other M. avium subspecies. In the present study, a monoclonal antibody was identified from mice immunized with a cell membrane fraction of M. avium subsp paratuberculosis strain K-10. This antibod...

  9. First Closed Genome Sequence of Campylobacter fetus subsp. venerealis bv. intermedius

    PubMed Central

    Yee, Emma; Bono, James L.; Rijnsburger, Martine; Campero, Carlos; Wagenaar, Jaap A.; Duim, Birgitta

    2014-01-01

    Campylobacter fetus subsp. venerealis bv. intermedius is a variant of C. fetus subsp. venerealis, the causative agent of bovine genital campylobacteriosis, a venereal disease associated with abortion and infertility in cattle. We report the first closed whole-genome sequence of this biovar. PMID:24503995

  10. Complete genome sequence of Mycobacterium avium subsp. paratuberculosis, isolated from human breast milk

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp paratuberculosis is the etiologic agent of Johne’s disease. We report the draft genome sequences of six M. avium subsp paratuberculosis isolates obtained from diverse hosts including bison, cattle and sheep. These sequences will deepen our understanding of host association ...

  11. Draft Genome Sequence of Photorhabdus luminescens subsp. laumondii HP88, an Entomopathogenic Bacterium Isolated from Nematodes

    PubMed Central

    Ghazal, Shimaa; Oshone, Rediet; Simpson, Stephen; Morris, Krystalynne; Abebe-Akele, Feseha; Thomas, W. Kelley; Khalil, Kamal M.

    2016-01-01

    Photorhabdus luminescens subsp. laumondii HP88 is an entomopathogenic bacterium that forms a symbiotic association with Heterorhabditis nematodes. We report here a 5.27-Mbp draft genome sequence for P. luminescens subsp. laumondii HP88, with a G+C content of 42.4% and containing 4,243 candidate protein-coding genes. PMID:26988056

  12. Complete Genome Sequence of Type Strain Campylobacter fetus subsp. venerealis NCTC 10354T

    PubMed Central

    Stynen, Ana Paula Reinato; Lage, Andrey Pereira; Moore, Robert J.; Rezende, Antonio Mauro; de Resende, Vivian D'Afonseca da Silva; Ruy, Patricia de Cássia; Daher, Nesley; Resende, Daniela de Melo; de Almeida, Sintia Silva; Soares, Siomar de Castro; de Abreu, Vinicius Augusto Carvalho; Rocha, Aryane Aparecida C. Magalhães; dos Santos, Anderson Rodrigues; Barbosa, Eudes Guilherme Vieira; Costa, Danielle Fonseca; Dorella, Fernanda Alves; Miyoshi, Anderson; de Lima, Alex Ranieri Jerônimo; Campos, Frederico Davi da Silva; de Sá, Pablo Gomes; Lopes, Thiago Souza; Rodrigues, Ryan Mauricio Araujo; Carneiro, Adriana Ribeiro; Leão, Thiago; Cerdeira, Louise Teixeira; Ramos, Rommel Thiago Jucá; Silva, Artur; Azevedo, Vasco; Ruiz, Jerônimo C.

    2011-01-01

    Campylobacter fetus subsp. venerealis is the etiologic agent of bovine genital campylobacteriosis, a sexually transmitted disease of cattle that is of worldwide importance. The complete sequencing and annotation of the genome of the type strain C. fetus subsp. venerealis NCTC 10354T are reported. PMID:21952544

  13. Genomic Sequence of Campylobacter jejuni subsp. jejuni HS:19 Penner Serotype Reference Strain RM3420

    PubMed Central

    Huynh, Steven; Heikema, Astrid P.

    2017-01-01

    ABSTRACT Campylobacter jejuni subsp. jejuni infections are a leading cause of foodborne gastroenteritis and the most prevalent antecedent to Guillain-Barré syndrome (GBS). Penner serotype HS:19 is among several capsular types shown to be markers for GBS. This study describes the genome of C. jejuni subsp. jejuni HS:19 Penner reference strain RM3420. PMID:28232429

  14. Draft Genome Sequence of Lactococcus lactis subsp. lactis Strain YF11

    PubMed Central

    Du, Yuhui; Song, Lifu; Feng, Wenjing; Pei, Guangsheng; Zheng, Ping; Yu, Zhichao; Sun, Jibin

    2013-01-01

    Lactococcus lactis subsp. lactis strain YF11 is a food preservative bacterium with a high capacity to produce nisin. Here, we announce the draft genome sequence of Lactococcus lactis subsp. lactis YF11 (2,527,433 bp with a G+C content of 34.81%). PMID:23929487

  15. The evolution of tribospheny and the antiquity of mammalian clades.

    PubMed

    Woodburne, Michael O; Rich, Thomas H; Springer, Mark S

    2003-08-01

    The evolution of tribosphenic molars is a key innovation in the history of Mammalia. Tribospheny allows for both shearing and grinding occlusal functions. Marsupials and placentals are advanced tribosphenic mammals (i.e., Theria) that show additional modifications of the tribosphenic dentition including loss of the distal metacristid and development of double-rank postvallum/prevallid shear. The recent discovery of Eomaia [Nature 416 (2002) 816], regarded as the oldest eutherian mammal, implies that the marsupial-placental split is at least 125 million years old. The conventional scenario for the evolution of tribosphenic and therian mammals hypothesizes that each group evolved once, in the northern hemisphere, and is based on a predominantly Laurasian fossil record. With the recent discovery of the oldest tribosphenic mammal (Ambondro) from the Mesozoic of Gondwana, Flynn et al. [Nature 401 (1999) 57] suggested that tribospheny evolved in Gondwana rather than in Laurasia. Luo et al. [Nature 409 (2001) 53; Acta Palaeontol. Pol. 47 (2002) 1] argued for independent origins of tribospheny in northern (Boreosphenida) and southern (Australosphenida) hemisphere clades, with the latter including Ambondro, ausktribosphenids, and monotremes. Here, we present cladistic evidence for a single origin of tribosphenic molars. Further, Ambondro may be a stem eutherian, making the split between marsupials and placentals at least 167 m.y. old. To test this hypothesis, we used the relaxed molecular clock approach of Thorne/Kishino with amino acid data sets for BRCA1 [J. Mammal. Evol. 8 (2001) 239] and the IGF2 receptor [Mammal. Genome 12 (2001) 513]. Point estimates for the marsupial-placental split were 182-190 million years based on BRCA1 and 185-187 million years based on the IGF2 receptor. These estimates are fully compatible with the results of our cladistic analyses.

  16. Hylid frog phylogeny and sampling strategies for speciose clades.

    PubMed

    Wiens, John J; Fetzner, James W; Parkinson, Christopher L; Reeder, Tod W

    2005-10-01

    How should characters and taxa be sampled to resolve efficiently the phylogeny of ancient and highly speciose groups? We addressed this question empirically in the treefrog family Hylidae, which contains > 800 species and may be nonmonophyletic with respect to other anuran families. We sampled 81 species (54 hylids and 27 outgroups) for two mitochondrial genes (12S, ND1), two nuclear genes (POMC, c-myc), and morphology (144 characters) in an attempt to resolve higher-level relationships. We then added 117 taxa to the combined data set, many of which were sampled for only one gene (12S). Despite the relative incompleteness of the majority of taxa, the resulting trees placed all taxa in the expected higher-level clades with strong support, despite some taxa being > 90% incomplete. Furthermore, we found no relationship between the completeness of a taxon and the support (parsimony bootstrap or Bayesian posterior probabilities) for its localized placement on the tree. Separate analysis of the data set with the most taxa (12S) gives a somewhat problematic estimate of higher-level relationships, suggesting that data sets scored only for some taxa (ND1, nuclear genes, morphology) are important in determining the outcome of the combined analysis. The results show that hemiphractine hylids are not closely related to other hylids and should be recognized as a distinct family. They also show that the speciose genus Hyla is polyphyletic, but that its species can be arranged into three monophyletic genera. A new classification of hylid frogs is proposed. Several potentially misleading signals in the morphological data are discussed.

  17. Massive Mitochondrial Gene Transfer in a Parasitic Flowering Plant Clade

    PubMed Central

    Bradley, Robert K.; Sugumaran, M.; Marx, Christopher J.; Rest, Joshua S.; Davis, Charles C.

    2013-01-01

    Recent studies have suggested that plant genomes have undergone potentially rampant horizontal gene transfer (HGT), especially in the mitochondrial genome. Parasitic plants have provided the strongest evidence of HGT, which appears to be facilitated by the intimate physical association between the parasites and their hosts. A recent phylogenomic study demonstrated that in the holoparasite Rafflesia cantleyi (Rafflesiaceae), whose close relatives possess the world's largest flowers, about 2.1% of nuclear gene transcripts were likely acquired from its obligate host. Here, we used next-generation sequencing to obtain the 38 protein-coding and ribosomal RNA genes common to the mitochondrial genomes of angiosperms from R. cantleyi and five additional species, including two of its closest relatives and two host species. Strikingly, our phylogenetic analyses conservatively indicate that 24%–41% of these gene sequences show evidence of HGT in Rafflesiaceae, depending on the species. Most of these transgenic sequences possess intact reading frames and are actively transcribed, indicating that they are potentially functional. Additionally, some of these transgenes maintain synteny with their donor and recipient lineages, suggesting that native genes have likely been displaced via homologous recombination. Our study is the first to comprehensively assess the magnitude of HGT in plants involving a genome (i.e., mitochondria) and a species interaction (i.e., parasitism) where it has been hypothesized to be potentially rampant. Our results establish for the first time that, although the magnitude of HGT involving nuclear genes is appreciable in these parasitic plants, HGT involving mitochondrial genes is substantially higher. This may represent a more general pattern for other parasitic plant clades and perhaps more broadly for angiosperms. PMID:23459037

  18. Massive mitochondrial gene transfer in a parasitic flowering plant clade.

    PubMed

    Xi, Zhenxiang; Wang, Yuguo; Bradley, Robert K; Sugumaran, M; Marx, Christopher J; Rest, Joshua S; Davis, Charles C

    2013-01-01

    Recent studies have suggested that plant genomes have undergone potentially rampant horizontal gene transfer (HGT), especially in the mitochondrial genome. Parasitic plants have provided the strongest evidence of HGT, which appears to be facilitated by the intimate physical association between the parasites and their hosts. A recent phylogenomic study demonstrated that in the holoparasite Rafflesia cantleyi (Rafflesiaceae), whose close relatives possess the world's largest flowers, about 2.1% of nuclear gene transcripts were likely acquired from its obligate host. Here, we used next-generation sequencing to obtain the 38 protein-coding and ribosomal RNA genes common to the mitochondrial genomes of angiosperms from R. cantleyi and five additional species, including two of its closest relatives and two host species. Strikingly, our phylogenetic analyses conservatively indicate that 24%-41% of these gene sequences show evidence of HGT in Rafflesiaceae, depending on the species. Most of these transgenic sequences possess intact reading frames and are actively transcribed, indicating that they are potentially functional. Additionally, some of these transgenes maintain synteny with their donor and recipient lineages, suggesting that native genes have likely been displaced via homologous recombination. Our study is the first to comprehensively assess the magnitude of HGT in plants involving a genome (i.e., mitochondria) and a species interaction (i.e., parasitism) where it has been hypothesized to be potentially rampant. Our results establish for the first time that, although the magnitude of HGT involving nuclear genes is appreciable in these parasitic plants, HGT involving mitochondrial genes is substantially higher. This may represent a more general pattern for other parasitic plant clades and perhaps more broadly for angiosperms.

  19. Comparing the effects of symbiotic algae (Symbiodinium) clades C1 and D on early growth stages of Acropora tenuis.

    PubMed

    Yuyama, Ikuko; Higuchi, Tomihiko

    2014-01-01

    Reef-building corals switch endosymbiotic algae of the genus Symbiodinium during their early growth stages and during bleaching events. Clade C Symbiodinium algae are dominant in corals, although other clades - including A and D - have also been commonly detected in juvenile Acroporid corals. Previous studies have been reported that only molecular data of Symbiodinium clade were identified within field corals. In this study, we inoculated aposymbiotic juvenile polyps with cultures of clades C1 and D Symbiodinium algae, and investigated the different effect of these two clades of Symbiodinium on juvenile polyps. Our results showed that clade C1 algae did not grow, while clade D algae grew rapidly during the first 2 months after inoculation. Polyps associated with clade C1 algae exhibited bright green fluorescence across the body and tentacles after inoculation. The growth rate of polyp skeletons was lower in polyps associated with clade C1 algae than those associated with clade D algae. On the other hand, antioxidant activity (catalase) of corals was not significantly different between corals with clade C1 and clade D algae. Our results suggested that clade D Symbiodinium algae easily form symbiotic relationships with corals and that these algae could contribute to coral growth in early symbiosis stages.

  20. Comparing the Effects of Symbiotic Algae (Symbiodinium) Clades C1 and D on Early Growth Stages of Acropora tenuis

    PubMed Central

    Yuyama, Ikuko; Higuchi, Tomihiko

    2014-01-01

    Reef-building corals switch endosymbiotic algae of the genus Symbiodinium during their early growth stages and during bleaching events. Clade C Symbiodinium algae are dominant in corals, although other clades — including A and D — have also been commonly detected in juvenile Acroporid corals. Previous studies have been reported that only molecular data of Symbiodinium clade were identified within field corals. In this study, we inoculated aposymbiotic juvenile polyps with cultures of clades C1 and D Symbiodinium algae, and investigated the different effect of these two clades of Symbiodinium on juvenile polyps. Our results showed that clade C1 algae did not grow, while clade D algae grew rapidly during the first 2 months after inoculation. Polyps associated with clade C1 algae exhibited bright green fluorescence across the body and tentacles after inoculation. The growth rate of polyp skeletons was lower in polyps associated with clade C1 algae than those associated with clade D algae. On the other hand, antioxidant activity (catalase) of corals was not significantly different between corals with clade C1 and clade D algae. Our results suggested that clade D Symbiodinium algae easily form symbiotic relationships with corals and that these algae could contribute to coral growth in early symbiosis stages. PMID:24914677

  1. Biosystematic studies on Enicostema axillare (Lam.) A. Raynal subsp. Axillare (Gentianaceae) in peninsular India.

    PubMed

    Shahina, P M; Nampy, Santhosh

    2014-05-01

    The pantropical genus Enicostema (Gentianaceae) has three species and two sub species world over, namely, E. verticillatum (L.) Engl. (America), E. elizabethae Veldkamp (Madagascar) and E. axillare having 3 subsp. viz., subsp. axillare (Lam.) A. Raynal (India), subsp. latilobum (N.E. Br.) A. Raynal (East Africa) and subsp. littorale (Blume) A. Raynal (Indonesia). The present study aims to delimit the Indian taxa based on field and herbarium studies. Comparative morphology is studied using live as well as consulting wide range of specimens housed at various herbaria. The anatomy of leaf, stem, and root is studied using free hand sections and from epidermal peelings. The seed and pollen morphology are studied under SEM. Information on anatomy, palynology and seed micromorphology of E. axillare subsp. axillare is provided for the first time.

  2. Complete Genome and Methylome Sequences of Salmonella enterica subsp. enterica Serovar Panama (ATCC 7378) and Salmonella enterica subsp. enterica Serovar Sloterdijk (ATCC 15791)

    PubMed Central

    Yao, Kuan; Muruvanda, Tim; Roberts, Richard J.; Payne, Justin; Allard, Marc W.

    2016-01-01

    Salmonella enterica spp. are pathogenic bacteria commonly associated with food-borne outbreaks in human and animals. Salmonella enterica spp. are characterized into more than 2,500 different serotypes, which makes epidemiological surveillance and outbreak control more difficult. In this report, we announce the first complete genome and methylome sequences from two Salmonella type strains associated with food-borne outbreaks, Salmonella enterica subsp. enterica serovar Panama (ATCC 7378) and Salmonella enterica subsp. enterica serovar Sloterdijk (ATCC 15791). PMID:26988049

  3. Complete Genome and Methylome Sequences of Salmonella enterica subsp. enterica Serovar Panama (ATCC 7378) and Salmonella enterica subsp. enterica Serovar Sloterdijk (ATCC 15791).

    PubMed

    Yao, Kuan; Muruvanda, Tim; Roberts, Richard J; Payne, Justin; Allard, Marc W; Hoffmann, Maria

    2016-03-17

    Salmonella enterica spp. are pathogenic bacteria commonly associated with food-borne outbreaks in human and animals. Salmonella enterica spp. are characterized into more than 2,500 different serotypes, which makes epidemiological surveillance and outbreak control more difficult. In this report, we announce the first complete genome and methylome sequences from two Salmonella type strains associated with food-borne outbreaks, Salmonella enterica subsp. enterica serovar Panama (ATCC 7378) and Salmonella enterica subsp. enterica serovar Sloterdijk (ATCC 15791).

  4. Draft Genome Sequences of Three Pectobacterium Strains Causing Blackleg of Potato: P. carotovorum subsp. brasiliensis ICMP 19477, P. atrosepticum ICMP 1526, and P. carotovorum subsp. carotovorum UGC32

    PubMed Central

    Fiers, Mark W. E. J.; Lu, Ashley; Armstrong, Karen F.

    2015-01-01

    Blackleg is a disease caused by several species of Pectobacterium that results in losses to potato crops worldwide. Here, we report the draft genomes of three taxonomically and geographically distinct blackleg-causing strains of Pectobacterium: P. carotovorum subsp. brasiliensis ICMP 19477, P. atrosepticum ICMP 1526, and P. carotovorum subsp. carotovorum UGC32. Comparison of these genomes will support the identification of common traits associated with their capacity to cause blackleg. PMID:26251497

  5. Genetic diversity and phylogeography in two diploid ferns, Asplenium fontanum subsp. fontanum and A. petrarchae subsp. bivalens, in the western Mediterranean.

    PubMed

    Hunt, H V; Ansell, S W; Russell, S J; Schneider, H; Vogel, J C

    2009-12-01

    Asplenium fontanum subsp. fontanum and A. petrarchae subsp. bivalens are diploid rock ferns of limestone outcrops of the western Mediterranean region. Asplenium fontanum subsp. fontanum occurs from Valencia through northeastern Spain to the Alpes-Maritimes and Swiss Jura. Asplenium petrarchae subsp. bivalens occurs only on Majorca, in Valencia and possibly in southern Spain. We analysed allozyme and chloroplast genetic marker diversity in 75 populations of A. fontanum subsp. fontanum and 12 populations of A. petrarchae subsp. bivalens sampled from across their respective ranges. The two species show similar levels of species and population genetic diversity to one another and to other diploid European Asplenium taxa. Both are predominantly outbreeding, as indicated by F(IS) = 0.108 and 0.167 respectively. Substantial between-population differentiation results largely from differentiation between regions. Isolation by distance operates over limited geographic ranges, up to 50 km. In A. fontanum subsp. fontanum, the major geographical differentiation between Valencia and the rest of the taxon range probably represents an ancient range fragmentation. A less pronounced differentiation divides populations in the SW from those in the NE of the range, with evidence for a biogeographic link between the eastern Pyrenees and southeastern France. High diversity in the Pyrenees may either represent ancient population differentiation, or a suture zone. In A. petrarchae subsp. bivalens, populations on Majorca exhibit a subset of the genetic diversity present in Valencia, although the two regions are strongly differentiated by differing allele frequencies. Dispersal from the mainland may have founded Majorcan populations, although a role for in situ island survival cannot be excluded.

  6. Draft Genome Sequences of Three Pectobacterium Strains Causing Blackleg of Potato: P. carotovorum subsp. brasiliensis ICMP 19477, P. atrosepticum ICMP 1526, and P. carotovorum subsp. carotovorum UGC32.

    PubMed

    Panda, Preetinanda; Fiers, Mark W E J; Lu, Ashley; Armstrong, Karen F; Pitman, Andrew R

    2015-08-06

    Blackleg is a disease caused by several species of Pectobacterium that results in losses to potato crops worldwide. Here, we report the draft genomes of three taxonomically and geographically distinct blackleg-causing strains of Pectobacterium: P. carotovorum subsp. brasiliensis ICMP 19477, P. atrosepticum ICMP 1526, and P. carotovorum subsp. carotovorum UGC32. Comparison of these genomes will support the identification of common traits associated with their capacity to cause blackleg.

  7. Analysis by gas chromatography-mass spectrometry of the volatiles from the fruits of Ammodaucus leucotrichus subsp. leucotrichus and subsp. nanocarpus grown in North Africa and the Canary Islands, respectively.

    PubMed

    Velasco-Negueruela, A; Pérez-Alonso, M J; Pérez de Paz, P L; Palá-Paúl, J; Sanz, J

    2006-03-10

    The volatiles from the fruits of Ammodaucus leucotrichus subsp. leucotrichus and subsp. nanocarpus (two endemic species, the first from North Africa and the second from the Canary Islands, Spain) were studied by gas chromatography and gas chromatography-mass spectrometry. The major components of the volatiles of subsp. nanocarpus were found to be, beta-pinene (22.2-33.6%), bornyl angelate (20.6-21.8%) and camphor (8.3-11.7%) whereas in the fruits of subsp. leucotrichus, the main constituents were perillaldehyde (63.6%) and limonene (26.8%). We also suggest that subsp. nanocarpus should have the status of species and should be named Ammodaucus nanocarpus.

  8. Assessing the Inactivation of Mycobacterium avium subsp. paratuberculosis during Composting of Livestock Carcasses

    PubMed Central

    Tkachuk, Victoria L.; Krause, Denis O.; McAllister, Tim A.; Buckley, Katherine E.; Reuter, Tim; Hendrick, Steve

    2013-01-01

    Mycobacterium avium subsp. paratuberculosis causes Johne's disease (JD) in ruminants, with substantial economic impacts on the cattle industry. Johne's disease is known for its long latency period, and difficulties in diagnosis are due to insensitivities of current detection methods. Eradication is challenging as M. avium subsp. paratuberculosis can survive for extended periods within the environment, resulting in new infections in naïve animals (W. Xu et al., J. Environ. Qual. 38:437-450, 2009). This study explored the use of a biosecure, static composting structure to inactivate M. avium subsp. paratuberculosis. Mycobacterium smegmatis was also assessed as a surrogate for M. avium subsp. paratuberculosis. Two structures were constructed to hold three cattle carcasses each. Naturally infected tissues and ground beef inoculated with laboratory-cultured M. avium subsp. paratuberculosis and M. smegmatis were placed in nylon and plastic bags to determine effects of temperature and compost environment on viability over 250 days. After removal, samples were cultured and growth of both organisms was assessed after 12 weeks. After 250 days, M. avium subsp. paratuberculosis was still detectable by PCR, while M. smegmatis was not detected after 67 days of composting. Furthermore, M. avium subsp. paratuberculosis remained viable in both implanted nylon and plastic bags over the composting period. As the compost never reached a homogenous thermophilic (55 to 65°C) state throughout each structure, an in vitro experiment was conducted to examine viability of M. avium subsp. paratuberculosis after exposure to 80°C for 90 days. Naturally infected lymph tissues were mixed with and without compost. After 90 days, M. avium subsp. paratuberculosis remained viable despite exposure to temperatures typically higher than that achieved in compost. In conclusion, it is unlikely composting can be used as a means of inactivating M. avium subsp. paratuberculosis associated with cattle

  9. Phylogeny, evolutionary trends and classification of the Spathelia–Ptaeroxylon clade: morphological and molecular insights

    PubMed Central

    Appelhans, M. S.; Smets, E.; Razafimandimbison, S. G.; Haevermans, T.; van Marle, E. J.; Couloux, A.; Rabarison, H.; Randrianarivelojosia, M.; Keßler, P. J. A.

    2011-01-01

    Background and Aims The Spathelia–Ptaeroxylon clade is a group of morphologically diverse plants that have been classified together as a result of molecular phylogenetic studies. The clade is currently included in Rutaceae and recognized at a subfamilial level (Spathelioideae) despite the fact that most of its genera have traditionally been associated with other families and that there are no obvious morphological synapomorphies for the clade. The aim of the present study is to construct phylogenetic trees for the Spathelia–Ptaeroxylon clade and to investigate anatomical characters in order to decide whether it should be kept in Rutaceae or recognized at the familial level. Anatomical characters were plotted on a cladogram to help explain character evolution within the group. Moreover, phylogenetic relationships and generic limits within the clade are also addressed. Methods A species-level phylogenetic analysis of the Spathelia–Ptaeroxylon clade based on five plastid DNA regions (rbcL, atpB, trnL–trnF, rps16 and psbA–trnH) was conducted using Bayesian, maximum parsimony and maximum likelihood methods. Leaf and seed anatomical characters of all genera were (re)investigated by light and scanning electron microscopy. Key Results With the exception of Spathelia, all genera of the Spathelila–Ptaeroxylon clade are monophyletic. The typical leaf and seed anatomical characters of Rutaceae were found. Further, the presence of oil cells in the leaves provides a possible synapomorphy for the clade. Conclusions The Spathelia–Ptaeroxylon clade is well placed in Rutaceae and it is reasonable to unite the genera into one subfamily (Spathelioideae). We propose a new tribal classification of Spathelioideae. A narrow circumscription of Spathelia is established to make the genus monophyletic, and Sohnreyia is resurrected to accommodate the South American species of Spathelia. The most recent common ancestor of Spathelioideae probably had leaves with secretory cavities

  10. Antigenicity and Immunogenicity of a Trimeric Envelope Protein from an Indian Clade C HIV-1 Isolate*

    PubMed Central

    Sneha Priya, Rangasamy; Veena, Menon; Kalisz, Irene; Whitney, Stephen; Priyanka, Dhopeshwarkar; LaBranche, Celia C.; Sri Teja, Mullapudi; Montefiori, David C.; Pal, Ranajit; Mahalingam, Sundarasamy; Kalyanaraman, Vaniambadi S.

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) isolates from India mainly belong to clade C and are quite distinct from clade C isolates from Africa in terms of their phylogenetic makeup, serotype, and sensitivity to known human broadly neutralizing monoclonal antibodies. Because many of these properties are associated with the envelope proteins of HIV-1, it is of interest to study the envelope proteins of Indian clade C isolates as part of the ongoing efforts to develop a vaccine against HIV-1. To this end, we purified trimeric uncleaved gp145 of a CCR5 tropic Indian clade C HIV-1 (93IN101) from the conditioned medium of 293 cells. The purified protein was shown to be properly folded with stable structure by circular dichroism. Conformational integrity was further demonstrated by its high affinity binding to soluble CD4, CD4 binding site antibodies such as b12 and VRC01, quaternary epitope-specific antibody PG9, and CD4-induced epitope-specific antibody 17b. Sera from rabbits immunized with gp145 elicited high titer antibodies to various domains of gp120 and neutralized a broad spectrum of clade B and clade C HIV-1 isolates. Similar to other clade B and clade C envelope immunogens, most of the Tier 1 neutralizing activity could be absorbed with the V3-specific peptide. Subsequent boosting of these rabbits with a clade B HIV-1 Bal gp145 resulted in an expanded breadth of neutralization of HIV-1 isolates. The present study strongly supports the inclusion of envelopes from Indian isolates in a future mixture of HIV-1 vaccines. PMID:25691567

  11. Evolution of fruit and seed characters in the Diervilla and Lonicera clades (Caprifoliaceae, Dipsacales)

    PubMed Central

    Jacobs, Bart; Lens, Frederic; Smets, Erik

    2009-01-01

    Background and Aims The Diervilla and Lonicera clades are members of the family Caprifoliaceae (Dipsacales sensu Donoghue et al., 2001, Harvard Papers in Botany 6: 459–479). So far, the intergeneric relationships of the Lonicera clade and the systematic position of Heptacodium remain equivocal. By studying fruit and seed morphology and anatomy, an attempt is made to clarify these issues. In addition, this study deals with the evolution of fruit and seed characters of the Diervilla and Lonicera clades with reference to allied taxa. Methods Light and scanning electron microscopy were used for the morphological and anatomical investigations. Phylogenetic analyses were carried out by applying the parsimony and Bayesian inference optimality criteria. Character evolution was studied by means of parsimony optimization and stochastic character mapping. Key Results Diervilla and Weigela (Diervilla clade) are characterized by several unique traits in Dipsacales, including capsules with numerous seeds, seed coats without sclerified outer tangential exotestal cell walls, and dehiscent fruits. Seeds with completely sclerified exotestal cells and fleshy fruits characterize the Lonicera clade. Leycesteria and Lonicera have berries, ovaries without sterile carpels and several seeds per locule, whereas Symphoricarpos and Triosteum have drupes, ovaries with one or two sterile carpels and a single seed per locule. Heptacodium shares several characteristics with members of the Linnina clade, e.g. achenes, single-seeded fruits and a compressed, parenchymatous seed coat. Conclusions The results confirm the monophyly of the Diervilla and Lonicera clades and allow us to hypothesize a close relationship between Leycesteria and Lonicera and between Symphoricarpos and Triosteum. Fruit and seed morphology and anatomy point to a sister relationship of Heptacodium with the Linnina clade, rather than with the Lonicera clade. PMID:19502353

  12. Horizontal gene transfer and recombination in Streptococcus dysgalactiae subsp. equisimilis

    PubMed Central

    McNeilly, Celia L.; McMillan, David J.

    2014-01-01

    Streptococcus dysgalactiae subsp. equisimilis (SDSE) is a human pathogen that colonizes the skin or throat, and causes a range of diseases from relatively benign pharyngitis to potentially fatal invasive diseases. While not as virulent as the close relative Streptococcus pyogenes the two share a number of virulence factors and are known to coexist in a human host. Both pre- and post-genomic studies have revealed that horizontal gene transfer (HGT) and recombination occurs between these two organisms and plays a major role in shaping the population structure of SDSE. This review summarizes our current knowledge of HGT and recombination in the evolution of SDSE. PMID:25566202

  13. Stilbenes and flavonoids from Artocarpus nitidus subsp. lingnanensis.

    PubMed

    Ti, Huihui; Wu, Ping; Lin, Lidong; Wei, Xiaoyi

    2011-06-01

    The first stilbene possessing a γ-aminobutyric acid lactam function, artocarpene (1), and a new flavanone, 2-hydroxynaringenin 4'-O-β-d-glucopyranoside (2), were isolated from the stems of Artocarpus nitidus subsp. lingnanensis along with four known compounds, 2-hydroxynaringenin (3), oxyresveratrol (4), 3,4',5-trihydroxy-3'-prenylstilbene (5) and norartocarpetin (6). Their structures were elucidated on the basis of spectroscopic data. Compounds 1 exhibited weak antioxidant activity and 2 displayed weak cytotoxicity against human lung cancer A549 cell line.

  14. Steroidal saponins from the roots of Smilax aspera subsp. mauritanica.

    PubMed

    Belhouchet, Zineddine; Sautour, Marc; Miyamoto, Tomofumi; Lacaille-Dubois, Marie-Aleth

    2008-09-01

    Two new steroidal saponins (1, 2) were isolated from the roots of Smilax aspera subsp. mauritanica (POIR.) ARCANG. (Liliaceae), together with the known curillin G (3), asparagoside E (4), asparoside A (5), asparoside B (6) and the phenolic compound resveratrol (7). Their structures were established mainly on the basis of 600 MHz 2D-NMR spectral data. 3 exhibited antifungal activity against the human pathogenic yeasts Candida albicans, C. glabrata and C. tropicalis (minimum inhibitory concentrations of 25, 25 and 50 microg/ml, respectively) whereas the other compounds were inactive.

  15. Updating the Vibrio clades defined by multilocus sequence phylogeny: proposal of eight new clades, and the description of Vibrio tritonius sp. nov.

    PubMed Central

    Sawabe, Tomoo; Ogura, Yoshitoshi; Matsumura, Yuta; Feng, Gao; Amin, AKM Rohul; Mino, Sayaka; Nakagawa, Satoshi; Sawabe, Toko; Kumar, Ramesh; Fukui, Yohei; Satomi, Masataka; Matsushima, Ryoji; Thompson, Fabiano L.; Gomez-Gil, Bruno; Christen, Richard; Maruyama, Fumito; Kurokawa, Ken; Hayashi, Tetsuya

    2013-01-01

    To date 142 species have been described in the Vibrionaceae family of bacteria, classified into seven genera; Aliivibrio, Echinimonas, Enterovibrio, Grimontia, Photobacterium, Salinivibrio and Vibrio. As vibrios are widespread in marine environments and show versatile metabolisms and ecologies, these bacteria are recognized as one of the most diverse and important marine heterotrophic bacterial groups for elucidating the correlation between genome evolution and ecological adaptation. However, on the basis of 16S rRNA gene phylogeny, we could not find any robust monophyletic lineages in any of the known genera. We needed further attempts to reconstruct their evolutionary history based on multilocus sequence analysis (MLSA) and/or genome wide taxonomy of all the recognized species groups. In our previous report in 2007, we conducted the first broad multilocus sequence analysis (MLSA) to infer the evolutionary history of vibrios using nine housekeeping genes (the 16S rRNA gene, gapA, gyrB, ftsZ, mreB, pyrH, recA, rpoA, and topA), and we proposed 14 distinct clades in 58 species of Vibrionaceae. Due to the difficulty of designing universal primers that can amplify the genes for MLSA in every Vibrionaceae species, some clades had yet to be defined. In this study, we present a better picture of an updated molecular phylogeny for 86 described vibrio species and 10 genome sequenced Vibrionaceae strains, using 8 housekeeping gene sequences. This new study places special emphasis on (1) eight newly identified clades (Damselae, Mediterranei, Pectenicida, Phosphoreum, Profundum, Porteresiae, Rosenbergii, and Rumoiensis); (2) clades amended since the 2007 proposal with recently described new species; (3) orphan clades of genomospecies F6 and F10; (4) phylogenetic positions defined in 3 genome-sequenced strains (N418, EX25, and EJY3); and (5) description of V. tritonius sp. nov., which is a member of the “Porteresiae” clade. PMID:24409173

  16. Description of Teunomyces gen. nov. for the Candida kruisii clade, Suhomyces gen. nov. for the Candida tanzawaensis clade and Suhomyces kilbournensis sp. nov.

    PubMed

    Kurtzman, Cletus P; Robnett, Christie J; Blackwell, Meredith

    2016-08-01

    DNA sequence analysis has shown that species of the Candida kruisii clade and species of the C. tanzawaensis clade represent phylogenetically circumscribed genera, which are described as Teunomyces gen. nov., type species T kruisii, and Suhomyces gen. nov., type species S tanzawaensis Many of the species are distributed worldwide and they are often isolated from fungus-feeding insects and their habitats. Included is the description of S. kilbournensis (type strain NRRL Y-17864, CBS 14276), a species found almost exclusively on maize kernels (Zea mays) in IL, USA.

  17. Efficient Inactivation of Burkholderia pseudomallei or Francisella tularensis in infected Cells for Safe Removal from Biosafety Level 3 Containment Laboratories

    PubMed Central

    Emery, Felicia D.; Stabenow, Jennifer M.; Miller, Mark A.

    2014-01-01

    Working with infectious agents that require BSL-3 level containment agents offers many challenges for researchers. BSL-3 containment laboratories are usually not equipped with expensive specialty equipment that is needed for studies such as flow cytometric analysis, microscopy, and proteomic analyses. Therefore, for most researchers that are working with BSL-3 level infectious agents, removal of samples from BSL-3 labs for these types of studies is necessary, and methods for complete and dependable inactivation of the samples are required. In this report we have done a thorough characterization of the effectiveness of paraformaldehyde fixation for inactivation of cell samples infected with the intracellular bacterial agents Burkholderia pseudomallei (Bp) and Francisella tularensis (Ft), both of which are Tier 1 select agent pathogens that require BSL-3 containment. We have demonstrated that cells infected with these pathogens are completely inactivated via 5-minute treatment with 4% paraformaldehyde. Moreover, a 15-minute treatment with 2% paraformaldehyde completely sterilized both Bp- and Ft-infected cells. These studies also revealed that Bp is significantly more sensitive to paraformaldehyde treatment than Ft. Our findings have clearly demonstrated that a 15-minute treatment of Bp- or Ft-infected cells with 4% paraformaldehyde solution will allow for safe removal of the cell samples from BSL-3 labs for downstream studies. PMID:24449562

  18. Metabolism-directed structure optimization of benzimidazole-based Francisella tularensis enoyl-reductase (FabI) inhibitors.

    PubMed

    Zhang, Yan-Yan; Liu, Yong; Mehboob, Shahila; Song, Jin-Hua; Boci, Teuta; Johnson, Michael E; Ghosh, Arun K; Jeong, Hyunyoung

    2014-05-01

    1. FabI is a potential antibiotic target against Francisella tularensis, which has been classified as a Category A biowarfare agent of high risk to public health. Our previous work demonstrated that N-benzyl benzimidazole compounds possess promising FabI inhibitory activity, but their druggability properties, including metabolic stability, are unknown. 2. The objective of this study was to characterize structure-metabolism relationships of a series of N-benzyl benzimidazole compounds to guide chemical optimization for better metabolic stability. To this end, metabolic stability data were obtained for 22 initial lead compounds using mouse hepatic microsomes. 3. Metabolic hotspots on the benzimidazole core structure as well as the benzyl ring were identified and verified by metabolite identification studies of four model compounds. Interestingly, the proposed structure-metabolism relationships did not apply to nine newly synthesized cyclopentane or oxacyclopentane derivatives of N-benzyl benzimidazole. 4. Subsequently, in silico quantitative structure-property relationship models were developed. Four molecular descriptors representing molecular polarity/polarisability, symmetry and size were identified to best explain variability in metabolic stability of different compounds. Multi-linear and non-linear regression models based on the selected molecular descriptors were developed and validated. 5. The structure-metabolism relationships for N-benzyl benzimidazole compounds should help optimization of N-benzyl benzimidazole compounds for better pharmacokinetic behavior.

  19. Metabolism-Directed Structure Optimization of Benzimidazole-Based Francisella tularensis Enoyl-Reductase (FabI) Inhibitors

    PubMed Central

    Zhang, Yan-Yan; Liu, Yong; Mehboob, Shahila; Song, Jin-Hua; Boci, Teuta; Johnson, Michael E.; Ghosh, Arun K.; Jeong, Hyunyoung

    2015-01-01

    FabI is a potential antibiotic target against Francisella tularensis, which has been classified as a Category A biowarfare agent of high risk to public health. Our previous work demonstrated that N-benzyl benzimidazole compounds possess promising FabI inhibitory activity, but their druggability properties including metabolic stability are unknown. The objective of this study was to characterize structure-metabolism relationships of a series of N-benzyl benzimidazole compounds to guide chemical optimization for better metabolic stability. To this end, metabolic stability data were obtained for 22 initial lead compounds using mouse hepatic microsomes. Metabolic hotspots on the benzimidazole core structure as well as the benzyl ring were identified and verified by metabolite identification studies of 4 model compounds. Interestingly, the proposed structure-metabolism relationships did not apply to 9 newly synthesized cyclopentane or oxacyclopentane derivatives of N-benzyl benzimidazole. Subsequently, in silico quantitative structure-property relationship models were developed. Four molecular descriptors representing molecular polarity/polarisability, symmetry and size were identified to best explain variability in metabolic stability of different compounds. Multi-linear and nonlinear regression models based on the selected molecular descriptors were developed and validated. The structure-metabolism relationships for N-benzyl benzimidazole compounds should help optimization of N-benzyl benzimidazole compounds for better pharmacokinetic behavior. PMID:24171690

  20. Visualization of murine intranasal dosing efficiency using luminescent Francisella tularensis: effect of instillation volume and form of anesthesia.

    PubMed

    Miller, Mark A; Stabenow, Jennifer M; Parvathareddy, Jyothi; Wodowski, Andrew J; Fabrizio, Thomas P; Bina, Xiaowen R; Zalduondo, Lillian; Bina, James E

    2012-01-01

    Intranasal instillation is a widely used procedure for pneumonic delivery of drugs, vaccine candidates, or infectious agents into the respiratory tract of research mice. However, there is a paucity of published literature describing the efficiency of this delivery technique. In this report we have used the murine model of tularemia, with Francisella tularensis live vaccine strain (FTLVS) infection, to evaluate the efficiency of pneumonic delivery via intranasal dosing performed either with differing instillation volumes or different types of anesthesia. FTLVS was rendered luminescent via transformation with a reporter plasmid that constitutively expressed the Photorhabdus luminescens lux operon from a Francisella promoter. We then used an IVIS Spectrum whole animal imaging system to visualize FT dissemination at various time points following intranasal instillation. We found that instillation of FT in a dose volume of 10 µl routinely resulted in infection of the upper airways but failed to initiate infection of the pulmonary compartment. Efficient delivery of FT into the lungs via intranasal instillation required a dose volume of 50 µl or more. These studies also demonstrated that intranasal instillation was significantly more efficient for pneumonic delivery of FTLVS in mice that had been anesthetized with inhaled (isoflurane) vs. parenteral (ketamine/xylazine) anesthesia. The collective results underscore the need for researchers to consider both the dose volume and the anesthesia type when either performing pneumonic delivery via intranasal instillation, or when comparing studies that employed this technique.

  1. Visualization of Murine Intranasal Dosing Efficiency Using Luminescent Francisella tularensis: Effect of Instillation Volume and Form of Anesthesia

    PubMed Central

    Miller, Mark A.; Stabenow, Jennifer M.; Parvathareddy, Jyothi; Wodowski, Andrew J.; Fabrizio, Thomas P.; Bina, Xiaowen R.; Zalduondo, Lillian; Bina, James E.

    2012-01-01

    Intranasal instillation is a widely used procedure for pneumonic delivery of drugs, vaccine candidates, or infectious agents into the respiratory tract of research mice. However, there is a paucity of published literature describing the efficiency of this delivery technique. In this report we have used the murine model of tularemia, with Francisella tularensis live vaccine strain (FTLVS) infection, to evaluate the efficiency of pneumonic delivery via intranasal dosing performed either with differing instillation volumes or different types of anesthesia. FTLVS was rendered luminescent via transformation with a reporter plasmid that constitutively expressed the Photorhabdus luminescens lux operon from a Francisella promoter. We then used an IVIS Spectrum whole animal imaging system to visualize FT dissemination at various time points following intranasal instillation. We found that instillation of FT in a dose volume of 10 µl routinely resulted in infection of the upper airways but failed to initiate infection of the pulmonary compartment. Efficient delivery of FT into the lungs via intranasal instillation required a dose volume of 50 µl or more. These studies also demonstrated that intranasal instillation was significantly more efficient for pneumonic delivery of FTLVS in mice that had been anesthetized with inhaled (isoflurane) vs. parenteral (ketamine/xylazine) anesthesia. The collective results underscore the need for researchers to consider both the dose volume and the anesthesia type when either performing pneumonic delivery via intranasal instillation, or when comparing studies that employed this technique. PMID:22384012

  2. Slow-Onset Inhibition of the FabI Enoyl Reductase from Francisella tularensis: Residence Time and in Vivo Activity

    SciTech Connect

    Lu, H.; England, K; Ende, C; Truglio, J; Luckner, S; Reddy, B; Marlenee, N; Knudson, S; Knudson, D; et. al.

    2009-01-01

    Francisella tularensis is a highly virulent and contagious Gram-negative intracellular bacterium that causes the disease tularemia in mammals. The high infectivity and the ability of the bacterium to survive for weeks in a cool, moist environment have raised the possibility that this organism could be exploited deliberately as a potential biological weapon. Fatty acid biosynthesis (FAS-II) is essential for bacterial viability and has been validated as a target for the discovery of novel antibacterials. The FAS-II enoyl reductase ftuFabI has been cloned and expressed, and a series of diphenyl ethers have been identified that are subnanomolar inhibitors of the enzyme with MIC90 values as low as 0.00018 ?g mL-1. The existence of a linear correlation between the Ki and MIC values strongly suggests that the antibacterial activity of the diphenyl ethers results from direct inhibition of ftuFabI within the cell. The compounds are slow-onset inhibitors of ftuFabI, and the residence time of the inhibitors on the enzyme correlates with their in vivo activity in a mouse model of tularemia infection. Significantly, the rate of breakdown of the enzyme-inhibitor complex is a better predictor of in vivo activity than the overall thermodynamic stability of the complex, a concept that has important implications for the discovery of novel chemotherapeutics that normally rely on equilibrium measurements of potency.

  3. Modulation of hepatic PPAR expression during Ft LVS LPS-induced protection from Francisella tularensis LVS infection

    PubMed Central

    2010-01-01

    Background It has been shown previously that administration of Francisella tularensis (Ft) Live Vaccine Strain (LVS) lipopolysaccharide (LPS) protects mice against subsequent challenge with Ft LVS and blunts the pro-inflammatory cytokine response. Methods To further investigate the molecular mechanisms that underlie Ft LVS LPS-mediated protection, we profiled global hepatic gene expression following Ft LVS LPS or saline pre-treatment and subsequent Ft LVS challenge using Affymetrix arrays. Results A large number of genes (> 3,000) were differentially expressed at 48 hours post-infection. The degree of modulation of inflammatory genes by infection was clearly attenuated by pre-treatment with Ft LVS LPS in the surviving mice. However, Ft LVS LPS alone had a subtle effect on the gene expression profile of the uninfected mice. By employing gene set enrichment analysis, we discovered significant up-regulation of the fatty acid metabolism pathway, which is regulated by peroxisome proliferator activated receptors (PPARs). Conclusions We hypothesize that the LPS-induced blunting of pro-inflammatory response in mouse is, in part, mediated by PPARs (α and γ). PMID:20082697

  4. Benefits from living together? Clades whose species use similar habitats may persist as a result of eco-evolutionary feedbacks.

    PubMed

    Prinzing, Andreas; Ozinga, Wim A; Brändle, Martin; Courty, Pierre-Emmanuel; Hennion, Françoise; Labandeira, Conrad; Parisod, Christian; Pihain, Mickael; Bartish, Igor V

    2017-01-01

    Contents 66 I. 67 II. 68 III. 69 IV. 70 V. 73 VI. 75 VII. 77 78 References 78 SUMMARY: Recent decades have seen declines of entire plant clades while other clades persist despite changing environments. We suggest that one reason why some clades persist is that species within these clades use similar habitats, because such similarity may increase the degree of co-occurrence of species within clades. Traditionally, co-occurrence among clade members has been suggested to be disadvantageous because of increased competition and enemy pressure. Here, we hypothesize that increased co-occurrence among clade members promotes mutualist exchange, niche expansion or hybridization, thereby helping species avoid population decline from environmental change. We review the literature and analyse published data for hundreds of plant clades (genera) within a well-studied region and find major differences in the degree to which species within clades occupy similar habitats. We tentatively show that, in clades for which species occupy similar habitats, species tend to exhibit increased co-occurrence, mutualism, niche expansion, and hybridization - and rarely decline. Consistently, throughout the geological past, clades whose species occupied similar habitats often persisted through long time-spans. Overall, for many plant species, the occupation of similar habitats among fellow clade members apparently reduced their vulnerability to environmental change. Future research should identify when and how this previously unrecognized eco-evolutionary feedback operates.

  5. Transmission of a heterologous clade C Symbiodinium in a model anemone infection system via asexual reproduction

    PubMed Central

    Chen, Wan-Nan U.; Hsiao, Ya-Ju; Mayfield, Anderson B.; Young, Ryan; Hsu, Ling-Lan

    2016-01-01

    Anemones of genus Exaiptasia are used as model organisms for the study of cnidarian-dinoflagellate (genus Symbiodinium) endosymbiosis. However, while most reef-building corals harbor Symbiodinium of clade C, Exaiptasia spp. anemones mainly harbor clade B Symbiodinium (ITS2 type B1) populations. In this study, we reveal for the first time that bleached Exaiptasia pallida anemones can establish a symbiotic relationship with a clade C Symbiodinium (ITS2 type C1). We further found that anemones can transmit the exogenously supplied clade C Symbiodinium cells to their offspring by asexual reproduction (pedal laceration). In order to corroborate the establishment of stable symbiosis, we used microscopic techniques and genetic analyses to examine several generations of anemones, and the results of these endeavors confirmed the sustainability of the system. These findings provide a framework for understanding the differences in infection dynamics between homologous and heterologous dinoflagellate types using a model anemone infection system. PMID:27635330

  6. Dispersal and niche evolution jointly shape the geographic turnover of phylogenetic clades across continents

    PubMed Central

    Eiserhardt, Wolf L.; Svenning, Jens-Christian; Baker, William J.; Couvreur, Thomas L. P.; Balslev, Henrik

    2013-01-01

    The turnover of phylogenetic clades across space is a fundamental biodiversity pattern that may depend on long-term evolutionary processes, and that has downstream effects on other aspects of diversity including species richness and community structure. Limited niche evolution and limited dispersal are two major processes causing spatial restriction, and thus turnover, of clades. We studied the determinants of clade turnover within the World's richest floristic kingdom, the Neotropics, using the palm family (Arecaceae) as a model. We show that continental-scale clade turnover is driven by a combination of limited niche evolution — with respect to temperature and soil tolerances — and limited dispersal. These findings are consistent with strong dispersal barriers within the Neotropics, and the observation that some palm lineages are most diverse in certain biomes or climates. The importance of such deep-time effects suggest that palms might be slow to adapt or disperse in response to anthropogenic climate change. PMID:23383367

  7. Mosaic clade M human immunodeficiency virus type 1 (HIV-1) envelope immunogens

    DOEpatents

    Korber, Bette T.; Fischer, William; Liao, Hua-Xin; Haynes, Barton F.; Letvin, Norman; Hahn; Beatrice H.

    2011-05-31

    The present invention relates to mosaic clade M HIV-1 Env polypeptides and to compositions comprising same. The polypeptides of the invention are suitable for use in inducing an immune response to HIV-1 in a human.

  8. Transmission of a heterologous clade C Symbiodinium in a model anemone infection system via asexual reproduction.

    PubMed

    Chen, Wan-Nan U; Hsiao, Ya-Ju; Mayfield, Anderson B; Young, Ryan; Hsu, Ling-Lan; Peng, Shao-En

    2016-01-01

    Anemones of genus Exaiptasia are used as model organisms for the study of cnidarian-dinoflagellate (genus Symbiodinium) endosymbiosis. However, while most reef-building corals harbor Symbiodinium of clade C, Exaiptasia spp. anemones mainly harbor clade B Symbiodinium (ITS2 type B1) populations. In this study, we reveal for the first time that bleached Exaiptasia pallida anemones can establish a symbiotic relationship with a clade C Symbiodinium (ITS2 type C1). We further found that anemones can transmit the exogenously supplied clade C Symbiodinium cells to their offspring by asexual reproduction (pedal laceration). In order to corroborate the establishment of stable symbiosis, we used microscopic techniques and genetic analyses to examine several generations of anemones, and the results of these endeavors confirmed the sustainability of the system. These findings provide a framework for understanding the differences in infection dynamics between homologous and heterologous dinoflagellate types using a model anemone infection system.

  9. Isolation of an aerobic sulfur oxidizer from the SUP05/Arctic96BD-19 clade.

    PubMed

    Marshall, Katharine T; Morris, Robert M

    2013-02-01

    Bacteria from the uncultured SUP05/Arctic96BD-19 clade of gamma proteobacterial sulfur oxidizers (GSOs) have the genetic potential to oxidize reduced sulfur and fix carbon in the tissues of clams and mussels, in oxygen minimum zones and throughout the deep ocean (>200 m). Here, we report isolation of the first cultured representative from this GSO clade. Closely related cultures were obtained from surface waters in Puget Sound and from the deep chlorophyll maximum in the North Pacific gyre. Pure cultures grow aerobically on natural seawater media, oxidize sulfur, and reach higher final cell densities when glucose and thiosulfate are added to the media. This suggests that aerobic sulfur oxidation enhances organic carbon utilization in the oceans. The first isolate from the SUP05/Arctic96BD-19 clade was given the provisional taxonomic assignment 'Candidatus: Thioglobus singularis', alluding to the clade's known role in sulfur oxidation and the isolate's planktonic lifestyle.

  10. Strong genetic differentiation between North American and European populations of Phytophthora alni subsp. uniformis.

    PubMed

    Aguayo, Jaime; Adams, Gerard C; Halkett, Fabien; Catal, Mursel; Husson, Claude; Nagy, Zoltán Á; Hansen, Everett M; Marçais, Benoît; Frey, Pascal

    2013-02-01

    Alder decline caused by Phytophthora alni has been one of the most important diseases of natural ecosystems in Europe during the last 20 years. The emergence of P. alni subsp. alni -the pathogen responsible for the epidemic-is linked to an interspecific hybridization event between two parental species: P. alni subsp. multiformis and P. alni subsp. uniformis. One of the parental species, P. alni subsp. uniformis, has been isolated in several European countries and, recently, in North America. The objective of this work was to assess the level of genetic diversity, the population genetic structure, and the putative reproduction mode and mating system of P. alni subsp. uniformis. Five new polymorphic microsatellite markers were used to contrast both geographical populations. The study comprised 71 isolates of P. alni subsp. uniformis collected from eight European countries and 10 locations in North America. Our results revealed strong differences between continental populations (Fst = 0.88; Rst = 0.74), with no evidence for gene flow. European isolates showed extremely low genetic diversity compared with the North American collection. Selfing appears to be the predominant mating system in both continental collections. The results suggest that the European P. alni subsp. uniformis population is most likely alien and derives from the introduction of a few individuals, whereas the North American population probably is an indigenous population.

  11. Are Clade Specific HIV Vaccines a Necessity? An Analysis Based on Mathematical Models.

    PubMed

    Dimitrov, Dobromir; Kublin, James G; Ramsey, Scott; Corey, Lawrence

    2015-12-01

    As HIV-1 envelope immune responses are critical to vaccine related protection, most candidate HIV vaccines entering efficacy trials are based upon a clade specific design. This need for clade specific vaccine prototypes markedly reduces the implementation of potentially effective HIV vaccines. We utilized a mathematical model to determine the effectiveness of immediate roll-out of a non-clade matched vaccine with reduced efficacy compared to constructing clade specific vaccines, which would take considerable time to manufacture and test in safety and efficacy trials. We simulated the HIV epidemic in San Francisco (SF) and South Africa (SA) and projected effectiveness of three vaccination strategies: i) immediate intervention with a 20-40% vaccine efficacy (VE) non-matched vaccine, ii) delayed intervention by developing a 50% VE clade-specific vaccine, and iii) immediate intervention with a non-matched vaccine replaced by a clade-specific vaccine when developed. Immediate vaccination with a non-clade matched vaccine, even with reduced efficacy, would prevent thousands of new infections in SF and millions in SA over 30 years. Vaccination with 50% VE delayed for five years needs six and 12 years in SA to break-even with immediate 20 and 30% VE vaccination, respectively, while not able to surpass the impact of immediate 40% VE vaccination over 30 years. Replacing a 30% VE with a 50% VE vaccine after 5 years reduces the HIV acquisition by 5% compared to delayed vaccination. The immediate use of an HIV vaccine with reduced VE in high risk communities appears desirable over a short time line but higher VE should be the pursued to achieve strong long-term impact. Our analysis illustrates the importance of developing surrogate markers (correlates of protection) to allow bridging types of immunogenicity studies to support more rapid assessment of clade specific vaccines.

  12. Co-circulation of Clade C New World Arenaviruses: New geographic distribution and host species.

    PubMed

    Fernandes, Jorlan; de Oliveira, Renata Carvalho; Guterres, Alexandro; de Carvalho Serra, Fabiana; Bonvicino, Cibele Rodrigues; D'Andrea, Paulo Sergio; Cunha, Rivaldo Venâncio; Levis, Silvana; de Lemos, Elba Regina Sampaio

    2015-07-01

    Clade C, of the New World Arenaviruses, is composed of only the Latino and Oliveros viruses and, besides the geographic range of their rodent reservoirs, the distribution of these viruses has been restricted to Bolivia and Argentina. In this study, the genetic detection and phylogenetic analysis of the complete S segment sequences of sympatric arenaviruses from Brazil revealed a new geographic distribution of clade C arenaviruses, as well as the association of Oliveros virus with a new rodent reservoir.

  13. Genome Sequences of Three Strains of the Pseudomonas aeruginosa PA7 Clade

    PubMed Central

    Marti, Romain; Cournoyer, Benoit

    2015-01-01

    Draft genome sequences of three P. aeruginosa strains from the PA7 clade are presented here. Their lengths are 6.36 (EML528), 6.44 (EML545), and 6.33 Mb (EML548). Comparisons with the PA7 genome showed 5,113 conserved coding sequences (CDSs), and significant numbers of strain-specific CDSs. Their analysis will improve our understanding of this highly divergent clade. PMID:26586898

  14. Comparative genomics of two 'Candidatus Accumulibacter' clades performing biological phosphorus removal.

    PubMed

    Flowers, Jason J; He, Shaomei; Malfatti, Stephanie; del Rio, Tijana Glavina; Tringe, Susannah G; Hugenholtz, Philip; McMahon, Katherine D

    2013-12-01

    Members of the genus Candidatus Accumulibacter are important in many wastewater treatment systems performing enhanced biological phosphorus removal (EBPR). The Accumulibacter lineage can be subdivided phylogenetically into multiple clades, and previous work showed that these clades are ecologically distinct. The complete genome of Candidatus Accumulibacter phosphatis strain UW-1, a member of Clade IIA, was previously sequenced. Here, we report a draft genome sequence of Candidatus Accumulibacter spp. strain UW-2, a member of Clade IA, assembled following shotgun metagenomic sequencing of laboratory-scale bioreactor sludge. We estimate the genome to be 80-90% complete. Although the two clades share 16S rRNA sequence identity of >98.0%, we observed a remarkable lack of synteny between the two genomes. We identified 2317 genes shared between the two genomes, with an average nucleotide identity (ANI) of 78.3%, and accounting for 49% of genes in the UW-1 genome. Unlike UW-1, the UW-2 genome seemed to lack genes for nitrogen fixation and carbon fixation. Despite these differences, metabolic genes essential for denitrification and EBPR, including carbon storage polymer and polyphosphate metabolism, were conserved in both genomes. The ANI from genes associated with EBPR was statistically higher than that from genes not associated with EBPR, indicating a high selective pressure in EBPR systems. Further, we identified genomic islands of foreign origins including a near-complete lysogenic phage in the Clade IA genome. Interestingly, Clade IA appeared to be more phage susceptible based on it containing only a single Clustered Regularly Interspaced Short Palindromic Repeats locus as compared with the two found in Clade IIA. Overall, the comparative analysis provided a genetic basis to understand physiological differences and ecological niches of Accumulibacter populations, and highlights the importance of diversity in maintaining system functional resilience.

  15. Phylogeny of Helieae (Gentianaceae): Resolving taxonomic chaos in a Neotropical clade.

    PubMed

    Calió, Maria Fernanda; Lepis, Katherine B; Pirani, José Rubens; Struwe, Lena

    2017-01-01

    The monophyletic and Neotropical tribe Helieae of the worldwide family Gentianaceae (Gentianales, Asterids, Angiospermae) is well known for its problematic generic classifications. An initial phylogenetic analysis of Helieae shed light onto the relationships between genera, and indicated that traditional generic limits did not correspond to monophyletic groups. In order to obtain a more thorough understanding of generic relationships within the group, we enhanced sampling within the so-called Symbolanthus clade and performed phylogenetic analyses from DNA sequences from one plastid region (matK) and two nuclear regions (ITS and 5S-NTS), plus 112 morphological characters, which were analyzed separately and in combination, using parsimony and Bayesian approaches. A total of 83 individuals representing 20 genera and 51 species of Helieae were sampled; 13 species were included in this study solely based on their morphological characters. Ancestral character reconstructions were performed to identify potential synapomorphies of clades and patterns of homoplasy in the morphological dataset. Our results demonstrate that Prepusa is sister to the remainder of Helieae. Furthermore, the Macrocarpaea clade, the Irlbachia clade and the Symbolanthus clade were also recovered. Within the Symbolanthus clade, our results confirm that Calolisianthus and Chelonanthus are not monophyletic, and also contest the monophyly of Irlbachia as currently circumscribed. Specifically, two species of Calolisianthus group with the type species of Chelonanthus, while the other Calolisianthus species are more closely related to Tetrapollinia and Symbolanthus. Moreover, the green-white-flowered Chelonanthus species and Adenolisianthus are undoubtedly related to Helia and several analyses support Irlbachia pratensis as more closely related to the lineage including the type species of Chelonanthus described above The addition of new characters and taxa led to higher confidence in the relative position

  16. Clade-specific morphological diversification and adaptive radiation in Hawaiian songbirds.

    PubMed Central

    Lovette, Irby J; Bermingham, Eldredge; Ricklefs, Robert E

    2002-01-01

    The Hawaiian honeycreepers are a dramatic example of adaptive radiation but contrast with the four other songbird lineages that successfully colonized the Hawaiian archipelago and failed to undergo similar diversification. To explore the processes that produced the diversity dichotomy in this insular fauna, we compared clade age and morphological diversity between the speciose honeycreepers and the comparatively depauperate Hawaiian thrushes. Mitochondrial-DNA-based genetic distances between these Hawaiian clades and their continental sister taxa indicate that the ancestral thrush colonized the Hawaiian Islands as early as the common ancestor of the honeycreepers. This similar timing of colonization indicates that the marked difference in diversity between the Hawaiian honeycreeper and thrush clades is unlikely to result from differences in these clades' tenures within the archipelago. If time cannot explain the contrasting diversities of these taxa, then an intrinsic, clade-specific trait may have fostered the honeycreeper radiation. As the honeycreepers have diversified most dramatically in morphological characters related to resource utilization, we used principal components analyses of bill characters to compare the magnitudes of morphological variation in the ancestral clades from which the Hawaiian honeycreeper and thrush lineages are derived, the Carduelini and Turdinae respectively. Although the Carduelini share a more recent common ancestor and have a lower species diversity than the Turdinae, these finch-like relatives of the honeycreepers exhibit significantly greater variation in bill morphology than do the continental relatives of the Hawaiian thrushes. The higher magnitude of morphological variation in the non-Hawaiian Carduelini suggests that the honeycreepers fall within a clade exhibiting a generally high evolutionary flexibility in bill morphology. Accordingly, although the magnitude of bill variation among the honeycreepers is similar to that of

  17. Neurocognitive Impairment in HIV-1 Clade C versus B Infected Individuals in Southern Brazil

    PubMed Central

    de Almeida, Sergio Monteiro; Ribeiro, Clea Elisa; de Pereira, Ana Paula; Badiee, Jayraan; Cherner, Mariana; Smith, Davey; Maich, Ingrid; Raboni, Sonia Mara; Rotta, Indianara; Barbosa, Francisco Jaime; Heaton, Robert K.; Umlauf, Anya; Ellis, Ronald J.

    2014-01-01

    HIV-1 clade C isolates show reduced Tat protein chemoattractant activity compared with clade B. This might influence neuropathogenesis by altering trafficking of monocytes into the CNS. A previous study suggested low rates of HIV-associated dementia in clade C infected individuals. The present study evaluated neurocognitive impairment rates in clade B- and C-infected individuals from the same local population. HIV+ and HIV- participants were recruited from the same geographic region in southern Brazil. We evaluated neuropsychological (NP) impairment using a screening instrument (the International HIV Dementia Scale; IHDS), as well as a Brazilian Portuguese adaptation of a comprehensive battery that has demonstrated sensitivity to HIV associated neurocognitive disorders (HAND) internationally. NP performance in controls was used to generate T-scores and impairment ratings by the global deficit score (GDS) method. Clade assignments were ascertained by sequencing pol and env. Blood and cerebrospinal fluid (CSF) were collected from all HIV+ participants. HIV+ and HIV- participants were comparable on demographic characteristics. HIV+ participants overall were more likely to be impaired than HIV- by the IHDS and the GDS. Clade B and C infected individuals were demographically similar and did not differ significantly in rates of impairment. The prevalence of pleocytosis, a marker of intrathecal cellular chemotaxis, also did not differ between clade B and C infections. Clade B and C HIV-infected individuals from the same geographic region, when ascertained using comparable methods, did not differ in their rates of neurocognitive impairment, and there was no evidence of differences in CNS chemotaxis. PMID:24277437

  18. Neurocognitive impairment in HIV-1 clade C- versus B-infected individuals in Southern Brazil.

    PubMed

    de Almeida, Sergio Monteiro; Ribeiro, Clea Elisa; de Pereira, Ana Paula; Badiee, Jayraan; Cherner, Mariana; Smith, Davey; Maich, Ingrid; Raboni, Sonia Mara; Rotta, Indianara; Barbosa, Francisco Jaime; Heaton, Robert K; Umlauf, Anya; Ellis, Ronald J

    2013-12-01

    HIV-1 clade C isolates show reduced Tat protein chemoattractant activity compared with clade B. This might influence neuropathogenesis by altering trafficking of monocytes into the CNS. A previous study suggested low rates of HIV-associated dementia in clade C-infected individuals. The present study evaluated neurocognitive impairment rates in clade B- and C-infected individuals from the same local population. HIV+ and HIV- participants were recruited from the same geographic region in Southern Brazil. We evaluated neuropsychological (NP) impairment using a screening instrument (the International HIV Dementia Scale (IHDS)), as well as a Brazilian Portuguese adaptation of a comprehensive battery that has demonstrated sensitivity to HIV-associated neurocognitive disorders (HAND) internationally. NP performance in controls was used to generate T scores and impairment ratings by the global deficit score (GDS) method. Clade assignments were ascertained by sequencing pol and env. Blood and cerebrospinal fluid were collected from all HIV+ participants. HIV+ and HIV- participants were comparable on demographic characteristics. HIV+ participants overall were more likely to be impaired than HIV- by the IHDS and the GDS. Clade B- and C-infected individuals were demographically similar and did not differ significantly in rates of impairment. The prevalence of pleocytosis, a marker of intrathecal cellular chemotaxis, also did not differ between clade B and C infections. Clade B and C HIV-infected individuals from the same geographic region, when ascertained using comparable methods, did not differ in their rates of neurocognitive impairment, and there was no evidence of differences in CNS chemotaxis.

  19. Systematics within Gyps vultures: a clade at risk

    PubMed Central

    Johnson, Jeff A; Lerner, Heather RL; Rasmussen, Pamela C; Mindell, David P

    2006-01-01

    Background Populations of the Oriental White-backed Vulture (Gyps bengalensis) have declined by over 95% within the past decade. This decline is largely due to incidental consumption of the non-steroidal anti-inflammatory veterinary pharmaceutical diclofenac, commonly used to treat domestic livestock. The conservation status of other Gyps vultures in southern Asia is also of immediate concern, given the lack of knowledge regarding status of their populations and the continuing existence of taxonomic uncertainties. In this study, we assess phylogenetic relationships for all recognized species and the majority of subspecies within the genus Gyps. The continuing veterinary use of diclofenac is an unknown but potential risk to related species with similar feeding habits to Gyps bengalensis. Therefore, an accurate assessment of the phylogenetic relationships among Gyps vultures should aid in their conservation by clarifying taxonomic uncertainties, and enabling inference of their respective relatedness to susceptible G. bengalensis. Results Phylogenetic results using mitochondrial cytB, ND2 and control region sequence data indicate a recent and rapid diversification within the genus Gyps. All recognized species formed monophyletic groups with high statistical support, with the exception of the Eurasian Vulture, for which specimens identified as subspecies G. fulvus fulvescens appear closely related to the Himalayan Vulture (G. himalayensis). In all analyses, the earliest divergence separated the Oriental White-backed Vulture from other Gyps taxa, with the next diverging taxon being either the African White-backed Vulture (G. africanus), or the Himalayan Vulture. All analyses supported a sister relationship between the Eurasian Vulture (G. f. fulvus), and Rüppell's Vulture (G. rueppellii), with this clade being sister to another consisting of the two taxa of "Long-billed" Vulture (G. indicus indicus and G. i. tenuirostris), and the Cape Vulture (G. coprotheres). These

  20. Development of Quantitative Real-time PCR Assays for Different Clades of “Candidatus Accumulibacter”

    NASA Astrophysics Data System (ADS)

    Zhang, An Ni; Mao, Yanping; Zhang, Tong

    2016-05-01

    We designed novel quantitative real-time polymerase chain reaction (qPCR) primers for the polyphosphate kinase 1 (ppk1) gene, targeting eight individual “Candidatus Accumulibacter” (referred to as Accumulibacter) clades. An evaluation of primer sets was conducted regarding the coverage, specificity, and PCR efficiency. (i) All primer sets were designed to cover all available sequences of the target clade. (ii) The phylogenetic analysis of the sequences retrieved from the qPCR products by each primer set demonstrated a high level of specificity. (iii) All calibration curves presented high PCR efficiencies in the range of 85–112% (R2 = 0.962–0.998). In addition, the possible interference of non-target amplicons was individually examined using the qPCR assay for 13 Accumulibacter clades, which were either undetected or showed negligible detection. With the primers designed by other research groups, a highly selective and sensitive qPCR-based method was developed to quantify all Accumulibacter clades, with the exception of Clade IE, in one assay, which enables more comprehensive insights into the community dynamics. The applicability to environmental samples was demonstrated by profiling the Accumulibacter clades in activated sludge samples of nine full-scale wastewater treatment plants.

  1. Global Escherichia coli Sequence Type 131 Clade with blaCTX-M-27 Gene

    PubMed Central

    Pitout, Johann D.D.; Gomi, Ryota; Matsuda, Tomonari; Noguchi, Taro; Yamamoto, Masaki; Peirano, Gisele; DeVinney, Rebekah; Bradford, Patricia A.; Motyl, Mary R.; Tanaka, Michio; Nagao, Miki; Takakura, Shunji; Ichiyama, Satoshi

    2016-01-01

    The Escherichia coli sequence type (ST) 131 C2/H30Rx clade with the blaCTX-M-15 gene had been most responsible for the global dissemination of extended-spectrum β-lactamase (ESBL)–producing E. coli. ST131 C1/H30R with blaCTX-M-27 emerged among ESBL-producing E. coli in Japan during the late 2000s. To investigate the possible expansion of a single clade, we performed whole-genome sequencing for 43 Japan and 10 global ST131 isolates with blaCTX-M-27 (n = 16), blaCTX-M-14 (n = 16), blaCTX-M-15 (n = 13), and others (n = 8). We also included 8 ST131 genomes available in public databases. Core genome-based analysis of 61 isolates showed that ST131 with blaCTX-M-27 from 5 countries formed a distinct cluster within the C1/H30R clade, named C1-M27 clade. Accessory genome analysis identified a unique prophage-like region, supporting C1-M27 as a distinct clade. Our findings indicate that the increase of ESBL-producing E. coli in Japan is due mainly to emergence of the C1-M27 clade. PMID:27767006

  2. Global Escherichia coli Sequence Type 131 Clade with blaCTX-M-27 Gene.

    PubMed

    Matsumura, Yasufumi; Pitout, Johann D D; Gomi, Ryota; Matsuda, Tomonari; Noguchi, Taro; Yamamoto, Masaki; Peirano, Gisele; DeVinney, Rebekah; Bradford, Patricia A; Motyl, Mary R; Tanaka, Michio; Nagao, Miki; Takakura, Shunji; Ichiyama, Satoshi

    2016-11-01

    The Escherichia coli sequence type (ST) 131 C2/H30Rx clade with the blaCTX-M-15 gene had been most responsible for the global dissemination of extended-spectrum β-lactamase (ESBL)-producing E. coli. ST131 C1/H30R with blaCTX-M-27 emerged among ESBL-producing E. coli in Japan during the late 2000s. To investigate the possible expansion of a single clade, we performed whole-genome sequencing for 43 Japan and 10 global ST131 isolates with blaCTX-M-27 (n = 16), blaCTX-M-14 (n = 16), blaCTX-M-15 (n = 13), and others (n = 8). We also included 8 ST131 genomes available in public databases. Core genome-based analysis of 61 isolates showed that ST131 with blaCTX-M-27 from 5 countries formed a distinct cluster within the C1/H30R clade, named C1-M27 clade. Accessory genome analysis identified a unique prophage-like region, supporting C1-M27 as a distinct clade. Our findings indicate that the increase of ESBL-producing E. coli in Japan is due mainly to emergence of the C1-M27 clade.

  3. Onshore-offshore trends in benthic faunal change: drive by clade origination

    SciTech Connect

    Bottjer, D.J.; Jablonski, D.

    1985-01-01

    Higher taxa in Phanerozoic benthic communities appear to arise preferentially in nearshore habitats and expand outwards across the continental shelf. This pattern is demonstrably independent of sea-level fluctuations, and could be driven by a variety of evolutionary processes, including: 1) differential origination of novelties nearshore; and 2) equal occurrence of innovations across the shelf, but with offshore novelties more extinction-prone than onshore novelties in the early phases of diversification. To test these mechanism, it is necessary to analyze the pattern on a clade-by-clade basis, rather than leaving the clades embedded within their communities. Review and synthesis of the literature indicates that a number of clades have their first species originating onshore with a subsequent history that shows either (A) migration offshore while losing onshore components or (B) expansion offshore while persisting onshore. In a detailed study of pattern A the authors have compiled presence/absence data for the crinoid order Isocrinida in 70 post-Paleozoic benthic communities, which shows migration across the shelf in the mid-Mesozoic. These studies show that different, contemporaneous clades proceeded across the shelf at different times and rates; no evidence is found for cohesive community migration. The supposed pattern of community migration may be an epiphenomenon, underlain by individualistic clade histories that appear to act in concert when viewed on a coarse time scale.

  4. Clade Replacements in Dengue Virus Serotypes 1 and 3 Are Associated with Changing Serotype Prevalence†

    PubMed Central

    Zhang, Chunlin; Mammen, Mammen P.; Chinnawirotpisan, Piyawan; Klungthong, Chonticha; Rodpradit, Prinyada; Monkongdee, Patama; Nimmannitya, Suchitra; Kalayanarooj, Siripen; Holmes, Edward C.

    2005-01-01

    The evolution of dengue virus (DENV) is characterized by phylogenetic trees that have a strong temporal structure punctuated by dramatic changes in clade frequency. To determine the cause of these large-scale phylogenetic patterns, we examined the evolutionary history of DENV serotype 1 (DENV-1) and DENV-3 in Thailand, where gene sequence and epidemiological data are relatively abundant over a 30-year period. We found evidence for the turnover of viral clades in both serotypes, most notably in DENV-1, where a major clade replacement event took place in genotype I during the mid-1990s. Further, when this clade replacement event was placed in the context of changes in serotype prevalence in Thailand, a striking pattern emerged; an increase in DENV-1 clade diversity was associated with an increase in the abundance of this serotype and a concomitant decrease in DENV-4 prevalence, while clade replacement was associated with a decline in DENV-1 prevalence and a rise of DENV-4. We postulate that intraserotypic genetic diversification proceeds at times of relative serotype abundance and that replacement events can result from differential susceptibility to cross-reactive immune responses. PMID:16306584

  5. Development of Quantitative Real-time PCR Assays for Different Clades of “Candidatus Accumulibacter”

    PubMed Central

    Zhang, An Ni; Mao, Yanping; Zhang, Tong

    2016-01-01

    We designed novel quantitative real-time polymerase chain reaction (qPCR) primers for the polyphosphate kinase 1 (ppk1) gene, targeting eight individual “Candidatus Accumulibacter” (referred to as Accumulibacter) clades. An evaluation of primer sets was conducted regarding the coverage, specificity, and PCR efficiency. (i) All primer sets were designed to cover all available sequences of the target clade. (ii) The phylogenetic analysis of the sequences retrieved from the qPCR products by each primer set demonstrated a high level of specificity. (iii) All calibration curves presented high PCR efficiencies in the range of 85–112% (R2 = 0.962–0.998). In addition, the possible interference of non-target amplicons was individually examined using the qPCR assay for 13 Accumulibacter clades, which were either undetected or showed negligible detection. With the primers designed by other research groups, a highly selective and sensitive qPCR-based method was developed to quantify all Accumulibacter clades, with the exception of Clade IE, in one assay, which enables more comprehensive insights into the community dynamics. The applicability to environmental samples was demonstrated by profiling the Accumulibacter clades in activated sludge samples of nine full-scale wastewater treatment plants. PMID:27142574

  6. Taxonomic evaluation of species in the Streptomyces hirsutus clade using multi-locus sequence analysis and proposals to reclassify several species in this clade

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous phylogenetic analyses of species of Streptomyces based on 16S rRNA gene sequences resulted in a statistically well-supported clade (100% bootstrap value) containing 8 species that exhibited very similar gross morphology in producing open looped (Retinaculum-Apertum) to spiral (Spira) chains...

  7. The binding sites of monoclonal antibodies to the non-reducing end of Francisella tularensis O-antigen accommodate mainly the terminal saccharide

    PubMed Central

    Lu, Zhaohua; Rynkiewicz, Michael J; Yang, Chiou-Ying; Madico, Guillermo; Perkins, Hillary M; Wang, Qi; Costello, Catherine E; Zaia, Joseph; Seaton, Barbara A; Sharon, Jacqueline

    2013-01-01

    We have previously described two types of protective B-cell epitopes in the O-antigen (OAg) of the Gram-negative bacterium Francisella tularensis: repeating internal epitopes targeted by the vast majority of anti-OAg monoclonal antibodies (mAbs), and a non-overlapping epitope at the non-reducing end targeted by the previously unique IgG2a mAb FB11. We have now generated and characterized three mAbs specific for the non-reducing end of F. tularensis OAg, partially encoded by the same variable region germline genes, indicating that they target the same epitope. Like FB11, the new mAbs, Ab63 (IgG3), N213 (IgG3) and N62 (IgG2b), had higher antigen-binding bivalent avidity than internally binding anti-OAg mAbs, and an oligosaccharide containing a single OAg repeat was sufficient for optimal inhibition of their antigen-binding. The X-ray crystal structure of N62 Fab showed that the antigen-binding site is lined mainly by aromatic amino acids that form a small cavity, which can accommodate no more than one and a third sugar residues, indicating that N62 binds mainly to the terminal Qui4NFm residue at the nonreducing end of OAg. In efficacy studies with mice infected intranasally with the highly virulent F. tularensis strain SchuS4, N62, N213 and Ab63 prolonged survival and reduced blood bacterial burden. These results yield insights into how antibodies to non-reducing ends of microbial polysaccharides can contribute to immune protection despite the smaller size of their target epitopes compared with antibodies to internal polysaccharide regions. PMID:23844703

  8. Proposal to reclassify Brenneria quercina (Hildebrand and Schroth 1967) Hauben et al. 1999 into a new genus, Lonsdalea gen. nov., as Lonsdalea quercina comb. nov., descriptions of Lonsdalea quercina subsp. quercina comb. nov., Lonsdalea quercina subsp. iberica subsp. nov. and Lonsdalea quercina subsp. britannica subsp. nov., emendation of the description of the genus Brenneria, reclassification of Dickeya dieffenbachiae as Dickeya dadantii subsp. dieffenbachiae comb. nov., and emendation of the description of Dickeya dadantii.

    PubMed

    Brady, Carrie L; Cleenwerck, Ilse; Denman, Sandra; Venter, Stephanus N; Rodríguez-Palenzuela, Pablo; Coutinho, Teresa A; De Vos, Paul

    2012-07-01

    Bacterial isolates from oak trees in Spain and Britain, showing symptoms of bark canker and Acute Oak Decline (AOD), respectively, were examined by a polyphasic approach. Both 16S rRNA gene sequencing and multilocus sequence analysis (MLSA), based on partial sequences of gyrB, rpoB, infB and atpD genes, revealed that the isolates were separated into two genetic groups according to their origin. Their closest phylogenetic relative was Brenneria quercina, the causal agent of drippy nut disease of oak, which clustered distant to the other species of the genus Brenneria. MLSA data for species of the genera Brenneria, Pectobacterium, Dickeya, Erwinia, Pantoea and Samsonia confirmed the polyphyletic nature of the genus Brenneria and indicated synonymy of Dickeya dadantii and Dickeya dieffenbachiae. DNA-DNA hybridization experiments confirmed this synonymy and also revealed DNA-DNA relatedness values of 58-73% between the new oak isolates and B. quercina. Phenotypic and/or chemotaxonomic methods allowed B. quercina and the two genetic groups of new oak isolates to be discriminated from other recognized species of the genus Brenneria and from members of the closely related genera Dickeya, Pectobacterium and Samsonia. Based on the data obtained, the following taxonomic proposals are made: (1) reclassification of B. quercina as the type species of a novel genus, Lonsdalea gen. nov., as Lonsdalea quercina comb. nov. (type strain LMG 2724(T)=ATCC 29281(T)=CCUG 48867(T)=CFBP 3617(T)=CIP 105201(T)=DSM 4561(T)=ICMP 1845(T)), (2) classification of the oak isolates as Lonsdalea quercina subsp. iberica subsp. nov. (type strain LMG26264(T)=NCPPB 4490(T)) and Lonsdalea quercina subsp. britannica subsp. nov. (type strain LMG 26267(T)=NCPPB 4481(T)) and leading to the automatic creation of Lonsdalea quercina subsp. quercina subsp. nov. (type strain LMG 2724(T)=ATCC 29281(T)), (3) emendation of the description of the genus Brenneria, and (4) reclassification of Dickeya dieffenbachiae as

  9. Broad Clade 2 Cross-Reactive Immunity Induced by an Adjuvanted Clade 1 rH5N1 Pandemic Influenza Vaccine

    PubMed Central

    Leroux-Roels, Isabel; Bernhard, Roger; Gérard, Pascal; Dramé, Mamadou; Hanon, Emmanuel; Leroux-Roels, Geert

    2008-01-01

    Background The availability of H5N1 vaccines that can elicit a broad cross-protective immunity against different currently circulating clade 2 H5N1 viruses is a pre-requisite for the development of a successful pre-pandemic vaccination strategy. In this regard, it has recently been shown that adjuvantation of a recombinant clade 1 H5N1 inactivated split-virion vaccine with an oil-in-water emulsion-based adjuvant system also promoted cross-immunity against a recent clade 2 H5N1 isolate (A/Indonesia/5/2005, subclade 2.1). Here we further analyse the cross-protective potential of the vaccine against two other recent clade 2 isolates (A/turkey/Turkey/1/2005 and A/Anhui/1/2005 which are, as defined by WHO, representatives of subclades 2.2 and 2.3 respectively). Methods and Findings Two doses of the recombinant A/Vietnam/1194/2004 (H5N1, clade 1) vaccine were administered 21 days apart to volunteers aged 18–60 years. We studied the cross-clade immunogenicity of the lowest antigen dose (3.8 µg haemagglutinin) given with (N = 20) or without adjuvant (N = 20). Immune responses were assessed at 21 days following the first and second vaccine doses and at 6 months following first vaccination. Vaccination with two doses of 3.8 µg of the adjuvanted vaccine induced four-fold neutralising seroconversion rates in 85% of subjects against A/turkey/Turkey/1/2005 (subclade 2.2) and 75% of subjects against A/Anhui/1/2005 (subclade 2.3) recombinant strains. There was no response induced against these strains in the non-adjuvanted group. At 6 months following vaccination, 70% and 60% of subjects retained neutralising antibodies against the recombinant subclade 2.2 and 2.3 strains, respectively and 40% of subjects retained antibodies against the recombinant subclade 2.1 A/Indonesia/5/2005 strain. Conclusions In addition to antigen dose-sparing, adjuvantation of inactivated split H5N1 vaccine promotes broad and persistent cross-clade immunity which is a pre-requisite for a pre

  10. Discrimination between Mycoplasma mycoides subsp. capri and Mycoplasma capricolum subsp. capricolum using PCR-RFLP and PCR.

    PubMed

    Cillara, Grazia; Manca, Maria Giovanna; Longheu, Carla; Tola, Sebastiana

    2015-09-01

    In this study, the dihydrolipoyl dehydrogenase (lpdA) gene was used to distinguish Mycoplasma mycoides subsp. capri (Mmc) from Mycoplasma capricolum subsp. capricolum (Mcc), two of four Mycoplasma species that cause contagious agalactia in sheep and goats. After alignment of nucleotide sequences of both species, specific primer sets were designed from unchanging and variable gene segments. The first primer set LPD-C1-F/LPD-C1-R was used to amplify a 911 bp fragment that was subsequently co-digested with FastDigest PstI, SspI, EcoRI and ClaI enzymes. The PCR-RFLP profiles differentiated the two mycoplasma species. The second primer set was used to distinguish Mmc from Mcc by single tube PCR. Both methods were further applied to identify 54 isolates collected from dairy herds from different provinces in Sardinia. The results of this study showed that PCR-RFLP and PCR could be used in routine diagnosis for rapid and specific simultaneous discrimination of Mmc and Mcc.

  11. A proposal to unify two subspecies of Staphylococcus equorum: Staphylococcus equorum subsp. equorum and Staphylococcus equorum subsp. linens.

    PubMed

    Jeong, Do-Won; Kim, Hye-Rim; Han, Seulhwa; Jeon, Che Ok; Lee, Jong-Hoon

    2013-12-01

    Twelve isolates from jeotgal, a Korean high-salt-fermented seafood, identified as Staphylococcus equorum were compared by phenotypic and genotypic methods to determine their precise taxonomic identities at the subspecies level. Four strains and three strains had complete 16S rRNA gene sequence matches with S. equorum subsp. equorum DSM 20674(T) and S. equorum subsp. linens DSM 15097(T), respectively. Five strains showed 99.9 % identity with the sequences of both type strains. In our DNA-DNA hybridization analyses among two type strains and two isolates, the similarities were over 72 % and were higher than the similarities presented at the subspecies proposal. Physiological characteristics such as sugar utilization, β-galactosidase activity, novobiocin resistance and salt tolerance, which were adopted for subspecies separation, could not be applied to assign the isolates to a taxonomic unit. Antibiotic susceptibility, hemolytic activity, biofilm formation and protein profiles did not present markers to divide the isolates into either of the subspecies. Multilocus sequence typing of the sequences of the 16S rRNA gene and five housekeeping genes did not produce any coherent relationship among the isolates and type strains. Repetitive element-PCR fingerprinting using ERIC (enterobacterial repetitive intergenic consensus) primers classified 12 isolates to three genotypes, and the genotypes of both type strains coincided with two isolates expressing different characteristics. Based on these phenotypic and genotypic analyses results, we propose to unify the present two subspecies of S. equorum into one species, S. equorum.

  12. Alcaligenes faecalis subsp. parafaecalis subsp. nov., a bacterium accumulating poly-beta-hydroxybutyrate from acetone-butanol bioprocess residues.

    PubMed

    Schroll, G; Busse, H J; Parrer, G; Rölleke, S; Lubitz, W; Denner, E B

    2001-04-01

    The authors have previously isolated a solvent tolerant bacterium, strain G(T), (T = type strain) capable to convert acetone-butanol bioprocess residues into poly-beta-hydroxybutyrate. Strain G(T) was initially identified as Alcaligenes spp by standard bacteriological tests. In this study the taxonomic position of the bacterium was investigated in detail. The 165 rDNA sequence analysis, the G + C content of DNA (56 mol%) and the presence of ubiquinone Q-8 confirmed strain G(T) as a representative of the genus Alcaligenes. In the polyamine pattern of the bacterium putrescine and cadaverine were detected, but only trace amounts of 2-hydroxyputrescine. The extremely low content of 2-hydroxyputrescine is remarkable, since this unique diamine is a common marker for beta-proteobacteria. Phylogenetic analyses of 16S rDNA demonstrated that Alcaligenes sp. G(T) is most closely related to the species Alcaligenes faecalis (99.6% sequence similarity to A. faecalis HR4 and 98.7% sequence similarity to A. faecalis [ATCC 8750T = DSM 30030T]. On the basis of DNA-DNA relatedness (56% similarity), the unique polyamine pattern, the physiological and biochemical differences strain G(T) could be distinguished from the species A. faecalis. Therefore, a new subspecies for the species Alcaligenes faecalis is proposed; Alcaligenes faecalis subsp. parafaecalis subsp. nov.

  13. Alcaligenes faecalis subsp. phenolicus subsp. nov. a phenol-degrading, denitrifying bacterium isolated from a graywater bioprocessor.

    PubMed

    Rehfuss, Marc; Urban, James

    2005-07-01

    A Gram (-) coccobacillary bacterium, J(T), was isolated from a graywater bioprocessor. 16S rRNA and biochemical analysis has revealed strain J(T) closely resembles Alcaligenes faecalis ATCC 8750T and A. faecalis subsp. parafaecalis DSM 13975T, but is a distinct, previously uncharacterized isolate. Strain J(T), along with the type strain of A. faecalis and its previously described subspecies share the ability to aerobically degrade phenol. The degradation rates of phenol for strain J(T) and reference phenol degrading bacteria were determined by photometrically measuring the change in optical density when grown on 0.1% phenol as the sole carbon source, followed by addition of Gibb's reagent to measure depletion of substrate. The phenol degradation rates of strain J(T) was found to exceed that of the phenol hydroxylase group III bacterium Pseudomonas pseudoalcaligenes, with isolate J(T) exhibiting a doubling time of 4.5 h. The presence of the large subunit of the multicomponent phenol hydroxylase gene in strain J(T) was confirmed by PCR. The presence of the nirK nitrite reductase gene as demonstrated by PCR as well as results obtained from nitrite media indicated denitrification at least to N2O. Based on phenotypic, phylogenetic, fatty acid analysis and results from DNA DNA hybridization, we propose assigning a novel subspecies of Alcaligenes faecalis, to be named Alcaligenes faecalis subsp. phenolicus with the type strain J(T) (= DSM 16503) (= NRRL B-41076).

  14. Cloning and expression of hyaluronate lyase genes of Streptococcus intermedius and Streptococcus constellatus subsp. constellatus(1).

    PubMed

    Takao, Ayuko

    2003-02-14

    Hyaluronate lyase (HAase) genes of Streptococcus intermedius and Streptococcus constellatus subsp. constellatus were isolated. In S. constellatus subsp. constellatus, the deduced amino acid sequence of HAase was most similar to that of S. intermedius (68%), whereas the enzyme of S. intermedius was most similar to that of S. pneumoniae (72%). Upstream of the HAase gene on the opposite strands, an open reading frame of a putative glutathione peroxidase started in S. intermedius, and this arrangement was similar to that in S. pneumoniae but unlike that in S. constellatus subsp. constellatus. Cell lysates of Escherichia coli carrying each streptococcal gene showed HAase activity, demonstrating that each cloned gene actually coded for HAase.

  15. Increased Replicative Fitness of a Dengue Virus 2 Clade in Native Mosquitoes: Potential Contribution to a Clade Replacement Event in Nicaragua

    PubMed Central

    Quiner, Claire A.; Parameswaran, Poornima; Ciota, Alexander T.; Ehrbar, Dylan J.; Dodson, Brittany L.; Schlesinger, Sondra; Kramer, Laura D.

    2014-01-01

    ABSTRACT The four dengue virus (DENV) serotypes (DENV serotype 1 [DENV-1] to DENV-4) are transmitted by Aedes aegypti and A. albopictus mosquitoes, causing up to 390 million DENV infections worldwide each year. We previously reported a clade replacement of the DENV-2 Asian-American genotype NI-1 clade by the NI-2B clade in Managua, Nicaragua. Here, we describe our studies of the replicative ability of NI-1 and NI-2B viruses in an A. aegypti cell line (Aag2) and A. aegypti mosquitoes reared from eggs collected in Managua. In coinfection experiments, several different pairs of NI-1 and NI-2B clinical isolates were used to infect Aag2 cells or blood-fed A. aegypti mosquitoes. Results consistently showed a significant replicative advantage of NI-2B over NI-1 viruses early after infection in vitro, and in mosquitoes, NI-2B viruses attained a higher replicative index than NI-1 isolates 3 to 7 days postinfection (dpi). At 7 dpi, NI-2B viruses displayed a significantly higher replicative index in legs and salivary glands; however, this advantage was lost by 14 and 21 dpi. We also found that the percentage of mosquitoes in which NI-2B viruses were dominant was significantly higher than that in which NI-1 viruses were dominant on day 7 but not at later time points. Taken together, these data demonstrate that clade NI-2B holds a replicative advantage over clade NI-1 early in infection that wanes at later time points. This early fitness advantage of NI-2B viruses over NI-1 viruses in the native vector, A. aegypti, suggests a shorter extrinsic incubation period for NI-2B viruses, which could have contributed to the clade replacement event in Managua. IMPORTANCE Dengue virus (DENV), one of the most medically important arthropod-borne viruses, is transmitted to humans by Aedes aegypti and A. albopictus mosquitoes in tropical and subtropical regions worldwide. Dengue epidemics continue to increase in frequency, geographic range, and severity and are a major public health concern

  16. Nanolipoprotein Particles (NLPs) as Versatile Vaccine Platforms for Co-delivery of Multiple Adjuvants with Subunit Antigens from Burkholderia spp. and F. tularensis - Technical Report

    SciTech Connect

    Fischer, N. O.

    2015-01-06

    The goal of this proposal is to demonstrate that colocalization of protein subunit antigens and adjuvants on nanolipoprotein particles (NLPs) can increase the protective efficacy of subunit antigens from Burkholderia spp. and Francisella tularensis against an aerosol challenge. In the second quarter of the third year, LLNL finalized all immunological assessments of NLP vaccine formulations in the F344 model. Battelle has immunized rats with three unique NLP formulations by either intramuscular or intranasal administration. All inoculations have been completed, and protective efficacy against an aerosolized challenge will begin at the end of October, 2014.

  17. Trading Capsule for Increased Cytotoxin Production: Contribution to Virulence of a Newly Emerged Clade of emm89 Streptococcus pyogenes

    PubMed Central

    Zhu, Luchang; Olsen, Randall J.; Nasser, Waleed; de la Riva Morales, Ivan

    2015-01-01

    ABSTRACT Strains of emm89 Streptococcus pyogenes have become one of the major causes of invasive infections worldwide in the last 10 years. We recently sequenced the genome of 1,125 emm89 strains and identified three major phylogenetic groups, designated clade 1, clade 2, and the epidemic clade 3. Epidemic clade 3 strains, which now cause the great majority of infections, have two distinct genetic features compared to clade 1 and clade 2 strains. First, all clade 3 organisms have a variant 3 nga promoter region pattern, which is associated with increased production of secreted cytolytic toxins SPN (S. pyogenes NADase) and SLO (streptolysin O). Second, all clade 3 strains lack the hasABC locus mediating hyaluronic acid capsule synthesis, whereas this locus is intact in clade 1 and clade 2 strains. We constructed isogenic mutant strains that produce different levels of SPN and SLO toxins and capsule (none, low, or high). Here we report that emm89 strains with elevated toxin production are significantly more virulent than low-toxin producers. Importantly, we also show that capsule production is dispensable for virulence in strains that already produce high levels of SPN and SLO. Our results provide new understanding about the molecular mechanisms contributing to the rapid emergence and molecular pathogenesis of epidemic clade 3 emm89 S. pyogenes. PMID:26443457

  18. Szidat's rule re-tested: relationships between flea and host phylogenetic clade ranks in four biogeographic realms.

    PubMed

    Krasnov, Boris R; Kiefer, Daniel; Warburton, Elizabeth M; Khokhlova, Irina S

    2016-05-01

    We tested Szidat's rule (the more primitive the host, the more primitive the parasites it harbours) by analysing the relationships between phylogenetic clade ranks of fleas and their small mammalian hosts in four biogeographic realms (Afrotropics, Neotropics, Nearctic and Palearctic). From the host perspective, we tested the association between host clade rank and the mean clade rank of all fleas collected from this host. From the flea perspective, we tested the relationships between flea clade rank and the mean clade rank of hosts on which this flea was recorded. First, we tested whether the analysis of the relationships between host and flea clade ranks should be controlled for phylogenetic dependence among either host or flea species. Then, we tested for the associations between host and flea clade ranks separately for each realm using either a phylogenetic general least-squares analysis or an ordinary least-squares analysis. In all realms, the mean clade rank of fleas parasitic on a given host increased with an increase of this host's clade rank, and the mean clade rank of hosts recorded on a given flea increased with an increase of this flea's clade rank, suggesting that Szidat's rule, at least to some extent, holds for fleas.

  19. One test microbial diagnostic microarray for identification of Mycoplasma mycoides subsp. mycoides and other Mycoplasma species.

    PubMed

    Tonelli, A; Sacchini, F; Krasteva, I; Zilli, K; Scacchia, M; Beaurepaire, C; Nantel, A; Pini, A

    2012-11-01

    The present study describes the use of microarray technology for rapid identification and differentiation of Mycoplasma mycoides subsp. mycoides from other mycoplasmas that may be pathogenic to ruminants, including those of the Mycoplasma mycoides cluster, genetically and antigenically strictly correlated with Mycoplasma mycoides subsp. mycoides. A microarray containing genetic sequences of 55 different bacterial species from Acholeplasma, Mycoplasma, Spiroplasma and Ureaplasma genera was constructed. Sequences to genes of interest were collected in FASTA format from NCBI. The collected sequences were processed with OligoPicker software. Oligonucleotides were then checked for their selectivity with BLAST searches in GenBank. The microarray was tested with ATCC/NCTC strains of Mycoplasma spp. of veterinary importance in ruminants including Mycoplasma belonging to the mycoides cluster as well as Mycoplasma mycoides subsp. mycoides and Mycoplasma mycoides subsp. capri field strains. The results showed that but one ATCC/NCTC reference strains hybridized with their species-specific sequences showed a profile/signature different and distinct from each other. The heat-map of the hybridization results for the nine genes interrogated for Mycoplasma mycoides subsp. mycoides demonstrated that the reference strain Mycoplasma mycoides subsp mycoides PG1 was positive for all of the gene sequences spotted on the microarray. CBPP field, vaccine and reference strains were all typed to be M. mycoides subsp. mycoides, and seven of the nine strains gave positive hybridization results for all of the nine genes. Two Italian strains were negative for some of the genes. Comparison with non-Mycoplasma mycoides subsp. mycoides reference strains showed some positive signals or considerable homology to Mycoplasma mycoides subsp. mycoides genes. As expected, some correlations were observed between the strictly genetically and antigenically correlated Mycoplasma mycoides subsp. mycoides and

  20. The Type III Secretion System Effector SeoC of Salmonella enterica subsp. salamae and S. enterica subsp. arizonae ADP-Ribosylates Src and Inhibits Opsonophagocytosis

    PubMed Central

    Pollard, Dominic J.; Young, Joanna C.; Covarelli, Valentina; Herrera-León, Silvia; Connor, Thomas R.; Fookes, Maria; Walker, Danielle; Echeita, Aurora; Thomson, Nicholas R.; Berger, Cedric N.

    2016-01-01

    Salmonella species utilize type III secretion systems (T3SSs) to translocate effectors into the cytosol of mammalian host cells, subverting cell signaling and facilitating the onset of gastroenteritis. In this study, we compared a draft genome assembly of Salmonella enterica subsp. salamae strain 3588/07 against the genomes of S. enterica subsp. enterica serovar Typhimurium strain LT2 and Salmonella bongori strain 12419. S. enterica subsp. salamae encodes the Salmonella pathogenicity island 1 (SPI-1), SPI-2, and the locus of enterocyte effacement (LEE) T3SSs. Though several key S. Typhimurium effector genes are missing (e.g., avrA, sopB, and sseL), S. enterica subsp. salamae invades HeLa cells and contains homologues of S. bongori sboK and sboC, which we named seoC. SboC and SeoC are homologues of EspJ from enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively), which inhibit Src kinase-dependent phagocytosis by ADP-ribosylation. By screening 73 clinical and environmental Salmonella isolates, we identified EspJ homologues in S. bongori, S. enterica subsp. salamae, and Salmonella enterica subsp. arizonae. The β-lactamase TEM-1 reporter system showed that SeoC is translocated by the SPI-1 T3SS. All the Salmonella SeoC/SboC homologues ADP-ribosylate Src E310 in vitro. Ectopic expression of SeoC/SboC inhibited phagocytosis of IgG-opsonized beads into Cos-7 cells stably expressing green fluorescent protein (GFP)-FcγRIIa. Concurrently, S. enterica subsp. salamae infection of J774.A1 macrophages inhibited phagocytosis of beads, in a seoC-dependent manner. These results show that S. bongori, S. enterica subsp. salamae, and S. enterica subsp. arizonae share features of the infection strategy of extracellular pathogens EPEC and EHEC and shed light on the complexities of the T3SS effector repertoires of Enterobacteriaceae. PMID:27736780

  1. The in vitro effect of six antimicrobials against Mycoplasma putrefaciens, Mycoplasma mycoides subsp. mycoides LC and Mycoplasma capricolum subsp. capricolum isolated from sheep and goats in Jordan.

    PubMed

    Al-Momani, W; Nicholas, R A J; Janakat, S; Abu-Basha, E; Ayling, R D

    2006-01-01

    Respiratory disease in sheep and goats is a major problem in Jordan and is often associated with Mycoplasma species. Without effective vaccines, control is mainly by chemotherapy, but the uncontrolled use of antimicrobials has led to concerns about the potential development of antimicrobial resistance. The in vitro effect of chloramphenicol, florfenicol, enrofloxacin, tylosin, erythromycin and oxytetracycline was determined against 32 isolates of Mycoplasma species-M. mycoides subsp. mycoides LC (6), M. capricolum subsp. capricolum (8) and M. putrefaciens (18), all isolated from either nasal swabs or milk, from sheep and goats in different regions of Jordan. The antimicrobial susceptibility showed some Mycoplasma species-specific differences, with M. capricolum subsp. capricolum being more susceptible to tylosin and erythromycin. Chloramphenicol and florfenicol were the least effective for all three Mycoplasma species. No trends or significant differences in antimicrobial susceptibilities were observed between sheep and goat isolates, between milk or nasal swab isolates, or between isolates from different regions of Jordan. Some isolates of M. capricolum subsp. capricolum and M. putrefaciens showed higher MIC levels with oxytetracycline, as did two isolates of M. mycoides subsp. mycoides LC with tylosin, possibly indicating signs of development of antimicrobial resistance.

  2. Functional and Structural Characterization of Francisella tularensis O-Antigen Antibodies at the Low End of Antigen Reactivity

    PubMed Central

    Lu, Zhaohua; Rynkiewicz, Michael J.; Yang, Chiou-Ying; Madico, Guillermo; Perkins, Hillary M.; Roche, Marly I.; Seaton, Barbara A.

    2014-01-01

    The O-antigen (OAg) of the Gram-negative bacterium Francisella tularensis (Ft), which is both a capsular polysaccharide and a component of lipopolysaccharide, is comprised of tetrasaccharide repeats and induces antibodies mainly against repeating internal epitopes. We previously reported on several BALB/c mouse monoclonal antibodies (MAbs) that bind to internal Ft OAg epitopes and are protective in mouse models of respiratory tularemia. We now characterize three new internal Ft OAg IgG2a MAbs, N203, N77, and N24, with 10- to 100-fold lower binding potency than previously characterized internal-OAg IgG2a MAbs, despite sharing one or more variable region germline genes with some of them. In a mouse model of respiratory tularemia with the highly virulent Ft type A strain SchuS4, the three new MAbs reduced blood bacterial burden with potencies that mirror their antigen-binding strength; the best binder of the new MAbs, N203, prolonged survival in a dose-dependent manner, but was at least 10-fold less potent than the best previously characterized IgG2a MAb, Ab52. X-ray crystallographic studies of N203 Fab showed a flexible binding site in the form of a partitioned groove, which cannot provide as many contacts to OAg as does the Ab52 binding site. These results reveal structural features of antibodies at the low end of reactivity with multi-repeat microbial carbohydrates and demonstrate that such antibodies still have substantial protective effects against infection. PMID:25171003

  3. Draft Genome Sequence of Salmonella enterica subsp. enterica Serovar Bardo Strain CRJJGF_00099 (Phylum Gammaproteobacteria)

    PubMed Central

    Gupta, Sushim K.; McMillan, Elizabeth A.; Jackson, Charlene R.; Desai, Prerak T.; Porwollik, Steffen; McClelland, Michael; Hiott, Lari M.; Humayoun, Shaheen B.

    2016-01-01

    Here, we report a 4.87-Mbp draft genome sequence of the multidrug-resistant (MDR) Salmonella enterica subsp. enterica serovar Bardo strain CRJJGF_00099, isolated from dairy cattle in 2005. PMID:27634995

  4. Draft Genome Sequence of the Probiotic Bifidobacterium longum subsp. longum Strain MC-42

    PubMed Central

    Tupikin, Alexey E.; Kalmykova, Anna I.

    2016-01-01

    Here, we report the draft genome sequence of Bifidobacterium longum subsp. longum strain MC-42 isolated from the feces of a healthy infant, and which was used in the commercially available probiotic product Biovestin. PMID:27979954

  5. Draft Genome Sequence of Klebsiella pneumoniae subsp. pneumoniae ATCC 9621.

    PubMed

    Poehlein, Anja; Najdenski, Hristo; Simeonova, Diliana D

    2017-03-23

    We present here the 5.561-Mbp assembled draft genome sequence of Klebsiella pneumoniae subsp. pneumoniae ATCC 9621, a phosphite- and organophosphonate-assimilating Gammaproteobacterium. The genome harbors 5,179 predicted protein-coding genes.

  6. Composition and potency characterization of Mycobacterium avium subsp. paratuberculosis purified protein derivatives

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP) purified protein derivatives (PPDs) are immunologic reagents prepared from cultured filtrates of the type strain ATCC 19698. Traditional production consists of floating culture incubation at 37oC, organism inactivation by autoclaving, coarse filtrat...

  7. Comparative Genomics of Bifidobacterium animalis subsp. lactis Reveals a Strict Monophyletic Bifidobacterial Taxon

    PubMed Central

    Milani, Christian; Duranti, Sabrina; Lugli, Gabriele Andrea; Bottacini, Francesca; Strati, Francesco; Arioli, Stefania; Foroni, Elena; Turroni, Francesca; van Sinderen, Douwe

    2013-01-01

    Strains of Bifidobacterium animalis subsp. lactis are extensively exploited by the food industry as health-promoting bacteria, although the genetic variability of members belonging to this taxon has so far not received much scientific attention. In this article, we describe the complete genetic makeup of the B. animalis subsp. lactis Bl12 genome and discuss the genetic relatedness of this strain with other sequenced strains belonging to this taxon. Moreover, a detailed comparative genomic analysis of B. animalis subsp. lactis genomes was performed, which revealed a closely related and isogenic nature of all currently available B. animalis subsp. lactis strains, thus strongly suggesting a closed pan-genome structure of this bacterial group. PMID:23645200

  8. Complete genome sequence of Campylobacter fetus subsp. testudinum type strain 03-427T

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Campylobacter fetus subsp. testudinum has been isolated from reptiles and humans. This Campylobacter subspecies is genetically distinct from other C. fetus subspecies. Here we present the first whole genome sequence for this C. fetus subspecies....

  9. Novel fermentation media for production of Bacillus thuringiensis subsp. israelensis.

    PubMed

    Poopathi, Subbiah; Kumar, K Anup

    2003-08-01

    The production of Bacillus thuringiensis subsp. israelensis (deBarjac) (Bti) as a biopesticide is not cost-effective using existing fermentation technology. In this study, we explored the use of several less expensive alternative culture media (potato, common sugar, and Bengal gram) for the growth and production of Bti. Growth was obtained in all tested media and was comparable to that obtained in conventional medium (Luria-Bertani). Toxicity assays showed that the toxin produced from the novel growth media were effective in killing larvae of Culex quinquefasciatus, Anopheles stephensi, and Aedes aegypti and toxicity was comparable to that produced from Luria-Bertani medium. These observations suggest that potato can be used as a cheap source of culture medium for the production of Bti toxin in mosquito control programs.

  10. Volatile Components Emitted from the Liverwort Marchantia paleacea subsp. diptera.

    PubMed

    Sakurai, Kazutoshi; Tomiyama, Kenichi; Kawakami, Yukihiko; Ochiai, Nozomi; Yabe, Shigeki; Nakagawa, Tomomi; Asakawa, Yoshinori

    2016-02-01

    The volatile components from the thalloid liverwort, Marchantia paleacea subsp. diptera were investigated by HS-SPME-GC-MS analysis. The monocyclic monoterpene aldehyde, perillaldehyde was identified for the first time as the major component and its content was about 50% of the volatiles, along with β-pinene, limonene, β-caryophyllene, α-selinene and β-selinene as minor volatiles. Using MD (Multi-dimensional) GC-MS analysis equipped with a chiral column as the second column, the chirality was determined of both perillaldehyde and limonene, which was considered as the precursor of perillaldehyde. Both compounds were (S)-(-)-enantiomers (over 99.0 %) and (R)-enantiomers (less than 0.5 %). This is the first report of the existence of perillaldehyde in liverworts.

  11. Metabolic engineering of Propionibacterium freudenreichii subsp. shermanii for xylose fermentation.

    PubMed

    Wei, Peilian; Lin, Meng; Wang, Zhongqiang; Fu, Hongxin; Yang, Hopen; Jiang, Wenyan; Yang, Shang-Tian

    2016-11-01

    Propionibacterium freudenreichii cannot use xylose, the second most abundant sugar in lignocellulosic biomass. Although Propionibacterium acidipropionici can use xylose as a carbon source, it is difficult to genetically modify, impeding further improvement through metabolic engineering. This study identified three xylose catabolic pathway genes encoding for xylose isomerase (xylA), xylose transporter (xylT), and xylulokinase (xylB) in P. acidipropionici and overexpressed them in P. freudenreichii subsp. shermanii via an expression plasmid pKHEM01, enabling the mutant to utilize xylose efficiently even in the presence of glucose without glucose-induced carbon catabolite repression. The mutant showed similar fermentation kinetics with glucose, xylose, and the mixture of glucose and xylose, respectively, as carbon source, and with or without the addition of antibiotic for selection pressure. The engineered P. shermanii thus can provide a novel cell factory for industrial production of propionic acid and other value-added products from lignocellulosic biomass.

  12. Occurrence of Mycobacterium avium subsp. paratuberculosis in untreated water in Northern Ireland.

    PubMed

    Whan, Lynne; Ball, Hywel J; Grant, Irene R; Rowe, Michael T

    2005-11-01

    Mycobacterium avium subsp. paratuberculosis is the known cause of Johne's disease of both domestic and wild ruminants and has been implicated as a possible cause of Crohn's disease in humans. The organism is shed in the feces of infected animals and can survive for protracted periods in the environment and hence could be present in catchment areas receiving agricultural runoff. A limited survey was undertaken in Northern Ireland to test for M. avium subsp. paratuberculosis in untreated water entering nine water treatment works (WTWs) over a 1-year period. Three detection methods were employed, viz., immunomagnetic separation-PCR and culture on Herrold's egg yolk medium (HEYM) and BACTEC 12B medium, the latter both supplemented with mycobactins. Of the 192 untreated water samples tested, 15 (8%) tested M. avium subsp. paratuberculosis positive by one or more of the three detection methods. M. avium subsp. paratuberculosis was successfully isolated from eight untreated water samples, three by BACTEC culture and five by culture on HEYM. Although the highest incidence of M. avium subsp. paratuberculosis was found in spring, overall, there was no statistically significant difference between the seasons. No significant correlation was found between numbers of coliforms or fecal coliforms and the presence of M. avium subsp. paratuberculosis. In general, a higher incidence of M. avium subsp. paratuberculosis was found in untreated water entering those WTWs that had a high mean water pH value over the sampling period. This work indicates the need to determine the efficacy of water treatment processes to either kill or remove M. avium subsp. paratuberculosis from untreated water and the possible risks posed by contact with recreational water sources.

  13. Unusual outbreak of clinical mastitis in dairy sheep caused by Streptococcus equi subsp. zooepidemicus.

    PubMed

    Las Heras, Alfonso; Vela, Ana I; Fernández, Elena; Legaz, Emilio; Domínguez, Lucas; Fernández-Garayzábal, Jose F

    2002-03-01

    This work describes an outbreak of clinical mastitis affecting 13 of 58 lactating ewes due to Streptococcus equi subsp. zooepidemicus. S. equi subsp. zooepidemicus was isolated in pure culture from all milk samples. All the clinical isolates had identical biochemical profiles and antimicrobial susceptibility patterns and also exhibited indistinguishable macrorestriction patterns by pulsed-field gel electrophoresis, indicating that all cases of mastitis were produced by a single strain.

  14. Unusual Outbreak of Clinical Mastitis in Dairy Sheep Caused by Streptococcus equi subsp. zooepidemicus

    PubMed Central

    Las Heras, Alfonso; Vela, Ana I.; Fernández, Elena; Legaz, Emilio; Domínguez, Lucas; Fernández-Garayzábal, Jose F.

    2002-01-01

    This work describes an outbreak of clinical mastitis affecting 13 of 58 lactating ewes due to Streptococcus equi subsp. zooepidemicus. S. equi subsp. zooepidemicus was isolated in pure culture from all milk samples. All the clinical isolates had identical biochemical profiles and antimicrobial susceptibility patterns and also exhibited indistinguishable macrorestriction patterns by pulsed-field gel electrophoresis, indicating that all cases of mastitis were produced by a single strain. PMID:11880454

  15. Seed-associated subspecies of the genus Clavibacter are clearly distinguishable from Clavibacter michiganensis subsp. michiganensis.

    PubMed

    Yasuhara-Bell, Jarred; Alvarez, Anne M

    2015-03-01

    The genus Clavibacter contains one recognized species, Clavibacter michiganensis. Clavibacter michiganensis is subdivided into subspecies based on host specificity and bacteriological characteristics, with Clavibacter michiganensis subsp. michiganensis causing bacterial canker of tomato. Clavibacter michiganensis subsp. michiganensis is often spread through contaminated seed leading to outbreaks of bacterial canker in tomato production areas worldwide. The frequent occurrence of non-pathogenic Clavibacter michiganensis subsp. michiganensis-like bacteria (CMB) is a concern for seed producers because Clavibacter michiganensis subsp. michiganensis is a quarantine organism and detection of a non-pathogenic variant may result in destruction of an otherwise healthy seed lot. A thorough biological and genetic characterization of these seed-associated CMB strains was performed using standard biochemical tests, cell wall analyses, metabolic profiling using Biolog, and single-gene and multilocus sequence analyses. Combined, these tests revealed two distinct populations of seed-associated members of the genus Clavibacter that differed from each other, as well as from all other described subspecies of Clavibacter michiganensis. DNA-DNA hybridization values are 70 % or higher, justifying placement into the single recognized species, C. michiganensis, but other analyses justify separate subspecies designations. Additionally, strains belonging to the genus Clavibacter isolated from pepper also represent a distinct population and warrant separate subspecies designation. On the basis of these data we propose subspecies designations for separate non-pathogenic subpopulations of Clavibacter michiganensis: Clavibacter michiganensis subsp. californiensis subsp. nov. and Clavibacter michiganensis subsp. chilensis subsp. nov. for seed-associated strains represented by C55(T) ( = ATCC BAA-2691(T) = CFBP 8216(T)) and ZUM3936(T) ( = ATCC BAA-2690(T) = CFBP 8217(T

  16. Temporal Transcriptional Response during Infection of Type II Alveolar Epithelial Cells with Francisella tularensis Live Vaccine Strain (LVS) Supports a General Host Suppression and Bacterial Uptake by Macropinocytosis*

    PubMed Central

    Bradburne, Christopher E.; Verhoeven, Anne B.; Manyam, Ganiraju C.; Chaudhry, Saira A.; Chang, Eddie L.; Thach, Dzung C.; Bailey, Charles L.; van Hoek, Monique L.

    2013-01-01

    Pneumonic tularemia is caused by inhalation of Francisella tularensis, one of the most infectious microbes known. We wanted to study the kinetics of the initial and early interactions between bacterium and host cells in the lung. To do this, we examined the infection of A549 airway epithelial cells with the live vaccine strain (LVS) of F. tularensis. A549 cells were infected and analyzed for global transcriptional response at multiple time points up to 16 h following infection. At 15 min and 2 h, a strong transcriptional response was observed including cytoskeletal rearrangement, intracellular transport, and interferon signaling. However, at later time points (6 and 16 h), very little differential gene expression was observed, indicating a general suppression of the host response consistent with other reported cell lines and murine tissues. Genes for macropinocytosis and actin/cytoskeleton rearrangement were highly up-regulated and common to the 15 min and 2 h time points, suggesting the use of this method for bacterial entry into cells. We demonstrate macropinocytosis through the uptake of FITC-dextran and amiloride inhibition of Francisella LVS uptake. Our results suggest that macropinocytosis is a potential mechanism of intracellular entry by LVS and that the host cell response is suppressed during the first 2–6 h of infection. These results suggest that the attenuated Francisella LVS induces significant host cell signaling at very early time points after the bacteria's interaction with the cell. PMID:23322778

  17. Structure of the Francisella tularensis enoyl-acyl carrier protein reductase (FabI) in complex with NAD[superscript +] and triclosan

    SciTech Connect

    Mehboob, Shahila; Truong, Kent; Santarsiero, Bernard D.; Johnson, Michael E.

    2010-11-19

    Enoyl-acyl carrier protein reductase (FabI) catalyzes the last rate-limiting step in the elongation cycle of the fatty-acid biosynthesis pathway and has been validated as a potential antimicrobial drug target in Francisella tularensis. The development of new antibiotic therapies is important both to combat potential drug-resistant bioweapons and to address the broader societal problem of increasing antibiotic resistance among many pathogenic bacteria. The crystal structure of FabI from F. tularensis (FtuFabI) in complex with the inhibitor triclosan and the cofactor NAD{sup +} has been solved to a resolution of 2.1 {angstrom}. Triclosan is known to effectively inhibit FabI from different organisms. Precise characterization of the mode of triclosan binding is required to develop highly specific inhibitors. Comparison of our structure with the previously determined FtuFabI structure (PDB code 2jjy) which is bound to only NAD{sup +} reveals the conformation of the substrate-binding loop, electron density for which was missing in the earlier structure, and demonstrates a shift in the conformation of the NAD{sup +} cofactor. This shift in the position of the phosphate groups allows more room in the active site for substrate or inhibitor to bind and be better accommodated. This information will be crucial for virtual screening studies to identify novel scaffolds for development into new active inhibitors.

  18. Structural and biological evaluation of a novel series of benzimidazole inhibitors of Francisella tularensis enoyl-ACP reductase (FabI)

    SciTech Connect

    Mehboob, Shahila; Song, Jinhua; Hevener, Kirk E.; Su, Pin-Chih; Boci, Teuta; Brubaker, Libby; Truong, Lena; Mistry, Tina; Deng, Jiangping; Cook, James L.; Santarsiero, Bernard D.; Ghosh, Arun K.; Johnson, Michael E.

    2015-01-29

    Francisella tularensis, the causative agent of tularemia, presents a significant biological threat and is a Category A priority pathogen due to its potential for weaponization. In the bacterial FASII pathway we found it a viable target for the development of novel antibacterial agents treating Gram-negative infections. Here, we report the advancement of a promising series of benzimidazole FabI (enoyl-ACP reductase) inhibitors to a second-generation using a systematic, structure-guided lead optimization strategy, and the determination of several co-crystal structures that confirm the binding mode of designed inhibitors. Furthermore, these compounds display an improved low nanomolar enzymatic activity as well as promising low microgram/mL antibacterial activity against both F. tularensis and Staphylococcus aureus and its methicillin-resistant strain (MRSA). Finally, the improvements in activity accompanying structural modifications lead to a better understanding of the relationship between the chemical structure and biological activity that encompasses both enzymatic and whole-cell activity.

  19. Structural and biological evaluation of a novel series of benzimidazole inhibitors of Francisella tularensis enoyl-ACP reductase (FabI)

    DOE PAGES

    Mehboob, Shahila; Song, Jinhua; Hevener, Kirk E.; ...

    2015-01-29

    Francisella tularensis, the causative agent of tularemia, presents a significant biological threat and is a Category A priority pathogen due to its potential for weaponization. In the bacterial FASII pathway we found it a viable target for the development of novel antibacterial agents treating Gram-negative infections. Here, we report the advancement of a promising series of benzimidazole FabI (enoyl-ACP reductase) inhibitors to a second-generation using a systematic, structure-guided lead optimization strategy, and the determination of several co-crystal structures that confirm the binding mode of designed inhibitors. Furthermore, these compounds display an improved low nanomolar enzymatic activity as well as promisingmore » low microgram/mL antibacterial activity against both F. tularensis and Staphylococcus aureus and its methicillin-resistant strain (MRSA). Finally, the improvements in activity accompanying structural modifications lead to a better understanding of the relationship between the chemical structure and biological activity that encompasses both enzymatic and whole-cell activity.« less

  20. Revision of the Lima clade (Miconia sect. Lima, Miconieae, Melastomataceae) of the Greater Antilles

    PubMed Central

    Majure, Lucas C.; Bécquer, Eldis R.; Judd, Walter S.

    2016-01-01

    Abstract Miconia sect. Lima is an entirely Greater Antillean clade that consists of 19 known species of shrubs and small trees, which were previously recognized under the polyphyletic genera Leandra and Ossaea. The highest species richness in the clade is represented on Cuba (10 species), followed by Hispaniola (8 species) and then Jamaica (1 species). Here we present a taxonomic revision of the clade based on the study of species in the field, herbarium specimens, as well as a DNA-based phylogeny reconstruction. The Lima clade most likely originated on Cuba and then spread to Jamaica once and Hispaniola multiple times. Species of this clade can be recognized by the well developed bulla-based hairs of the adaxial leaf surface, as well as the clavate-dendritic hairs produced along the primary, secondary and tertiary veins of the adaxial leaf surface, mostly towards the leaf base, terminal inflorescences, acute petal apices, slightly bulla-based hairs produced subapically along the petal abaxial surface, and anthers with a dorso-basal appendage and a single, dorsally oriented pore. Descriptions, synonymies, along with distribution maps and illustrations/figures, are given for each species. Miconia pagnolensis sp. nov. is newly described in this revision. PMID:27829802

  1. Mitochondrial DNA variation reveals recent evolutionary history of main Boa constrictor clades.

    PubMed

    Hynková, Ivana; Starostová, Zuzana; Frynta, Daniel

    2009-09-01

    We sequenced a 1114-bp fragment of cytochrome b gene in six subspecies (115 samples) of Boa constrictor and detected 67 haplotypes. Our analyses revealed the presence of two distinct clades, one from Central America (CA) including the neighboring part of South America west of the Andes, and the other covering the rest of South America (SA). Sequence divergence between CA and SA clades is about 5-7%, which roughly corresponds to a separation at the time of uplift of the Colombian Andes following formation of the Panama Isthmus before 3.5 Myr Sequence divergence within the SA and CA clades is only 2-3%, suggesting a fairly recent spread of these clades Into their current geographic ranges. Thus, we may not be dealing with taxa with a markedly old evolutionary history. Because juveniles of B. constrictor feed mostly on small rodents, we hypothesized that spread of this species was allowed by a new food source represented by murold rodents that appeared after closure of the Panama portal. With respect to the taxonomy, B. c. imperator may be elevated to full species rank. Within the SA clade, a haplotype of Argentinian B. c. occidentalis is markedly distinct, while the remaining haplotype groups analyzed are distributed throughout large ranges and may all belong to a single nominotypic subspecies.

  2. The expansion of Phytophthora clade 8b: three new species associated with winter grown vegetable crops.

    PubMed

    Bertier, L; Brouwer, H; de Cock, A W A M; Cooke, D E L; Olsson, C H B; Höfte, M

    2013-12-01

    Despite its association with important agricultural crops, Phytophthora clade 8b is a poorly studied group of species. The clade currently consists of three officially described species (Phytophthora porri, P. brassicae and P. primulae) that are host-specific pathogens of leek, cabbages and Primula spp., respectively. However, over the past few decades, several other clade 8b-like Phytophthoras have been found on a variety of different host plants that were all grown at low temperatures in winter seasons. In this study, a collection of 30 of these isolates was subjected to a phylogenetic study using two loci (the rDNA ITS region and the mitochondrial cox1 gene). This analysis revealed a clear clustering of isolates according to their host plants. To verify whether these isolates belong to separate species, a detailed morphological study was conducted. On the basis of genetic and morphological differences and host specificity, we now present the official description of three new species in clade 8b: Phytophthora cichorii sp. nov., P. dauci sp. nov. and P. lactucae sp. nov. Two other groups of isolates (Phytophthora taxon castitis and Phytophthora taxon parsley) might also represent new species but the data available at this time are insufficient for an official description. This brings Phytophthora clade 8b to a group of six species that are all host-specific, slow-growing and specifically infect herbaceous crops at low temperatures.

  3. The role of clade competition in the diversification of North American canids

    PubMed Central

    Silvestro, Daniele; Antonelli, Alexandre; Salamin, Nicolas; Quental, Tiago B.

    2015-01-01

    The history of biodiversity is characterized by a continual replacement of branches in the tree of life. The rise and demise of these branches (clades) are ultimately determined by changes in speciation and extinction rates, often interpreted as a response to varying abiotic and biotic factors. However, understanding the relative importance of these factors remains a major challenge in evolutionary biology. Here we analyze the rich North American fossil record of the dog family Canidae and of other carnivores to tease apart the roles of competition, body size evolution, and climate change on the sequential replacement of three canid subfamilies (two of which have gone extinct). We develop a novel Bayesian analytic framework to show that competition from multiple carnivore clades successively drove the demise and replacement of the two extinct canid subfamilies by increasing their extinction rates and suppressing their speciation. Competitive effects have likely come from ecologically similar species from both canid and felid clades. These results imply that competition among entire clades, generally considered a rare process, can play a more substantial role than climate change and body size evolution in determining the sequential rise and decline of clades. PMID:26124128

  4. Revision of the Lima clade (Miconia sect. Lima, Miconieae, Melastomataceae) of the Greater Antilles.

    PubMed

    Majure, Lucas C; Bécquer, Eldis R; Judd, Walter S

    2016-01-01

    Miconia sect. Lima is an entirely Greater Antillean clade that consists of 19 known species of shrubs and small trees, which were previously recognized under the polyphyletic genera Leandra and Ossaea. The highest species richness in the clade is represented on Cuba (10 species), followed by Hispaniola (8 species) and then Jamaica (1 species). Here we present a taxonomic revision of the clade based on the study of species in the field, herbarium specimens, as well as a DNA-based phylogeny reconstruction. The Lima clade most likely originated on Cuba and then spread to Jamaica once and Hispaniola multiple times. Species of this clade can be recognized by the well developed bulla-based hairs of the adaxial leaf surface, as well as the clavate-dendritic hairs produced along the primary, secondary and tertiary veins of the adaxial leaf surface, mostly towards the leaf base, terminal inflorescences, acute petal apices, slightly bulla-based hairs produced subapically along the petal abaxial surface, and anthers with a dorso-basal appendage and a single, dorsally oriented pore. Descriptions, synonymies, along with distribution maps and illustrations/figures, are given for each species. Miconia pagnolensissp. nov. is newly described in this revision.

  5. Four New Vining Species of Solanum (Dulcamaroid Clade) from Montane Habitats in Tropical America

    PubMed Central

    Knapp, Sandra

    2010-01-01

    Background Solanum (Solanaceae), with approximately 1500 species, is one of the largest genera of flowering plants, and has a centre of diversity in the New World tropics. The genus is divided into 13 major clades, of which two, the Dulcamaroid clade and the “African Non-Spiny” clade, exhibit vine morphology with twining petioles. I am currently preparing a worldwide monograph of these two groups, comprising some 70 species. Methods I formally describe here four new species of Solanum from montane Mexico and South America all belonging to the Dulcamaroid clade (including the traditionally recognised section Jasminosolanum Bitter). Descriptions, discussions of closely related species and preliminary conservation assessments are provided for all species; all species are illustrated. This paper is also a test case for the electronic publication of new names in flowering plants. Conclusions These new species are all relatively rare, but not currently of conservation concern. Solanum aspersum sp. nov. is distributed in Colombia and Ecuador, S. luculentum sp. nov. in Colombia and Venezuela, S. sanchez-vegae sp. nov. is endemic to northern Peru and S. sousae sp. nov. to southern Mexico. Solanum luculentum has the morphology of a dioecious species; this is the first report of this breeding system in the Dulcamaroid clade. PMID:20463921

  6. Phylogenetic analysis of New Zealand earthworms (Oligochaeta: Megascolecidae) reveals ancient clades and cryptic taxonomic diversity.

    PubMed

    Buckley, Thomas R; James, Sam; Allwood, Julia; Bartlam, Scott; Howitt, Robyn; Prada, Diana

    2011-01-01

    We have constructed the first ever phylogeny for the New Zealand earthworm fauna (Megascolecinae and Acanthodrilinae) including representatives from other major continental regions. Bayesian and maximum likelihood phylogenetic trees were constructed from 427 base pairs from the mitochondrial large subunit (16S) rRNA gene and 661 base pairs from the nuclear large subunit (28S) rRNA gene. Within the Acanthodrilinae we were able to identify a number of well-supported clades that were restricted to continental landmasses. Estimates of nodal support for these major clades were generally high, but relationships among clades were poorly resolved. The phylogenetic analyses revealed several independent lineages in New Zealand, some of which had a comparable phylogenetic depth to monophyletic groups sampled from Madagascar, Africa, North America and Australia. These results are consistent with at least some of these clades having inhabited New Zealand since rifting from Gondwana in the Late Cretaceous. Within the New Zealand Acanthodrilinae, major clades tended to be restricted to specific regions of New Zealand, with the central North Island and Cook Strait representing major biogeographic boundaries. Our field surveys of New Zealand and subsequent identification has also revealed extensive cryptic taxonomic diversity with approximately 48 new species sampled in addition to the 199 species recognized by previous authors. Our results indicate that further survey and taxonomic work is required to establish a foundation for future biogeographic and ecological research on this vitally important component of the New Zealand biota.

  7. Experimental evolution reveals habitat-specific fitness dynamics among Wolbachia clades in Drosophila melanogaster.

    PubMed

    Versace, Elisabetta; Nolte, Viola; Pandey, Ram Vinay; Tobler, Ray; Schlötterer, Christian

    2014-02-01

    The diversity and infection dynamics of the endosymbiont Wolbachia can be influenced by many factors, such as transmission rate, cytoplasmic incompatibility, environment, selection and genetic drift. The interplay of these factors in natural populations can result in heterogeneous infection patterns with substantial differences between populations and strains. The causes of these heterogeneities are not yet understood, partly due to the complexity of natural environments. We present experimental evolution as a new approach to study Wolbachia infection dynamics in replicate populations exposed to a controlled environment. A natural Drosophila melanogaster population infected with strains of Wolbachia belonging to different clades evolved in two laboratory environments (hot and cold) for 1.5 years. In both treatments, the rate of Wolbachia infection increased until fixation. In the hot environment, the relative frequency of different Wolbachia clades remained stable over 37 generations. In the cold environment, however, we observed marked changes in the composition of the Wolbachia population: within 15 generations, one Wolbachia clade increased more than 50% in frequency, whereas the other two clades decreased in frequency, resulting in the loss of one clade. The frequency change was highly reproducible not only among replicates, but also when flies that evolved for 42 generations in the hot environment were transferred to the cold environment. These results document how environmental factors can affect the composition of Wolbachia in D. melanogaster. The high reproducibility of the pattern suggests that experimental evolution studies can efficiently determine the functional basis of habitat-specific fitness among Wolbachia strains.

  8. Maximizing Capture Efficiency and Specificity of Magnetic Separation for Mycobacterium avium subsp. paratuberculosis Cells ▿

    PubMed Central

    Foddai, Antonio; Elliott, Christopher T.; Grant, Irene R.

    2010-01-01

    In order to introduce specificity for Mycobacterium avium subsp. paratuberculosis prior to a phage amplification assay, various magnetic-separation approaches, involving either antibodies or peptides, were evaluated in terms of the efficiency of capture (expressed as a percentage) of M. avium subsp. paratuberculosis cells and the percentage of nonspecific binding by other Mycobacterium spp. A 50:50 mixture of MyOne Tosylactivated Dynabeads coated with the chemically synthesized M. avium subsp. paratuberculosis-specific peptides biotinylated aMp3 and biotinylated aMptD (i.e., peptide-mediated magnetic separation [PMS]) proved to be the best magnetic-separation approach for achieving 85 to 100% capture of M. avium subsp. paratuberculosis and minimal (<1%) nonspecific recovery of other Mycobacterium spp. (particularly if beads were blocked with 1% skim milk before use) from broth samples containing 103 to 104 CFU/ml. When PMS was coupled with a recently optimized phage amplification assay and used to detect M. avium subsp. paratuberculosis in 50-ml volumes of spiked milk, the mean 50% limit of detection (LOD50) was 14.4 PFU/50 ml of milk (equivalent to 0.3 PFU/ml). This PMS-phage assay represents a novel, rapid method for the detection and enumeration of viable M. avium subsp. paratuberculosis organisms in milk, and potentially other sample matrices, with results available within 48 h. PMID:20851966

  9. Decreased toxicity of Bacillus thuringiensis subsp. israelensis to mosquito larvae after contact with leaf litter.

    PubMed

    Tetreau, Guillaume; Stalinski, Renaud; Kersusan, Dylann; Veyrenc, Sylvie; David, Jean-Philippe; Reynaud, Stéphane; Després, Laurence

    2012-08-01

    Bacillus thuringiensis subsp. israelensis is a bacterium producing crystals containing Cry and Cyt proteins, which are toxic for mosquito larvae. Nothing is known about the interaction between crystal toxins and decaying leaf litter, which is a major component of several mosquito breeding sites and represents an important food source. In the present work, we investigated the behavior of B. thuringiensis subsp. israelensis toxic crystals sprayed on leaf litter. In the presence of leaf litter, a 60% decrease in the amount of Cyt toxin detectable by immunology (enzyme-linked immunosorbent assays [ELISAs]) was observed, while the respective proportions of Cry toxins were not affected. The toxicity of Cry toxins toward Aedes aegypti larvae was not affected by leaf litter, while the synergistic effect of Cyt toxins on all B. thuringiensis subsp. israelensis Cry toxins was decreased by about 20% when mixed with leaf litter. The toxicity of two commercial B. thuringiensis subsp. israelensis strains (VectoBac WG and VectoBac 12AS) and a laboratory-produced B. thuringiensis subsp. israelensis strain decreased by about 70% when mixed with leaf litter. Taken together, these results suggest that Cyt toxins interact with leaf litter, resulting in a decreased toxicity of B. thuringiensis subsp. israelensis in litter-rich environments and thereby dramatically reducing the efficiency of mosquitocidal treatments.

  10. Characterization of Pneumonia Due to Streptococcus equi subsp. zooepidemicus in Dogs▿

    PubMed Central

    Priestnall, Simon L.; Erles, Kerstin; Brooks, Harriet W.; Cardwell, Jacqueline M.; Waller, Andrew S.; Paillot, Romain; Robinson, Carl; Darby, Alistair C.; Holden, Matthew T. G.; Schöniger, Sandra

    2010-01-01

    Streptococcus equi subsp. zooepidemicus has been linked to cases of acute fatal pneumonia in dogs in several countries. Outbreaks can occur in kenneled dog populations and result in significant levels of morbidity and mortality. This highly contagious disease is characterized by the sudden onset of clinical signs, including pyrexia, dyspnea, and hemorrhagic nasal discharge. The pathogenesis of S. equi subsp. zooepidemicus infection in dogs is poorly understood. This study systematically characterized the histopathological changes in the lungs of 39 dogs from a large rehoming shelter in London, United Kingdom; the dogs were infected with S. equi subsp. zooepidemicus. An objective scoring system demonstrated that S. equi subsp. zooepidemicus caused pneumonia in 26/39 (66.7%) dogs, and most of these dogs (17/26 [65.4%]) were classified as severe fibrino-suppurative, necrotizing, and hemorrhagic. Three recently described superantigen genes (szeF, szeN, and szeP) were detected by PCR in 17/47 (36.2%) of the S. equi subsp. zooepidemicus isolates; however, there was no association between the presence of these genes and the histopathological score. The lungs of S. equi subsp. zooepidemicus-infected dogs with severe respiratory signs and lung pathology did however have significantly higher mRNA levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and interleukin 8 (IL-8) than in uninfected controls, suggesting a role for an exuberant host immune response in the pathogenesis of this disease. PMID:20861329

  11. Decreased Toxicity of Bacillus thuringiensis subsp. israelensis to Mosquito Larvae after Contact with Leaf Litter

    PubMed Central

    Stalinski, Renaud; Kersusan, Dylann; Veyrenc, Sylvie; David, Jean-Philippe; Reynaud, Stéphane; Després, Laurence

    2012-01-01

    Bacillus thuringiensis subsp. israelensis is a bacterium producing crystals containing Cry and Cyt proteins, which are toxic for mosquito larvae. Nothing is known about the interaction between crystal toxins and decaying leaf litter, which is a major component of several mosquito breeding sites and represents an important food source. In the present work, we investigated the behavior of B. thuringiensis subsp. israelensis toxic crystals sprayed on leaf litter. In the presence of leaf litter, a 60% decrease in the amount of Cyt toxin detectable by immunology (enzyme-linked immunosorbent assays [ELISAs]) was observed, while the respective proportions of Cry toxins were not affected. The toxicity of Cry toxins toward Aedes aegypti larvae was not affected by leaf litter, while the synergistic effect of Cyt toxins on all B. thuringiensis subsp. israelensis Cry toxins was decreased by about 20% when mixed with leaf litter. The toxicity of two commercial B. thuringiensis subsp. israelensis strains (VectoBac WG and VectoBac 12AS) and a laboratory-produced B. thuringiensis subsp. israelensis strain decreased by about 70% when mixed with leaf litter. Taken together, these results suggest that Cyt toxins interact with leaf litter, resulting in a decreased toxicity of B. thuringiensis subsp. israelensis in litter-rich environments and thereby dramatically reducing the efficiency of mosquitocidal treatments. PMID:22610426

  12. Safety and Immunogenicity of a Single Low Dose or High Dose of Clade 2 Influenza A(H5N1) Inactivated Vaccine in Adults Previously Primed With Clade 1 Influenza A(H5N1) Vaccine.

    PubMed

    Winokur, Patricia L; Patel, Shital M; Brady, Rebecca; Chen, Wilbur H; El-Kamary, Samer S; Edwards, Kathryn; Creech, C Buddy; Frey, Sharon; Keitel, Wendy A; Belshe, Robert; Walter, Emmanuel; Bellamy, Abbie; Hill, Heather

    2015-08-15

    Influenza A(H5N1) vaccination strategies that improve the speed of the immunological response and cross-clade protection are desired. We compared the immunogenicity of a single 15-μg or 90-μg dose of A/H5N1/Indonesia/05/05 (clade 2) vaccine in adults who were previously primed with A/H5N1/Vietnam/1203/2004 (clade 1) vaccine. High-dose vaccine resulted in significantly higher titers to both clade 1 and 2 antigens. Clade 2 titers were unaffected by the previous dose of clade 1 vaccine. Low-dose priming with a mismatched pandemic influenza A(H5N1) vaccine would improve the rapidity, magnitude, and cross-reactivity of the immunological response following a single high-dose, unadjuvanted, pandemic vaccine.

  13. Geobacter sulfurreducens subsp. ethanolicus, subsp. nov., an ethanol-utilizing dissimilatory Fe(III)-reducing bacterium from a lotus field.

    PubMed

    Viulu, Samson; Nakamura, Kohei; Kojima, Akihiro; Yoshiyasu, Yuki; Saitou, Sakiko; Takamizawa, Kazuhiro

    2013-01-01

    An ethanol-utilizing Fe(III)-reducing bacterial strain, OSK2A(T), was isolated from a lotus field in Aichi, Japan. Phylogenetic analysis of the 16S rRNA gene sequences of OSK2A(T) and related strains placed it within Geobacter sulfurreducens PCA(T). Strain OSK2A(T) was shown to be a Gram-negative, motile, rod-shaped bacterium, strictly anaerobic, 0.76-1.65 µm long and 0.28-0.45 μm wide. Its growth occurred at 20-40℃, pH 6.0-8.1, and it tolerated up to 1% NaCl. The G+C content of the genomic DNA was 61.2 mol% and DNA-DNA hybridization value with Geobacter sulfurreducens PCA(T) was 60.7%. The major respiratory quinone was MK-8. The major fatty acids were 16:1 ω7c, 16:0, 14:0, 15:0 iso, 16:1 ω5c, and 18:1 ω7c. Strain OSK2A(T) could utilize H2, ethanol, acetate, lactate, pyruvate, and formate as substrates with Fe(III)-citrate as electron acceptor. Amorphous Fe(III) hydroxide, Fe(III)-NTA, fumarate, malate, and elemental sulfur were utilized as electron acceptors with either acetate or ethanol as substrates. Results obtained from physiological, DNA-DNA hybridization, and chemotaxonomic tests support genotypic and phenotypic differentiation of strain OSK2A(T) from its closest relative. The isolate is assigned as a novel subspecies with the name Geobacter sulfurreducens subsp. ethanolicus, subsp. nov. (type strain OSK2A(T)=DSMZ 26126(T)=JCM 18752(T)).

  14. Postantifungal effect of caspofungin against the Candida albicans and Candida parapsilosis clades.

    PubMed

    Gil-Alonso, Sandra; Jauregizar, Nerea; Eraso, Elena; Quindós, Guillermo

    2016-10-01

    Killing and postantifungal effects could be relevant for the selection of optimal dosing schedules. This study aims to compare time-kill and postantifungal effects with caspofungin on Candida albicans (C. albicans, Candida dubliniensis, Candida africana) and Candida parapsilosis (C. parapsilosis, Candida metapsilosis, Candida orthopsilosis) clades. In the postantifungal effect experiments, strains were exposed to caspofungin for 1 h at concentrations 0.12-8 μg/mL. Time-kill experiments were conducted at the same concentrations. Caspofungin exhibited a significant and prolonged postantifungal effect (>37 h) with 2 μg/mL against the most strains of C. albicans clade. Against the C. parapsilosis clade, the postantifungal effect was <12 h at 8 μg/mL, except for two strains. Caspofungin was fungicidal against C. albicans, C. dubliniensis and C. metapsilosis.

  15. Phylogeny of the sea hares in the aplysia clade based on mitochondrial DNA sequence data

    SciTech Connect

    Medina, Monica; Collins, Timothy; Walsh, Patrick J.

    2004-02-20

    Sea hare species within the Aplysia clade are distributed worldwide. Their phylogenetic and biogeographic relationships are, however, still poorly known. New molecular evidence is presented from a portion of the mitochondrial cytochrome oxidase c subunit 1 gene (cox1) that improves our understanding of the phylogeny of the group. Based on these data a preliminary discussion of the present distribution of sea hares in a biogeographic context is put forward. Our findings are consistent with only some aspects of the current taxonomy and nomenclatural changes are proposed. The first, is the use of a rank free classification for the different Aplysia clades and subclades as opposed to previously used genus and subgenus affiliations. The second, is the suggestion that Aplysia brasiliana (Rang, 1828) is a junior synonym of Aplysia fasciata (Poiret, 1789). The third, is the elimination of Neaplysia since its only member is confirmed to be part of the large Varria clade.

  16. Impact of Clade, Geography, and Age of the Epidemic on HIV-1 Neutralization by Antibodies

    PubMed Central

    Hraber, Peter; Korber, Bette T.; Lapedes, Alan S.; Bailer, Robert T.; Seaman, Michael S.; Gao, Hongmei; Greene, Kelli M.; McCutchan, Francine; Williamson, Carolyn; Kim, Jerome H.; Tovanabutra, Sodsai; Hahn, Beatrice H.; Swanstrom, Ronald; Thomson, Michael M.; Gao, Feng; Harris, Linda; Giorgi, Elena; Hengartner, Nicholas; Bhattacharya, Tanmoy; Mascola, John R.

    2014-01-01

    ABSTRACT Neutralizing antibodies (nAbs) are a high priority for vaccines that aim to prevent the acquisition of HIV-1 infection. Vaccine effectiveness will depend on the extent to which induced antibodies neutralize the global diversity of circulating HIV-1 variants. Using large panels of genetically and geographically diverse HIV-1 Env-pseudotyped viruses and chronic infection plasma samples, we unambiguously show that cross-clade nAb responses are commonly induced in response to infection by any virus clade. Nonetheless, neutralization was significantly greater when the plasma clade matched the clade of the virus being tested. This within-clade advantage was diminished in older, more-diverse epidemics in southern Africa, the United States, and Europe compared to more recent epidemics in Asia. It was most pronounced for circulating recombinant form (CRF) 07_BC, which is common in China and is the least-divergent lineage studied; this was followed by the slightly more diverse Asian CRF01_AE. We found no evidence that transmitted/founder viruses are generally more susceptible to neutralization and are therefore easier targets for vaccination than chronic viruses. Features of the gp120 V1V2 loop, in particular, length, net charge, and number of N-linked glycans, were associated with Env susceptibility and plasma neutralization potency in a manner consistent with neutralization escape being a force that drives viral diversification and plasma neutralization breadth. The overall susceptibility of Envs and potencies of plasma samples were highly predictive of the neutralization outcome of any single virus-plasma combination. These findings highlight important considerations for the design and testing of candidate HIV-1 vaccines that aim to elicit effective nAbs. IMPORTANCE An effective HIV-1 vaccine will need to overcome the extraordinary variability of the virus, which is most pronounced in the envelope glycoproteins (Env), which are the sole targets for neutralizing

  17. The Estimation of Tree Posterior Probabilities Using Conditional Clade Probability Distributions

    PubMed Central

    Larget, Bret

    2013-01-01

    In this article I introduce the idea of conditional independence of separated subtrees as a principle by which to estimate the posterior probability of trees using conditional clade probability distributions rather than simple sample relative frequencies. I describe an algorithm for these calculations and software which implements these ideas. I show that these alternative calculations are very similar to simple sample relative frequencies for high probability trees but are substantially more accurate for relatively low probability trees. The method allows the posterior probability of unsampled trees to be calculated when these trees contain only clades that are in other sampled trees. Furthermore, the method can be used to estimate the total probability of the set of sampled trees which provides a measure of the thoroughness of a posterior sample. [Bayesian phylogenetics; conditional clade distributions; improved accuracy; posterior probabilities of trees.] PMID:23479066

  18. Molecular phylogenetics and character evolution of the "sacaca" clade: novel relationships of Croton section Cleodora (Euphorbiaceae).

    PubMed

    Caruzo, Maria Beatriz R; van Ee, Benjamin W; Cordeiro, Inês; Berry, Paul E; Riina, Ricarda

    2011-08-01

    Phylogenetic relationships of Croton section Cleodora (Klotzsch) Baill. were evaluated using the nuclear ribosomal ITS and the chloroplast trnL-F and trnH-psbA regions. Our results show a strongly supported clade containing most previously recognized section Cleodora species, plus some other species morphologically similar to them. Two morphological synapomorphies that support section Cleodora as a clade include pistillate flowers in which the sepals overlap to some degree, and styles that are connate at the base to varying degrees. The evolution of vegetative and floral characters that have previously been relied on for taxonomic decisions within this group are evaluated in light of the phylogenetic hypotheses. Within section Cleodora there are two well-supported clades, which are proposed here as subsections (subsection Sphaerogyni and subsection Spruceani). The resulting phylogenetic hypothesis identifies the closest relatives of the medicinally important and essential oil-rich Croton cajucara Benth. as candidates for future screening in phytochemical and pharmacological studies.

  19. Six genetically distinct clades of Palola (Eunicidae, Annelida) from Lizard Island, Great Barrier Reef, Australia.

    PubMed

    Schulze, Anja

    2015-09-18

    A total of 36 lots of Palola spp. (Eunicidae, Annelida) were collected during the Lizard Island Polychaete Workshop on Lizard Island, Great Barrier Reef, Queensland, Australia. Of these, 21 specimens were sequenced for a portion of the mitochondrial cytochrome c oxidase I gene. These sequences were analysed in conjunction with existing sequences of Palola spp. from other geographic regions. The samples from Lizard Island form six distinct clades, although none of them can clearly be assigned to any of the nominal species. Four of the six Lizard Island clades fall into species group A and the remaining two into species group B (which also includes the type species, Palola viridis). All sequenced specimens were characterized morphologically as far as possible and a dichotomous key was assembled. Based on this key, the remaining samples were identified as belonging to one of the clades.

  20. Implications of global and regional patterns of highly pathogenic avian influenza virus H5N1 clades for risk management.

    PubMed

    Pfeiffer, Dirk U; Otte, Martin J; Roland-Holst, David; Inui, Ken; Nguyen, Tung; Zilberman, David

    2011-12-01

    This paper analyses the publicly available data on the distribution and evolution of highly pathogenic avian influenza virus (HPAIV) H5N1 clades, whilst acknowledging the biases resulting from the non-random selection of isolates for gene sequencing. The data indicate molecular heterogeneity in the global distribution of HPAIV H5N1, in particular in different parts of East and Southeast Asia. Analysis of the temporal pattern of haemagglutinin clade data shows a progression from clade 0 (the 'dominant' clade between 1996 and 2002) to clade 1 (2003-2005) and then to clade 2.3.4 (2005 onwards). This process continuously produces variants, depending on the frequency of virus multiplication in the host population, which is influenced by geographical variation in poultry density, poultry production systems and also HPAI risk management measures such as vaccination. Increased multilateral collaboration needs to focus on developing enhanced disease surveillance and control targeted at evolutionary 'hotspots'.

  1. Strong purifying selection on the Odysseus gene in two clades of sibling species of the Drosophila montium species subgroup.

    PubMed

    Wen, Shuo-Yang; Shimada, Kimio; Kawai, Kuniko; Toda, Masanori J

    2006-05-01

    The Odysseus (OdsH) gene was duplicated from its ancestral neuron-expressed gene, unc-4, and then evolved very rapidly under strong positive Darwinian selection as a speciation gene causing hybrid-male sterility between closely related species of the Drosophila simulans clade. Has OdsH also experienced similar positive selection between Drosophila sibling species other than those of the simulans clade? We cloned and sequenced OdsH and unc-4 from two clades of the Drosophila montium species subgroup, the Drosophila lini and the Drosophila kikkawai clades. The ratios of Ka/Ks for OdsH were remarkably low between sibling species of these two clades, suggesting that OdsH has been subjected to strong purifying selection in these two clades.

  2. Denitrification of nitrate and nitrite by 'Candidatus Accumulibacter phosphatis' clade IC.

    PubMed

    Saad, Sondos A; Welles, Laurens; Abbas, Ben; Lopez-Vazquez, Carlos M; van Loosdrecht, Mark C M; Brdjanovic, Damir

    2016-11-15

    Phosphate accumulating organisms (PAO) are assumed to use nitrate as external electron acceptor, allowing an efficient integration of simultaneous nitrogen and phosphate removal with minimal organic carbon (COD) requirements. However, contradicting findings appear in literature regarding the denitrification capacities of PAO due to the lack of clade specific highly enriched PAO cultures. Whereas some studies suggest that only PAO clade I may be capable of using nitrate as external electron acceptor for anoxic P-uptake, other studies indicate that PAO clade II may be responsible for anoxic P-removal. In the present study, a highly enriched PAO clade IC culture (>99% according to FISH) was cultivated in an SBR operated under Anaerobic/Oxic conditions and subsequently exposed to Anaerobic/Anoxic/Oxic conditions using nitrate as electron acceptor. Before and after acclimatization to the presence of nitrate, the aerobic and anoxic (nitrate and nitrite) activities of the PAO I culture were assessed through the execution of batch tests using either acetate or propionate as electron donor. In the presence of nitrate, significant P-uptake by PAO I was not observed before or after acclimatization. Using nitrite as electron acceptor, limited nitrite removal rates were observed before acclimatization with lower rates in the acetate fed reactor without P-uptake and slightly higher in the propionate fed reactor with a marginal anoxic P-uptake. Only after acclimatization to nitrate, simultaneous P and nitrite removal was observed. This study suggests that PAO clade IC is not capable of using nitrate as external electron acceptor for anoxic P-removal. The elucidation of the metabolic capacities for individual PAO clades helps in better understanding and optimization of the relation between microbial ecology and process performance in enhanced biological phosphate removal processes.

  3. Phylogeny and taxonomy of the North American clade of the Ceratocystis fimbriata complex.

    PubMed

    Johnson, Jason A; Harrington, Thomas C; Engelbrecht, C J B

    2005-01-01

    Ceratocystis fimbriata is a widely distributed, plant pathogenic fungus that causes wilts and cankers on many woody hosts. Earlier phylogenetic analyses of DNA sequences revealed three geographic clades within the C. fimbriata complex that are centered respectively in North America, Latin America and Asia. This study looked for cryptic species within the North American clade. The internal transcribed spacer regions (ITS) of the rDNA were sequenced, and phylogenetic analysis indicated that most isolates from the North American clade group into four host-associated lineages, referred to as the aspen, hickory, oak and cherry lineages, which were isolated primarily from wounds or diseased trees of Populus, Carya, Quercus and Prunus, respectively. A single isolate collected from P. serotina in Wisconsin had a unique ITS sequence. Allozyme electromorphs also were highly polymorphic within the North American clade, and the inferred phylogenies from these data were congruent with the ITS-rDNA analyses. In pairing experiments isolates from the aspen, hickory, oak and cherry lineages were interfertile only with other isolates from their respective lineages. Inoculation experiments with isolates of the four host-associated groupings showed strong host specialization by isolates from the aspen and hickory lineages on Populus tremuloides and Carya illinoensis, respectively, but isolates from the oak and cherry lineages did not consistently reveal host specialization. Morphological features distinguish isolates in the North American clade from those of the Latin American clade (including C. fimbriata sensu stricto). Based on the phylogenetic evidence, interfertility, host specialization and morphology, the oak and cherry lineages are recognized as the earlier described C. variospora, the poplar lineage as C. populicola sp. nov., and the hickory lineage as C. caryae sp. nov. A new species associated with the bark beetle Scolytus quadrispinosus on Carya is closely related to C

  4. FISH-Flow: a quantitative molecular approach for describing mixed clade communities of Symbiodinium

    NASA Astrophysics Data System (ADS)

    McIlroy, S. E.; Smith, G. J.; Geller, J. B.

    2014-03-01

    Our understanding of reef corals and their fate in a changing climate is limited by our ability to monitor the diversity and abundance of the dinoflagellate endosymbionts that sustain them. This study combined two well-known methods in tandem: fluorescent in situ hybridization (FISH) for genotype-specific labeling of Symbiodinium and flow cytometry to quantify the abundance of each symbiont clade in a sample. This technique (FISH-Flow) was developed with cultured Symbiodinium representing four distinct clades (based on large subunit rDNA) and was used to distinguish and quantify these types with high efficiency and few false positives. This technique was also applied to freshly isolated symbionts of Orbicella faveolata and Orbicella annularis. Isolates from acutely bleached coral tissues had significantly lower labeling efficiency; however, isolates from healthy tissue had efficiencies comparable to cultured Symbiodinium trials. RNA degradation in bleaching samples may have interfered with labeling of cells. Nevertheless, we were able to determine that, with and without thermal stress, experimental columns of the coral O. annularis hosted a majority of clade B and B/C symbionts on the top and side of the coral column, respectively. We demonstrated that, for cultured Symbiodinium and Symbiodinium freshly isolated from healthy host tissues, the relative ratio of clades could be accurately determined for clades present at as low as 7 % relative abundance. While this method does not improve upon PCR-based techniques in identifying clades at background levels, FISH-Flow provides a high precision, flexible system for targeting, quantifying and isolating Symbiodinium genotypes of interest.

  5. Origin and diversification of the Milla Clade (Brodiaeoideae, Asparagaceae): a Neotropical group of six geophytic genera.

    PubMed

    Gándara, Etelvina; Specht, Chelsea D; Sosa, Victoria

    2014-06-01

    The Milla clade currently comprises six genera of geophytic plants distributed from Arizona to Guatemala. Three genera (Behria, Jaimehintonia and Petronymphe) are monotypic while the remaining genera (Bessera, Dandya and Milla) contain from two to ten (Milla) species. Parsimony, Maximum Likelihood and Bayesian Inference analyses were conducted with plastid and nuclear DNA sequences from a total of 181 plants belonging to 15 species in all six genera. Molecular dating was performed under a relaxed clock model. We examined the phylogenetic relationships of the genera and species, estimated origin-divergence times for the clade and genera and determined the ancestral distribution area of the clade by optimizing ancestral areas given current biogeographic distributions. The phylogenetic results suggest that final decisions on limits of the six genera in the Milla clade will have to be established until further taxonomic work is completed for Milla, in particular for the group of populations included under the name M. biflora. The later genus is rendered polyphyletic by other genera of the family. The origin of the Milla clade is estimated at 15.8Ma. Ancestral area of the clade most likely was located in the California Floristic Province and dispersal occurred most likely to the Chihuahuan-Coahuila Plateaus and the Trans-Mexican Volcanic Belt and from there to Baja California and the Sierra Madre del Sur. Two hypotheses that need further testing are proposed to explain complex relationships of genera and polyphyly of Milla, one in relation to fragmentation of populations and pollinator shifts and another suggesting that populations remained in refugia in the Trans-Mexican Volcanic Belt.

  6. Biotechnological potential of the fungal CTG clade species in the synthetic biology era.

    PubMed

    Papon, Nicolas; Courdavault, Vincent; Clastre, Marc

    2014-04-01

    Some of the fungal CTG clade species represent attractive yeast models in many aspects of biotechnology. Their particular codon usage has hindered the development of genetic approaches for exploring and exploiting their biotechnological potential. CTG clade yeast biotechnology now benefits from the establishment of versatile molecular toolboxes. In addition, a large range of rapidly evolving genomic and postgenomic approaches has recently enhanced the understanding of the architecture of CTG species metabolic networks. These represent essential prerequisites for further successful development of metabolic engineering in CTG yeasts by facilitating the design of synthetic pathways.

  7. Streamlining and core genome conservation among highly divergent members of the SAR11 clade.

    PubMed

    Grote, Jana; Thrash, J Cameron; Huggett, Megan J; Landry, Zachary C; Carini, Paul; Giovannoni, Stephen J; Rappé, Michael S

    2012-01-01

    SAR11 is an ancient and diverse clade of heterotrophic bacteria that are abundant throughout the world's oceans, where they play a major role in the ocean carbon cycle. Correlations between the phylogenetic branching order and spatiotemporal patterns in cell distributions from planktonic ocean environments indicate that SAR11 has evolved into perhaps a dozen or more specialized ecotypes that span evolutionary distances equivalent to a bacterial order. We isolated and sequenced genomes from diverse SAR11 cultures that represent three major lineages and encompass the full breadth of the clade. The new data expand observations about genome evolution and gene content that previously had been restricted to the SAR11 Ia subclade, providing a much broader perspective on the clade's origins, evolution, and ecology. We found small genomes throughout the clade and a very high proportion of core genome genes (48 to 56%), indicating that small genome size is probably an ancestral characteristic. In their level of core genome conservation, the members of SAR11 are outliers, the most conserved free-living bacteria known. Shared features of the clade include low GC content, high gene synteny, a large hypervariable region bounded by rRNA genes, and low numbers of paralogs. Variation among the genomes included genes for phosphorus metabolism, glycolysis, and C1 metabolism, suggesting that adaptive specialization in nutrient resource utilization is important to niche partitioning and ecotype divergence within the clade. These data provide support for the conclusion that streamlining selection for efficient cell replication in the planktonic habitat has occurred throughout the evolution and diversification of this clade. IMPORTANCE The SAR11 clade is the most abundant group of marine microorganisms worldwide, making them key players in the global carbon cycle. Growing knowledge about their biochemistry and metabolism is leading to a more mechanistic understanding of organic carbon

  8. Genetic characterization of clade 2.3.2.1 avian influenza A(H5N1) viruses, Indonesia, 2012.

    PubMed

    Dharmayanti, Ni Luh Putu Indi; Hartawan, Risza; Wibawa, Hendra; Balish, Amanda; Donis, Ruben; Davis, C Todd; Samaan, Gina

    2014-04-01

    After reports of unusually high mortality rates among ducks on farms in Java Island, Indonesia, in September 2012, influenza A(H5N1) viruses were detected and characterized. Sequence analyses revealed all genes clustered with contemporary clade 2.3.2.1 viruses, rather than enzootic clade 2.1.3 viruses, indicating the introduction of an exotic H5N1 clade into Indonesia.

  9. Single- and multiple-clade influenza A H5N1 vaccines induce cross protection in ferrets

    PubMed Central

    Forrest, Heather L.; Khalenkov, Alexey M.; Govorkova, Elena A.; Kim, Jeong-Ki; Giudice, Giuseppe Del; Webster, Robert G.

    2009-01-01

    The rapid evolution, genetic diversity, broad host range, and increasing human infection with avian influenza A (H5N1) viruses highlight the need for an efficacious cross-clade vaccine. Using the ferret model, we compared induction of cross-reactive immunity and protective efficacy of three single-clade H5N1 vaccines and a novel multiple-clade H5N1 vaccine, with and without MF59 adjuvant. Reverse genetics (rg) was used to generate vaccine viruses containing the hemagglutinin (HA) and neuraminidase genes of wild-type H5N1 viruses. Ferrets received 2 doses of inactivated whole-virus vaccine separated by 3 weeks. Single-clade vaccines (7.5 μg HA per dose) included rg-A/Vietnam/1203/04 (clade 1), rg-A/Hong Kong/213/03 (clade 1), and rg-A/Japanese white eye/Hong Kong/1038/06 (clade 2.3). The multiple-clade vaccine contained 3.75 μg HA per dose of each single-clade vaccine and of rg-A/Whooper Swan/Mongolia/244/05 (clade 2.2). Two doses of vaccine were required to substantially increase anti-HA and virus neutralizing antibody titers to H5N1 viruses. MF59 adjuvant enhanced induction of clade-specific and cross-clade serum antibody responses, reduced frequency of infection (as determined by upper respiratory tract virus shedding and seroconversion data), and eliminated disease signs. The rg-A/Hong Kong/213/03 vaccine induced the highest antibody titers to homologous and heterologous H5N1 viruses, while rg-A/Japanese white eye/Hong Kong/1038/06 vaccine induced the lowest. The multiple-clade vaccine was broadly immunogenic against clade 1 and 2 viruses. The rg-A/Vietnam/1203/04 vaccine (the currently stockpiled H5N1 vaccine) most effectively reduced upper respiratory tract virus shedding after challenge with clade 1 and 2 viruses. Importantly, all vaccines protected against lethal challenge with A/Vietnam/1203/04 virus and provided cross-clade protection. PMID:19406182

  10. Clade age and diversification rate variation explain disparity in species richness among water scavenger beetle (Hydrophilidae) lineages.

    PubMed

    Bloom, Devin D; Fikáček, Martin; Short, Andrew E Z

    2014-01-01

    Explaining the disparity of species richness across the tree of life is one of the great challenges in evolutionary biology. Some lineages are exceptionally species rich, while others are relatively species poor. One explanation for heterogeneity among clade richness is that older clades are more species rich because they have had more time to accrue diversity than younger clades. Alternatively, disparity in species richness may be due to among-lineage diversification rate variation. Here we investigate diversification in water scavenger beetles (Hydrophilidae), which vary in species richness among major lineages by as much as 20 fold. Using a time-calibrated phylogeny and comparative methods, we test for a relationship between clade age and species richness and for shifts in diversification rate in hydrophilids. We detected a single diversification rate increase in Megasternini, a relatively young and species rich clade whose diversity might be explained by the stunning diversity of ecological niches occupied by this clade. We find that Amphiopini, an old clade, is significantly more species poor than expected, possibly due to its restricted geographic range. The remaining lineages show a correlation between species richness and clade age, suggesting that both clade age and variation in diversification rates explain the disparity in species richness in hydrophilids. We find little evidence that transitions between aquatic, semiaquatic, and terrestrial habitats are linked to shifts in diversification rates.

  11. Clade Age and Diversification Rate Variation Explain Disparity in Species Richness among Water Scavenger Beetle (Hydrophilidae) Lineages

    PubMed Central

    Bloom, Devin D.; Fikáček, Martin; Short, Andrew E. Z.

    2014-01-01

    Explaining the disparity of species richness across the tree of life is one of the great challenges in evolutionary biology. Some lineages are exceptionally species rich, while others are relatively species poor. One explanation for heterogeneity among clade richness is that older clades are more species rich because they have had more time to accrue diversity than younger clades. Alternatively, disparity in species richness may be due to among-lineage diversification rate variation. Here we investigate diversification in water scavenger beetles (Hydrophilidae), which vary in species richness among major lineages by as much as 20 fold. Using a time-calibrated phylogeny and comparative methods, we test for a relationship between clade age and species richness and for shifts in diversification rate in hydrophilids. We detected a single diversification rate increase in Megasternini, a relatively young and species rich clade whose diversity might be explained by the stunning diversity of ecological niches occupied by this clade. We find that Amphiopini, an old clade, is significantly more species poor than expected, possibly due to its restricted geographic range. The remaining lineages show a correlation between species richness and clade age, suggesting that both clade age and variation in diversification rates explain the disparity in species richness in hydrophilids. We find little evidence that transitions between aquatic, semiaquatic, and terrestrial habitats are linked to shifts in diversification rates. PMID:24887453

  12. Microbiota of Minas cheese as influenced by the nisin producer Lactococcus lactis subsp. lactis GLc05.

    PubMed

    Perin, Luana Martins; Dal Bello, Barbara; Belviso, Simona; Zeppa, Giuseppe; de Carvalho, Antônio Fernandes; Cocolin, Luca; Nero, Luís Augusto

    2015-12-02

    Minas cheese is a popular dairy product in Brazil that is traditionally produced using raw or pasteurized cow milk. This study proposed an alternative production of Minas cheese using raw goat milk added of a nisin producer Lactococcus lactis subsp. lactis GLc05. An in situ investigation was carried on to evaluate the interactions between the L. lactis subsp. lactis GLc05 and the autochthonous microbiota of a Minas cheese during the ripening; production of biogenic amines (BAs) was assessed as a safety aspect. Minas cheese was produced in two treatments (A, by adding L. lactis subsp. lactis GLc05, and B, without adding this strain), in three independent repetitions (R1, R2, and R3). Culture dependent (direct plating) and independent (rep-PCR and PCR-DGGE) methods were employed to characterize the microbiota and to assess the possible interferences caused by L. lactis subsp. lactis GLc05. BA amounts were measured using HPLC. A significant decrease in coagulase-positive cocci was observed in the cheeses produced by adding L. lactis subsp. lactis GLc05 (cheese A). The rep-PCR and PCR-DGGE highlighted the differences in the microbiota of both cheeses, separating them into two different clusters. Lactococcus sp. was found as the main microorganism in both cheeses, and the microbiota of cheese A presented a higher number of species. High concentrations of tyramine were found in both cheeses and, at specific ripening times, the BA amounts in cheese B were significantly higher than in cheese A (p<0.05). The interaction of nisin producer L. lactis subsp. lactis GLc05 was demonstrated in situ, by demonstration of its influence in the complex microbiota naturally present in a raw goat milk cheese and by controlling the growth of coagulase-positive cocci. L. lactis subsp. lactis GLc05 influenced also the production of BA determining that their amounts in the cheeses were maintained at acceptable levels for human consumption.

  13. Effects of Pistacia atlantica subsp. kurdica on Growth and Aflatoxin Production by Aspergillus parasiticus

    PubMed Central

    Khodavaisy, Sadegh; Rezaie, Sassan; Noorbakhsh, Fatemeh; Baghdadi, Elham; Sharifynia, Somayeh; Aala, Farzad

    2016-01-01

    Background Aflatoxins are highly toxic secondary metabolites mainly produced by Aspergillus parasiticus. This species can contaminate a wide range of agricultural commodities, including cereals, peanuts, and crops in the field. In recent years, research on medicinal herbs, such as Pistacia atlantica subsp. kurdica, have led to reduced microbial growth, and these herbs also have a particular effect on the production of aflatoxins as carcinogenic compounds. Objectives In this study, we to examine P. atlantica subsp. kurdica as a natural compound used to inhibit the growth of A. parasiticus and to act as an anti-mycotoxin. Materials and Methods In vitro antifungal susceptibility testing of P. atlantica subsp. kurdica for A. parasiticus was performed according to CLSI document M38-A2. The rate of aflatoxin production was determined using the HPLC technique after exposure to different concentrations (62.5 - 125 mg/mL) of the gum. The changes in expression levels of the aflR gene were analyzed with a quantitative real-time PCR assay. Results The results showed that P. atlantica subsp. kurdica can inhibit A. parasiticus growth at a concentration of 125 mg/mL. HPLC results revealed a significant decrease in aflatoxin production with 125 mg/mL of P. atlantica subsp. kurdica, and AFL-B1 production was entirely inhibited. Based on quantitative real-time PCR results, the rate of aflR gene expression was significantly decreased after treatment with P. atlantica subsp. kurdica. Conclusions Pistacia atlantica subsp. kurdica has anti-toxic properties in addition to an inhibitory effect on A. parasiticus growth, and is able to decrease aflatoxin production effectively in a dose-dependent manner. Therefore, this herbal extract maybe considered a potential anti-mycotoxin agent in medicine or industrial agriculture. PMID:27800127

  14. Bifidobacterium animalis subsp. lactis ATCC 27673 Is a Genomically Unique Strain within Its Conserved Subspecies

    PubMed Central

    Loquasto, Joseph R.; Barrangou, Rodolphe; Dudley, Edward G.; Stahl, Buffy; Chen, Chun

    2013-01-01

    Many strains of Bifidobacterium animalis subsp. lactis are considered health-promoting probiotic microorganisms and are commonly formulated into fermented dairy foods. Analyses of previously sequenced genomes of B. animalis subsp. lactis have revealed little genetic diversity, suggesting that it is a monomorphic subspecies. However, during a multilocus sequence typing survey of Bifidobacterium, it was revealed that B. animalis subsp. lactis ATCC 27673 gave a profile distinct from that of the other strains of the subspecies. As part of an ongoing study designed to understand the genetic diversity of this subspecies, the genome of this strain was sequenced and compared to other sequenced genomes of B. animalis subsp. lactis and B. animalis subsp. animalis. The complete genome of ATCC 27673 was 1,963,012 bp, contained 1,616 genes and 4 rRNA operons, and had a G+C content of 61.55%. Comparative analyses revealed that the genome of ATCC 27673 contained six distinct genomic islands encoding 83 open reading frames not found in other strains of the same subspecies. In four islands, either phage or mobile genetic elements were identified. In island 6, a novel clustered regularly interspaced short palindromic repeat (CRISPR) locus which contained 81 unique spacers was identified. This type I-E CRISPR-cas system differs from the type I-C systems previously identified in this subspecies, representing the first identification of a different system in B. animalis subsp. lactis. This study revealed that ATCC 27673 is a strain of B. animalis subsp. lactis with novel genetic content and suggests that the lack of genetic variability observed is likely due to the repeated sequencing of a limited number of widely distributed commercial strains. PMID:23995933

  15. Stability of the delta-endotoxin gene from Bacillus thuringiensis subsp. kurstaki in a recombinant strain of Clavibacter xyli subsp. cynodontis.

    PubMed

    Turner, J T; Lampel, J S; Stearman, R S; Sundin, G W; Gunyuzlu, P; Anderson, J J

    1991-12-01

    Deletion of chromosomally inserted gene sequences from Clavibacter xyli subsp. cynodontis, a xylem-inhabiting endophyte, was studied in vitro and in planta. We found that nonreplicating plasmid pCG610, which conferred resistance to kanamycin and tetracycline and contained segments of C. xyli subsp. cynodontis genomic DNA, integrated into a homologous sequence in the bacterial chromosome. In addition, pCG610 contains two copies of the gene encoding the CryIA(c) insecticidal protein of Bacillus thuringiensis subsp. kurstaki HD73. Using drug resistance phenotypes and specific DNA probes, we found that the loss of all three genes arose both in vitro under nonselective conditions and in planta. The resulting segregants are probably formed by recombination between the repeated DNA sequences flanking pCG610 that resulted from the integration event into the chromosome. Eventually, segregants predominated in the bacterial population. The loss of the integrated plasmid from C. xyli subsp. cynodontis revealed a possible approach for decreasing the environmental consequences of recombinant bacteria for agricultural use.

  16. In vitro studies of Lactobacillus delbrueckii subsp. lactis in Atlantic salmon (Salmo salar L.) foregut: tissue responses and evidence of protection against Aeromonas salmonicida subsp. salmonicida epithelial damage.

    PubMed

    Salinas, Irene; Myklebust, Reidar; Esteban, Maria Angeles; Olsen, Rolf Erik; Meseguer, José; Ringø, Einar

    2008-04-01

    Probiotic bacteria increase the host health status and protect mucosal tissue against pathogen-caused damage in mammalian models. Using an in vitro (intestinal sac) method this study aimed to address (a) the in vitro ability of Lactobacillus delbrueckii subsp. lactis to remain in the gastrointestinal tract of Atlantic salmon (Salmo salar L.) and (b) its ability to prevent cellular damage caused by successive incubation with Aeromonas salmonicida subsp. salmonicida the causative agent of furunculosis. Short in vitro incubation of salmon foregut with (TRITC)-labelled L. delbrueckii subsp. lactis showed that the probiont was able to colonize the enterocyte surface as studied by confocal microscopy. Furthermore, foregut incubated with the probiotic bacteria only, resulted in a healthy intestinal barrier whereas exposure to A. salmonicida disrupted its integrity. However, pre-treatment of salmon intestine with L. delbrueckii subsp. lactis prevented Aeromonas damaging effects. These results are promising in the context of the use of non-autochthonous probiotic bacteria as prophylactic agents against fish bacterial infections in the gastrointestinal tract.

  17. Production of sophorolipids biosurfactants by multiple species of the Starmerella (Candida) bombicola yeast clade

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sophorolipid production was tested for 26 strains representing 19 species of the Starmerella yeast clade, including S. bombicola and Candida apicola, which were previously reported to produce sophorolipids. Five of the 19 species tested showed significant production of sophorolipids: S. bombicola, ...

  18. A novel sister clade to the enterobacteria microviruses (family Microviridae) identified in methane seep sediments.

    PubMed

    Bryson, Samuel Joseph; Thurber, Andrew R; Correa, Adrienne M S; Orphan, Victoria J; Vega Thurber, Rebecca

    2015-10-01

    Methane seep microbial communities perform a key ecosystem service by consuming the greenhouse gas methane prior to its release into the hydrosphere, minimizing the impact of marine methane sources on our climate. Although previous studies have examined the ecology and biochemistry of these communities, none has examined viral assemblages associated with these habitats. We employed virus particle purification, genome amplification, pyrosequencing and gene/genome reconstruction and annotation on two metagenomic libraries, one prepared for ssDNA and the other for all DNA, to identify the viral community in a methane seep. Similarity analysis of these libraries (raw and assembled) revealed a community dominated by phages, with a significant proportion of similarities to the Microviridae family of ssDNA phages. We define these viruses as the Eel River Basin Microviridae (ERBM). Assembly and comparison of 21 ERBM closed circular genomes identified five as members of a novel sister clade to the Microvirus genus of Enterobacteria phages. Comparisons among other metagenomes and these Microviridae major-capsid sequences indicated that this clade of phages is currently unique to the Eel River Basin sediments. Given this ERBM clade's relationship to the Microviridae genus Microvirus, we define this sister clade as the candidate genus Pequeñovirus.

  19. Early Tertiary mammals from North Africa reinforce the molecular Afrotheria clade

    PubMed Central

    Tabuce, Rodolphe; Marivaux, Laurent; Adaci, Mohammed; Bensalah, Mustapha; Hartenberger, Jean-Louis; Mahboubi, Mohammed; Mebrouk, Fateh; Tafforeau, Paul; Jaeger, Jean-Jacques

    2007-01-01

    The phylogenetic pattern and timing of the radiation of mammals, especially the geographical origins of major crown clades, are areas of controversy among molecular biologists, morphologists and palaeontologists. Molecular phylogeneticists have identified an Afrotheria clade, which includes several taxa as different as tenrecs (Tenrecidae), golden moles (Chrysochloridae), elephant-shrews (Macroscelididae), aardvarks (Tubulidentata) and paenungulates (elephants, sea cows and hyracoids). Molecular data also suggest a Cretaceous African origin for Afrotheria within Placentalia followed by a long period of endemic evolution on the Afro-Arabian continent after the mid-Cretaceous Gondwanan breakup (approx. 105–25 Myr ago). However, there was no morphological support for such a natural grouping so far. Here, we report new dental and postcranial evidence of Eocene stem hyrax and macroscelidid from North Africa that, for the first time, provides a congruent phylogenetic view with the molecular Afrotheria clade. These new fossils imply, however, substantial changes regarding the historical biogeography of afrotheres. Their long period of isolation in Africa, as assumed by molecular inferences, is now to be reconsidered inasmuch as Eocene paenungulates and elephant-shrews are here found to be related to some Early Tertiary Euramerican ‘hyopsodontid condylarths’ (archaic hoofed mammals). As a result, stem members of afrotherian clades are not strictly African but also include some Early Paleogene Holarctic mammals. PMID:17329227

  20. Poly (beta-L-malic acid) production by diverse phylogenetic clades of Aureobasidium pullulans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Poly (beta-L-malic acid) (PMA) is a natural biopolyester that has pharmaceutical applications and other potential uses. Here we examine PMA production by genetically diverse phylogenetic clades of the fungus A. pullulans. Thirty-six strains of A. pullulans were isolated for this study from various...

  1. Placement of the unclassified Cyranomonas australis Lee 2002 within a novel clade of Cercozoa.

    PubMed

    Lee, Won Je; Park, Jong Soo

    2016-10-01

    Two heterotrophic flagellate strains were isolated from marine sediment samples off eastern Canada and Korea. These new isolates are indistinguishable by light microscopy from the unclassified protist Cyranomonas australis. The organisms are ovoid-shaped cells, 3.5-6μm long, laterally compressed, and somewhat flexible. They have two unequal flagella, about 1.1-2.5 times body length. Typically, the cells show a gliding motility and do not exhibit any amoeboid form or pseudopodia. 18S rDNA phylogenies clearly indicate that the isolates can be assigned to the taxon Filosa, within Cercozoa. The isolates are closest to an environmental sequence (CYSGM-16; 99% identity). Cyranomonas, CYSGM-16, and uncultured eukaryote RM1-SGM46 form a clade with strong statistical supports, here called novel clade CU (Cyranomonas plus Uncultured eukaryotes). This clade may be sister to the order Marimonadida. The novel clade CU and the Marimonadida have been detected only in marine habitats. Our findings suggest that C. australis may not belong to any previously described family within Filosa and Cercozoa.

  2. Mitogenomic circumscription of a novel percomorph fish clade mainly comprising "Syngnathoidei" (Teleostei).

    PubMed

    Song, Ha Yeun; Mabuchi, Kohji; Satoh, Takashi P; Moore, Jon A; Yamanoue, Yusuke; Miya, Masaki; Nishida, Mutsumi

    2014-06-01

    Percomorpha, comprising about 60% of modern teleost fishes, has been described as the "(unresolved) bush at the top" of the tree, with its intrarelationships still being ambiguous owing to huge diversity (>15,000 species). Recent molecular phylogenetic studies based on extensive taxon and character sampling, however, have revealed a number of unexpected clades of Percomorpha, and one of which is composed of Syngnathoidei (seahorses, pipefishes, and their relatives) plus several groups distributed across three different orders. To circumscribe the clade more definitely, we sampled several candidate taxa with reference to the previous studies and newly determined whole mitochondrial genome (mitogenome) sequences for 16 percomorph species across syngnathoids, dactylopterids, and their putatively closely-related fishes (Mullidae, Callionymoidei, Malacanthidae). Unambiguously aligned sequences (13,872 bp) from those 16 species plus 78 percomorphs and two outgroups (total 96 species) were subjected to partitioned Bayesian and maximum likelihood analyses. The resulting trees revealed a highly supported clade comprising seven families in Syngnathoidei (Gasterosteiformes), Dactylopteridae (Scorpaeniformes), Mullidae in Percoidei and two families in Callionymoidei (Perciformes). We herein proposed to call this clade "Syngnathiformes" following the latest nuclear DNA studies with some revisions on the included families.

  3. Glucosylation and other biotransformations of T-2 toxin by yeasts of the Trichomonascus clade

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Twenty-five yeasts assigned to the Trichomonascus clade (Saccharomycotina, Ascomycota), including three Trichomonascus species and 22 anamorphic species presently classified in Blastobotrys, were tested for their ability to convert T-2 toxin, a Fusarium trichothecene mycotoxin, to less toxic product...

  4. Characterization of Virulence-Related Phenotypes in Candida Species of the CUG Clade

    PubMed Central

    Priest, Shelby J.

    2015-01-01

    Candida species cause a variety of mucosal and invasive infections and are, collectively, the most important human fungal pathogens in the developed world. The majority of these infections result from a few related species within the “CUG clade,” so named because they use a nonstandard translation for that codon. Some members of the CUG clade, such as Candida albicans, present significant clinical problems, whereas others, such as Candida (Meyerozyma) guilliermondii, are uncommon in patients. The differences in incidence rates are imperfectly correlated with virulence in animal models of infection, but comparative analyses that might provide an explanation for why some species are effective pathogens and others are not have been rare or incomplete. To better understand the phenotypic basis for these differences, we characterized eight CUG clade species—C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis, Clavispora lusitaniae, M. guilliermondii, Debaryomyces hansenii, and Lodderomyces elongisporus—for host-relevant phenotypes, including nutrient utilization, stress tolerance, morphogenesis, interactions with phagocytes, and biofilm formation. Two species deviated from expectations based on animal studies and human incidence. C. dubliniensis was quite robust, grouping in nearly all assays with the most virulent species, C. albicans and C. tropicalis, whereas C. parapsilosis was substantially less fit than might be expected from its clinical importance. These findings confirm the utility of in vitro measures of virulence and provide insight into the evolution of virulence in the CUG clade. PMID:26150417

  5. A divergent clade of circular single-stranded DNA viruses from pig feces

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using metagenomics and molecular cloning methods, we characterized five novel small circular viral genomes from pig feces distantly related to chimpanzee and porcine stool-associated circular viruses, (ChiSCV and PoSCV1). Phylogenetic analysis placed these viruses into a new, highly divergent, clade...

  6. Genetic analysis reveals candidate species in the Scinax catharinae clade (Amphibia: Anura) from Central Brazil

    PubMed Central

    Nogueira, Lídia; Solé, Mirco; Siqueira, Sérgio; Affonso, Paulo Roberto Antunes de Mello; Strüssmann, Christine; Sampaio, Iracilda

    2016-01-01

    Abstract Scinax (Anura: Hylidae) is a species-rich genus of amphibians (113 spp.), divided into five species groups by morphological features. Cladistic analyses however revealed only two monophyletic clades in these groups: Scinax catharinae and Scinax ruber. Most species from the S. catharinae clade are found in Atlantic rainforest, except for Scinax canastrensis,S. centralis, S. luizotavioi, S. machadoi,S. pombali and S. skaios. In the present work, specimens of Scinax collected in Chapada dos Guimarães, central Brazil, were morphologically compatible with species from theS. catharinae group. On the other hand, genetic analysis based on mitochondrial (16S and 12S) and nuclear (rhodopsin) sequences revealed a nucleotide divergence of 6 to 20% between Scinax sp. and other congeners from the Brazilian savannah (Cerrado). Accordingly, Bayesian inference placed Scinax sp. in the S. catharinae clade with high support values. Hence, these findings strongly indicate the presence of a new species in the S. catharinae clade from the southwestern portion of the Brazilian savannah. To be properly validated as a novel species, detailed comparative morphological and bioacustic studies with other taxa from Brazil such asS. canastrensis, S. centralis, S. luizotavioi, S. machadoi, S. pombali and S. skaios are required. PMID:27007898

  7. Genetic analysis reveals candidate species in the Scinax catharinae clade (Amphibia: Anura) from Central Brazil.

    PubMed

    Nogueira, Lídia; Solé, Mirco; Siqueira, Sérgio; Affonso, Paulo Roberto Antunes de Mello; Strüssmann, Christine; Sampaio, Iracilda

    2016-03-01

    Scinax (Anura: Hylidae) is a species-rich genus of amphibians (113 spp.), divided into five species groups by morphological features. Cladistic analyses however revealed only two monophyletic clades in these groups: Scinax catharinae and Scinax ruber. Most species from the S. catharinae clade are found in Atlantic rainforest, except for Scinax canastrensis,S. centralis, S. luizotavioi, S. machadoi,S. pombali and S. skaios. In the present work, specimens of Scinax collected in Chapada dos Guimarães, central Brazil, were morphologically compatible with species from theS. catharinae group. On the other hand, genetic analysis based on mitochondrial (16S and 12S) and nuclear (rhodopsin) sequences revealed a nucleotide divergence of 6 to 20% between Scinax sp. and other congeners from the Brazilian savannah (Cerrado). Accordingly, Bayesian inference placed Scinax sp. in the S. catharinae clade with high support values. Hence, these findings strongly indicate the presence of a new species in the S. catharinae clade from the southwestern portion of the Brazilian savannah. To be properly validated as a novel species, detailed comparative morphological and bioacustic studies with other taxa from Brazil such asS. canastrensis, S. centralis, S. luizotavioi, S. machadoi, S. pombali and S. skaios are required.

  8. Developing Clade-Specific Microsatellite Markers: A Case Study in the Filamentous Fungal Genus Aspergillus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microsatellite markers are highly variable and very commonly used in population genetics studies. However, microsatellite loci are typically poorly conserved and cannot be used in distant related species. Thus, development of clade-specific microsatellite markers would increase efficiency and allow ...

  9. Testing Evolutionary Hypotheses in the Classroom with MacClade Software.

    ERIC Educational Resources Information Center

    Codella, Sylvio G.

    2002-01-01

    Introduces MacClade which is a Macintosh-based software package that uses the techniques of cladistic analysis to explore evolutionary patterns. Describes a novel and effective exercise that allows undergraduate biology majors to test a hypothesis about behavioral evolution in insects. (Contains 13 references.) (Author/YDS)

  10. Full-Genome Sequence of a Novel Varicella-Zoster Virus Clade Isolated in Mexico

    PubMed Central

    Rodríguez-Castillo, Araceli; Ortiz-Alcántara, Joanna María; Gonzalez-Durán, Elizabeth; Segura-Candelas, José Miguel; Pérez-Agüeros, Sandra Ivette; Escobar-Escamilla, Noé; Méndez-Tenorio, Alfonso; Diaz-Quiñonez, José Alberto

    2015-01-01

    Varicella-zoster virus (VZV) is a member of the Herpesviridae family, which causes varicella (chicken pox) and herpes zoster (shingles) in humans. Here, we report the complete genome sequence of varicella-zoster virus, isolated from a vesicular fluid sample, revealing the circulation of VZV clade VIII in Mexico. PMID:26159533

  11. Nucleic acids encoding mosaic clade M human immunodeficiency virus type 1 (HIV-1) envelope immunogens

    DOEpatents

    Korber, Bette T; Fischer, William; Liao, Hua-Xin; Haynes, Barton F; Letvin, Norman; Hahn, Beatrice H

    2015-04-21

    The present invention relates to nucleic acids encoding mosaic clade M HIV-1 Env polypeptides and to compositions and vectors comprising same. The nucleic acids of the invention are suitable for use in inducing an immune response to HIV-1 in a human.

  12. Phylogeny and biogeography of the most diverse clade of South American gymnophthalmid lizards (Squamata, Gymnophthalmidae, Cercosaurinae).

    PubMed

    Torres-Carvajal, Omar; Lobos, Simón E; Venegas, Pablo J; Chávez, Germán; Aguirre-Peñafiel, Vanessa; Zurita, Daniel; Echevarría, Lourdes Y

    2016-06-01

    Nearly 50% of the diversity of the speciose Neotropical lizard clade Gymnophthalmidae is nested within the subclade Cercosaurinae. The taxonomy of Cercosaurinae lizards has been historically confusing because many diagnostic characters of those clades traditionally ranked as genera do not represent true diagnostic apomorphies. Even though molecular phylogenies of several 'genera' have been presented in the last few years, some of them remain poorly sampled (e.g., Anadia, Echinosaura, Potamites, Riama). In this paper we present a more comprehensive phylogeny of Cercosaurinae lizards with emphasis on Andean taxa from Ecuador and Peru, as well as a time-calibrated phylogeny with reconstruction of ancestral areas. Our analysis includes 52% of all recognized species of Cercosaurinae (67 species) and 1914 characters including three mitochondrial and one nuclear gene. We find that Anadia, Echinosaura, Euspondylus, Potamites, Proctoporus, and Riama are not monophyletic: the Tepuian Anadia mcdiarmidi is not sister to Andean species of Anadia; Echinosaura sulcarostrum is not included in the same clade formed by other species of Echinosaura and their more recent common ancestor; Teuchocercus is nested within Echinosaura; species of Euspondylus included in this study are nested within Proctoporus; Riama laudahnae is included in Proctoporus; and Potamites is paraphyletic and split in two separate clades, one of which we name Gelanesaurus, also a new genus-group name. Within Potamites, P. ecpleopus is paraphyletic, and P. strangulatus strangulatus and P. strangulatus trachodus are recognized as two distinct species. We also identify three unnamed clades (i.e., not nested within any of the recognized 'genera') from Andean populations in Ecuador and Peru. The estimated age of the clade Cercosaurinae (∼60Ma) corresponds to the early stages of the northern Andes. Even though the distribution of the most recent common ancestor of Cercosaurinae remains equivocal, our analysis shows

  13. Identification of immunoreactive proteins of Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Piras, Cristian; Soggiu, Alessio; Bonizzi, Luigi; Greco, Viviana; Ricchi, Matteo; Arrigoni, Norma; Bassols, Anna; Urbani, Andrea; Roncada, Paola

    2015-02-01

    Mycobacterium avium subsp. paratuberculosis (MAP) is the cause of a chronic enteritis of ruminants (bovine paratuberculosis (PTB)--Johne's disease) that is associated with enormous worldwide economic losses for the animal production. Diagnosis is based on observation of clinical signs, the detection of antibodies in milk or serum, or evaluation of bacterial culture from feces. The limit of these methods is that they are not able to detect the disease in the subclinical stage and are applicable only when the disease is already advanced. For this reason, the main purpose of this study is to use the MAP proteome to detect novel immunoreactive proteins that may be helpful for PTB diagnoses. 2DE and 2D immunoblotting of MAP proteins were performed using sera of control cattle and PTB-infected cattle in order to highlight the specific immunoreactive proteins. Among the assigned identifiers to immunoreactive spots it was found that most of them correspond to surface-located proteins while three of them have never been described before as antigens. The identification of these proteins improves scientific knowledge that could be useful for PTB diagnoses. The sequence of the identified protein can be used for the synthesis of immunoreactive peptides that could be screened for their immunoreaction against bovine sera infected with MAP. All MS data have been deposited in the ProteomeXchange consortium with identifier PXD001159 and DOI 10.6019/PXD001159.

  14. Histidine biosynthesis genes in Lactococcus lactis subsp. lactis.

    PubMed Central

    Delorme, C; Ehrlich, S D; Renault, P

    1992-01-01

    The genes of Lactococcus lactis subsp. lactis involved in histidine biosynthesis were cloned and characterized by complementation of Escherichia coli and Bacillus subtilis mutants and DNA sequencing. Complementation of E. coli hisA, hisB, hisC, hisD, hisF, hisG, and hisIE genes and the B. subtilis hisH gene (the E. coli hisC equivalent) allowed localization of the corresponding lactococcal genes. Nucleotide sequence analysis of the 11.5-kb lactococcal region revealed 14 open reading frames (ORFs), 12 of which might form an operon. The putative operon includes eight ORFs which encode proteins homologous to enzymes involved in histidine biosynthesis. The operon also contains (i) an ORF encoding a protein homologous to the histidyl-tRNA synthetases but lacking a motif implicated in synthetase activity, which suggests that it has a role different from tRNA aminoacylation, and (ii) an ORF encoding a protein that is homologous to the 3'-aminoglycoside phosphotransferases but does not confer antibiotic resistance. The remaining ORFs specify products which have no homology with proteins in the EMBL and GenBank data bases. PMID:1400209

  15. Development of vaccines to Mycobacterium avium subsp. paratuberculosis infection

    PubMed Central

    2016-01-01

    Johne's disease or paratuberculosis is a chronic debilitating disease in ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). The disease causes significant economic losses in livestock industries worldwide. There are no effective control measures to eradicate the disease because there are no appropriate diagnostic methods to detect subclinically infected animals. Therefore, it is very difficult to control the disease using only test and cull strategies. Vaccination against paratuberculosis has been considered as an alternative strategy to control the disease when combined with management interventions. Understanding host-pathogen interactions is extremely important to development of vaccines. It has long been known that Th1-mediated cellular immune responses are play a crucial role in protection against MAP infection. However, recent studies suggested that innate immune responses are more closely related to protective effects than adaptive immunity. Based on this understanding, several attempts have been made to develop vaccines against paratuberculosis. A variety of ideas for designing novel vaccines have emerged, and the tests of the efficacy of these vaccines are conducted constantly. However, no effective vaccines are commercially available. In this study, studies of the development of vaccines for MAP were reviewed and summarized. PMID:27489800

  16. Anti-tumour activity of Digitalis purpurea L. subsp. heywoodii.

    PubMed

    López-Lázaro, Miguel; Palma De La Peña, Nieves; Pastor, Nuria; Martín-Cordero, Carmen; Navarro, Eduardo; Cortés, Felipe; Ayuso, María Jesús; Toro, María Victoria

    2003-08-01

    Recent research has shown the anticancer effects of digitalis compounds suggesting their possible use in medical oncology. Four extracts obtained from the leaves of Digitalis purpurea subsp. heywoodii have been assessed for cytotoxic activity against three human cancer cell lines, using the SRB assay. All of them showed high cytotoxicity, producing IC50 values in the 0.78 - 15 microg/mL range with the methanolic extract being the most active, in non toxic concentrations. Steroid glycosides (gitoxigenin derivatives) were detected in this methanolic extract. Gitoxigenin and gitoxin were evaluated in the SRB assay using the three human cancer cell lines, showing IC50 values in the 0.13 - 2.8 microM range, with the renal adenocarcinoma cancer cell line (TK-10) being the most sensitive one. Morphological apoptosis evaluation of the methanolic extract and both compounds on the TK-10 cell line showed that their cytotoxicity was mediated by an apoptotic effect. Finally, possible mechanisms involved in apoptosis induction by digitalis compounds are discussed.

  17. Control of Mycobacterium avium subsp. paratuberculosis infection in agricultural species.

    PubMed

    Kennedy, D J; Benedictus, G

    2001-04-01

    Paratuberculosis or Johne's disease is a chronic intestinal disease caused by Mycobacterium avium subsp. paratuberculosis, which continues to spread in agricultural species. Control of paratuberculosis is challenging and should not be underestimated. Due to the long incubation period of the infection, disease is largely subclinical in domesticated livestock. Hence, direct effects on animal productivity and welfare are often masked and may appear insufficient to justify large investments in control programmes by individual farmers, livestock industries or governments. Furthermore, in some countries the main effects of the disease are indirect, resulting from the impact of market discrimination against herds and flocks known to be infected, or from the control measures enforced to reduce transmission. In such circumstances, producers may be unwilling to co-operate with surveillance that may detect infection in herds or flocks. As control programmes are rarely successful in eliminating the infection from a herd or flock in the short term without an aggressive and costly programme, financial and community support assists producers to deal with the challenge. Successful prevention and control depends on animal health authorities and livestock industries acquiring a good understanding of the nature and epidemiology of infection, and of the application of tools for diagnosis and control. Building support for control programmes under the leadership of the affected livestock industries is critical, as programmes are unlikely to be successful without ongoing political will, supported by funding for research, surveillance and control.

  18. Molecular Characterization of Invasive Streptococcus dysgalactiae subsp. equisimilis, Japan.

    PubMed

    Wajima, Takeaki; Morozumi, Miyuki; Hanada, Shigeo; Sunaoshi, Katsuhiko; Chiba, Naoko; Iwata, Satoshi; Ubukata, Kimiko

    2016-02-01

    We collected β-hemolytic streptococci (1,611 isolates) from patients with invasive streptococcal infections in Japan during April 2010-March 2013. Streptococcus dysgalactiae subsp. equisimilis (SDSE) was most common (n = 693); 99% of patients with SDSE infections were elderly (mean age 75 years, SD ±15 years). We aimed to clarify molecular and epidemiologic characteristics of SDSE isolates and features of patient infections. Bacteremia with no identified focus of origin and cellulitis were the most prevalent manifestations; otherwise, clinical manifestations resembled those of S. pyogenes infections. Clinical manifestations also differed by patient's age. SDSE isolates were classified into 34 emm types; stG6792 was most prevalent (27.1%), followed by stG485 and stG245. Mortality rates did not differ according to emm types. Multilocus sequence typing identified 46 sequence types and 12 novel types. Types possessing macrolide- and quinolone-resistance genes were 18.4% and 2.6%, respectively; none showed β-lactam resistance. Among aging populations, invasive SDSE infections are an increasing risk.

  19. Bacillus thuringiensis subsp. israelensis and Its Dipteran-Specific Toxins

    PubMed Central

    Ben-Dov, Eitan

    2014-01-01

    Bacillus thuringiensis subsp. israelensis (Bti) is the first Bacillus thuringiensis to be found and used as an effective biological control agent against larvae of many mosquito and black fly species around the world. Its larvicidal activity resides in four major (of 134, 128, 72 and 27 kDa) and at least two minor (of 78 and 29 kDa) polypeptides encoded respectively by cry4Aa, cry4Ba, cry11Aa, cyt1Aa, cry10Aa and cyt2Ba, all mapped on the 128 kb plasmid known as pBtoxis. These six δ-endotoxins form a complex parasporal crystalline body with remarkably high, specific and different toxicities to Aedes, Culex and Anopheles larvae. Cry toxins are composed of three domains (perforating domain I and receptor binding II and III) and create cation-selective channels, whereas Cyts are composed of one domain that acts as well as a detergent-like membrane perforator. Despite the low toxicities of Cyt1Aa and Cyt2Ba alone against exposed larvae, they are highly synergistic with the Cry toxins and hence their combinations prevent emergence of resistance in the targets. The lack of significant levels of resistance in field mosquito populations treated for decades with Bti-bioinsecticide suggests that this bacterium will be an effective biocontrol agent for years to come. PMID:24686769

  20. Bacillus thuringiensis subsp. israelensis and its dipteran-specific toxins.

    PubMed

    Ben-Dov, Eitan

    2014-03-28

    Bacillus thuringiensis subsp. israelensis (Bti) is the first Bacillus thuringiensis to be found and used as an effective biological control agent against larvae of many mosquito and black fly species around the world. Its larvicidal activity resides in four major (of 134, 128, 72 and 27 kDa) and at least two minor (of 78 and 29 kDa) polypeptides encoded respectively by cry4Aa, cry4Ba, cry11Aa, cyt1Aa, cry10Aa and cyt2Ba, all mapped on the 128 kb plasmid known as pBtoxis. These six δ-endotoxins form a complex parasporal crystalline body with remarkably high, specific and different toxicities to Aedes, Culex and Anopheles larvae. Cry toxins are composed of three domains (perforating domain I and receptor binding II and III) and create cation-selective channels, whereas Cyts are composed of one domain that acts as well as a detergent-like membrane perforator. Despite the low toxicities of Cyt1Aa and Cyt2Ba alone against exposed larvae, they are highly synergistic with the Cry toxins and hence their combinations prevent emergence of resistance in the targets. The lack of significant levels of resistance in field mosquito populations treated for decades with Bti-bioinsecticide suggests that this bacterium will be an effective biocontrol agent for years to come.