Reclassification of Wolbachia persica as Francisella persica comb. nov. and emended description of the family Francisellaceae.

The taxonomic status of the bacterium Wolbachia persica is described, and based on the evidence presented, transfer of this species to the genus Francisella as Francisella persica comb. nov. is proposed. This reclassification is supported by data generated from genomic comparisons of W. persica ATCC VR-331T ( = FSC845T = DSM 101678T) to other near neighbours, including Francisella tularensis subsp. novicida. The full-length 16S rRNA gene sequence of strain ATCC VR-331T had 98.5 % nucleotide identity to the cognate gene in F. tularensis, with the highest similarity to subspecies novicida. Phylogenetic trees of full-length 16S rRNA gene, gyrA and recA sequences from species of the genera Wolbachia (class Alphaproteobacteria) and Francisella (class Gammaproteobacteria) indicated that W. persica ATCC VR-331T was most closely related to members of the genus Francisella and not Wolbachia. Local collinear blocks within the chromosome of strain ATCC VR-331T had considerable similarity with F. tularensis subsp. novicida, but not with any Wolbachia strain. The genomes of strain ATCC VR-331T and F. tularensis subsp. novicida Utah 112T ( = ATCC 15482T) contained an average nucleotide identity mean of 88.72 % and median of 89.18 %. Importantly, the genome of strain ATCC VR-331T contained one Francisella Pathogenicity Island, similar to F. tularensis subsp. novicida, as well as the Francisella-specific gene fopA1 and F. tularensis-specific genes fopA2 and lpnA (also referred to as tul4). In contrast to the obligate intracellular genus Wolbachia, strain ATCC VR-331T and facultative intracellular Francisella can replicate in specialized cell-free media. Collectively, these results demonstrate that Wolbachia persica should be reclassified in the genus Francisella as Francisella persica comb. nov. The type strain of Francisella persica comb. nov. is ATCC VR-331T ( = FSC845T = DSM 101678T). An emended description of the family Francisellaceae is also provided.

Francisella tularensis molecular typing using differential insertion sequence amplification.

Tularemia is a potentially fatal disease that is caused by the highly infectious and zoonotic pathogen Francisella tularensis. Despite the monomorphic nature of sequenced F. tularensis genomes, there is a significant degree of plasticity in the organization of genetic elements. The observed variability in these genomes is due primarily to the transposition of direct repeats and insertion sequence (IS) elements. Since current methods used to genotype F. tularensis are time-consuming and require extensive laboratory resources, IS elements were investigated as a means to subtype this organism. The unique spatial location of specific IS elements provided the basis for the development of a differential IS amplification (DISA) assay to detect and distinguish the more virulent F. tularensis subsp. tularensis (subtypes A.I and A.II) and subsp. holarctica (type B) strains from F. tularensis subsp. novicida and other near neighbors, including Francisella philomiragia and Francisella-like endosymbionts found in ticks. Amplicon sizes and sequences derived from DISA showed heterogeneity within members of the subtype A.I and A.II isolates but not the type B strains. These differences were due to a 312-bp fragment derived from the IS element ISFtu1. Analysis of wild-type F. tularensis isolates by DISA correlated with pulsed-field gel electrophoresis genotyping utilizing two different restriction endonucleases and provided rapid results with minimal sample processing. The applicability of this molecular typing assay for environmental studies was demonstrated by the accurate identification and differentiation of tick-borne F. tularensis. The described approach to IS targeting and amplification provides new capability for epidemiological investigations and characterizations of tularemia source outbreaks.

Identification of Bacillus anthracis, Brucella spp., and Coxiella burnetii DNA signatures from bushmeat.

Meat from wildlife species (bushmeat) represents a major source of dietary protein in low- and middle-income countries where humans and wildlife live in close proximity. Despite the occurrence of zoonotic pathogens in wildlife, their prevalence in bushmeat remains unknown. To assess the risk of exposure to major pathogens in bushmeat, a total of 3784 samples, both fresh and processed, were collected from three major regions in Tanzania during both rainy and dry seasons, and were screened by real-time PCR for the presence of DNA signatures of Bacillus anthracis (B. anthracis), Brucella spp. (Brucella) and Coxiella burnetii (Coxiella). The analysis identified DNA signatures of B. anthracis (0.48%), Brucella (0.9%), and Coxiella (0.66%) in a total of 77 samples. Highest prevalence rates of B. anthracis, Brucella, and Coxiella were observed in wildebeest (56%), dik-dik (50%), and impala (24%), respectively. Fresh samples, those collected during the rainy season, and samples from Selous or Serengeti had a greater relative risk of being positive. Microbiome characterization identified Firmicutes and Proteobacteria as the most abundant phyla. The results highlight and define potential risks of exposure to endemic wildlife diseases from bushmeat and the need for future investigations to address the public health and emerging infectious disease risks associated with bushmeat harvesting, trade, and consumption.

Historical distribution and molecular diversity of Bacillus anthracis, Kazakhstan.

To map the distribution of anthrax outbreaks and strain subtypes in Kazakhstan during 1937-2005, we combined geographic information system technology and genetic analysis by using archived cultures and data. Biochemical and genetic tests confirmed the identity of 93 archived cultures in the Kazakhstan National Culture Collection as Bacillus anthracis. Multilocus variable number tandem repeat analysis genotyping identified 12 genotypes. Cluster analysis comparing these genotypes with previously published genotypes indicated that most (n = 78) isolates belonged to the previously described A1.a genetic cluster, 6 isolates belonged to the A3.b cluster, and 2 belonged to the A4 cluster. Two genotypes in the collection appeared to represent novel genetic sublineages; 1 of these isolates was from Krygystan. Our data provide a description of the historical, geographic, and genetic diversity of B. anthracis in this Central Asian region.

Comparative Proteomic Studies of Yersinia pestis Strains Isolated from Natural Foci in the Republic of Georgia.

Yersinia pestis, the causative agent of plague, is a highly virulent bacterium responsible for millions of human deaths throughout history. In the last decade, two natural plague foci have been described in the Republic of Georgia from which dozens of Y. pestis strains have been isolated. Analyses indicate that there are genetic differences between these strains, but it is not known if these differences are also reflected in protein expression. We chose four strains of Y. pestis (1390, 1853, 2944, and 8787) from the National Center for Disease Control and Public Health collection for proteomic studies based on neighbor-joining tree genetic analysis and geographical loci of strain origin. Proteomic expression was analyzed using two-dimensional gel electrophoresis and mass spectrometry. Select Y. pestis strains were grown under different physiological conditions and their proteomes were compared: (1) 28°C without calcium; (2) 28°C with calcium; (3) 37°C without calcium; and (4) 37°C with calcium. Candidate proteins were identified and the differences in expression of F1 antigen, tellurium-resistance protein, and outer membrane protein C, porin were validated by Western blotting. The in vitro cytotoxicity activity of these strains was also compared. The results indicate that protein expression and cytotoxic activities differ significantly among the studied strains; these differences could contribute to variations in essential physiological functions in these strains.

Characterization and performance evaluation of in vivo and in vitro produced monoclonal anti-TNT antibodies for the detection of TNT.

In this paper are the experimental results used to characterize four distinct monoclonal anti-TNT antibodies (in vivo and in vitro cloned) for potential use in a field-portable immunosensor. Direct and competitive enzyme-linked immunosorbent assays (ELISA) were performed to determine their affinity for TNT and a fluorescently labeled analog of TNT for use in an immunosensor. Effective concentrations (EC(50)), inhibition concentration (IC(50)) and cross-reactivity measurements to related nitroaromatics (e.g., 2,4,6-trinitrobenzene [TNB], methyl-2,4,6-trinitrophenyl nitramine [tetryl], 2-amino-4,6-dinitrotoluene [2A-4,6-DNT], 2,4-dinitrotoluene [2,4-DNT] and 1,3-dinitrotoluene [1,3-DNT]) were measured. Final characterization of the monoclonal antibodies was based on performance (measured by fluorescence dose response) using a fluorescence-based microcapillary displacement assay. Analytical techniques showed a high degree of affinity for TNT and varying degrees of cross-reactivity with each respective monoclonal antibody. Microcapillary displacement immunoassays with each of the antibodies resulted in detection capabilities at the lowest applied TNT concentration (10 ng/ml).

Francisella tularensis Subtype A.II Genomic Plasticity in Comparison with Subtype A.I.

Although Francisella tularensis is considered a monomorphic intracellular pathogen, molecular genotyping and virulence studies have demonstrated important differences within the tularensis subspecies (type A). To evaluate genetic variation within type A strains, sequencing and assembly of a new subtype A.II genome was achieved for comparison to other completed F. tularensis type A genomes. In contrast with the F. tularensis A.I strains (SCHU S4, FSC198, NE061598, and TI0902), substantial genomic variation was observed between the newly sequenced F. tularensis A.II strain (WY-00W4114) and the only other publically available A.II strain (WY96-3418). Genome differences between WY-00W4114 and WY96-3418 included three major chromosomal translocations, 1580 indels, and 286 nucleotide substitutions of which 159 were observed in predicted open reading frames and 127 were located in intergenic regions. The majority of WY-00W4114 nucleotide deletions occurred in intergenic regions, whereas most of the insertions and substitutions occurred in predicted genes. Of the nucleotide substitutions, 48 (30%) were synonymous and 111 (70%) were nonsynonymous. WY-00W4114 and WY96-3418 nucleotide polymorphisms were predominantly G/C to A/T allelic mutations, with WY-00W4114 having more A+T enrichment. In addition, the A.II genomes contained a considerably higher number of intact genes and longer repetitive sequences, including transposon remnants than the A.I genomes. Together these findings support the premise that F. tularensis A.II may have a fitness advantage compared to the A.I subtype due to the higher abundance of functional genes and repeated chromosomal sequences. A better understanding of the selective forces driving F. tularensis genetic diversity and plasticity is needed.

Application of chromosomal DNA and protein targeting for the identification of Yersinia pestis.

PURPOSE: A comprehensive strategy was developed and validated for the identification of pathogens from closely related near neighbors using both chromosomal and protein biomarkers, with emphasis on distinguishing Yersinia pestis from the ancestral bacterium Yersinia pseudotuberculosis. EXPERIMENTAL DESIGN: Computational analysis was used to discover chromosomal targets unique to Y. pestis. Locus identifier YPO1670 was selected for further validation and PCR was used to confirm that this biomarker was exclusively present in Y. pestis strains, while absent in other Yersinia species. RT-PCR and Western blot analyses were utilized to evaluate YPO1670 expression and MRM MS was performed to identify the YPO1670 protein within cell lysates. RESULTS: The described study validated that YPO1670 was exclusive to Y. pestis. PCR confirmed the locus to be unique to Y. pestis. The associated transcript and protein were produced throughout growth with the highest abundance occurring in stationary phase and MRM MS conclusively identified the YPO1670 protein in cell extracts. CONCLUSIONS AND CLINICAL RELEVANCE: These findings validated YPO1670 as a reliable candidate biomarker for Y. pestis and that a dual DNA and protein targeting approach is feasible for the development of next-generation assays to accurately differentiate pathogens from near neighbors.

Development of three quantitative real-time PCR assays for the detection of Rickettsia raoultii, Rickettsia slovaca, and Rickettsia aeschlimannii and their validation with ticks from the country of Georgia and the Republic of Azerbaijan.

A previous surveillance study of human pathogens within ticks collected in the country of Georgia showed a relatively high infection rate for Rickettsia raoultii, R. slovaca, and R. aeschlimannii. These 3 spotted fever group rickettsiae are human pathogens: R. raoultii and R. slovaca cause tick-borne lymphadenopathy (TIBOLA), and R. aeschlimannii causes an infection characterized by fever and maculopapular rash. Three quantitative real-time polymerase chain reaction (qPCR) assays, Rraoul, Rslov, and Raesch were developed and optimized to detect R. raoultii, R. slovaca, and R. aeschlimannii, respectively, by targeting fragments of the outer membrane protein B gene (ompB) using species-specific molecular beacon or TaqMan probes. The 3 qPCR assays showed 100% specificity when tested against a rickettsiae DNA panel (n=20) and a bacteria DNA panel (n=12). The limit of detection was found to be at least 3 copies per reaction for all assays. Validation of the assays using previously investigated tick nucleic acid preparations, which included Rickettsia-free tick samples, tick samples that contain R. raoultii, R. slovaca, R. aeschlimannii, and other Rickettsia spp., gave 100% sensitivity for all 3 qPCR assays. In addition, a total of 65 tick nucleic acid preparations (representing 259 individual ticks) collected from the country of Georgia and the Republic of Azerbaijan in 2009 was tested using the 3 qPCR assays. R. raoultii, R. slovaca, and R. aeschlimannii were not detected in any ticks (n=31) from the Republic of Azerbaijan, but in the ticks from the country of Georgia (n=228) the minimal infection rate for R. raoultii and R. slovaca in Dermacentor marginatus was 10% and 4%, respectively, and for R. aeschlimannii in Haemaphysalis sulcata and Hyalomma spp. it was 1.9% and 20%, respectively.

Large direct repeats flank genomic rearrangements between a new clinical isolate of Francisella tularensis subsp. tularensis A1 and Schu S4.

Francisella tularensis subspecies tularensis consists of two separate populations A1 and A2. This report describes the complete genome sequence of NE061598, an F. tularensis subspecies tularensis A1 isolated in 1998 from a human with clinical disease in Nebraska, United States of America. The genome sequence was compared to Schu S4, an F. tularensis subspecies tularensis A1a strain originally isolated in Ohio in 1941. It was determined that there were 25 nucleotide polymorphisms (22 SNPs and 3 indels) between Schu S4 and NE061598; two of these polymorphisms were in potential virulence loci. Pulsed-field gel electrophoresis analysis demonstrated that NE061598 was an A1a genotype. Other differences included repeat sequences (n = 11 separate loci), four of which were contained in coding sequences, and an inversion and rearrangement probably mediated by insertion sequences and the previously identified direct repeats I, II, and III. Five new variable-number tandem repeats were identified; three of these five were unique in NE061598 compared to Schu S4. Importantly, there was no gene loss or gain identified between NE061598 and Schu S4. Interpretation of these data suggests there is significant sequence conservation and chromosomal synteny within the A1 population. Further studies are needed to determine the biological properties driving the selective pressure that maintains the chromosomal structure of this monomorphic pathogen.

DNA assays for detection, identification, and individualization of select agent microorganisms.

The purpose of this article is to review the status of DNA assays used for the detection, identification, and individualization of Bacillus anthracis, Yersinia pestis, Francisella tularensis, Burkholderia mallei, and Brucella abortus. These select agent microorganisms are historically significant as they have either been used or experimented with as a bioweapon or as a terrorist agent and are the subject of intense research in the areas of biodefense and bioforensics. If the presence of a biological agent is suspected, sensitive and specific assays for rapid detection and identification are necessary. However, DNA methods for identification of the sample may also be applied in order to individualize the strain and potentially determine the source of the microorganism. Methods used at the Armed Forces Institute of Pathology (AFIP) for select agent microbial DNA analyses include DNA extraction, DNA quantitation, real-time polymerase chain reaction (real-time PCR) of genetic targets unique to the select agent microorganism, microbial 16S ribosomal RNA gene DNA sequencing, amplified fragment length polymorphism polymerase chain reaction (AFLP-PCR), and more recently, repetitive element polymerase chain reaction (REP-PCR) DNA fingerprinting. The methodologies of 16S ribosomal RNA gene DNA sequencing and DNA fingerprinting of microorganisms are well established within the field of diagnostic microbiology for DNA identification purposes, as well as DNA typing for epidemiological and genetic relatedness studies. 16S ribosomal RNA gene DNA sequencing and AFLP DNA fingerprinting have been validated at the Armed Force Institute of Pathology (AFIP) laboratory for identification purposes and can be used as a possible strain typing tool for Bacillus anthracis, Yersinia pestis, Francisella tularensis, as well as Brucella and Burkholderia species. The continued development and implementation of new DNA based methods with increased sensitivity and defined specificity will be particularly useful for the detection of residual microbial DNA signature in situations where the microorganism has been rendered nonviable by decontamination procedures or not able to be cultured on microbiological media.