Demonstration of a capsule plasmid in Bacillus anthracis

Green, B. D.; Battisti, L.; Koehler, Theresa M.; Thorne, Curtis B.; Ivins, Bruce E.

doi:10.1128/iai.49.2.291-297.1985

Cited by 459 publications

(177 citation statements)

References 17 publications

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“…The virulence of B. anthracis has been associated with two mega plasmids, pX01 and pX02 [20]. Plasmid pX02 (60 MDa) carries the genes required for synthesis of an antiphagocytic poly-D-glutamic acid capsule facilitating host immune system evasion [20][21][22][23][24][25]. The 110-MDa plasmid pX01 is required for synthesis of the three anthrax toxin proteins, edema factor (EF), lethal factor (LF), and protective antigen (PA).…”

Section: Discussionmentioning

confidence: 99%

Simultaneous Detection of CDC Category "A" DNA and RNA Bioterrorism Agents by Use of Multiplex PCR & RT-PCR Enzyme Hybridization Assays

Kraft

Fan

et al. 2009

Viruses

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Assays to simultaneously detect multiple potential agents of bioterrorism are limited. Two multiplex PCR and RT-PCR enzyme hybridization assays (mPCR-EHA, mRT-PCR-EHA) were developed to simultaneously detect many of the CDC category “A” bioterrorism agents. The “Bio T” DNA assay was developed to detect: Variola major (VM), Bacillus anthracis (BA), Yersinia pestis (YP), Francisella tularensis (FT) and Varicella zoster virus (VZV). The “Bio T” RNA assay (mRT-PCR-EHA) was developed to detect: Ebola virus (Ebola), Lassa fever virus (Lassa), Rift Valley fever (RVF), Hantavirus Sin Nombre species (HSN) and dengue virus (serotypes 1–4). Sensitivity and specificity of the 2 assays were tested by using genomic DNA, recombinant plasmid positive controls, RNA transcripts controls, surrogate (spiked) clinical samples and common respiratory pathogens. The analytical sensitivity (limit of detection (LOD)) of the DNA asssay for genomic DNA was 1×100∼1×102 copies/mL for BA, FT and YP. The LOD for VZV whole organism was 1×10−2 TCID50/mL. The LOD for recombinant controls ranged from 1×102∼1×103copies/mL for BA, FT, YP and VM. The RNA assay demonstrated LOD for RNA transcript controls of 1×104∼1×106 copies/mL without extraction and 1×105∼1×106 copies/mL with extraction for Ebola, RVF, Lassa and HSN. The LOD for dengue whole organisms was ∼1×10−4 dilution for dengue 1 and 2, 1×104 LD50/mL and 1×102 LD50/mL for dengue 3 and 4. The LOD without extraction for recombinant plasmid DNA controls was ∼1×103 copies/mL (1.5 input copies/reaction) for Ebola, RVF, Lassa and HSN. No cross-reactivity of primers and probes used in both assays was detected with common respiratory pathogens or between targeted analytes. Clinical sensitivity was estimated using 264 surrogate clinical samples tested with the BioT DNA assay and 549 samples tested with the BioT RNA assay. The clinical specificity is 99.6% and 99.8% for BioT DNA assay and BioT RNA assay, respectively. The surrogate sensitivities of these two assays were 100% (95%CI 83–100) for FT, BA (pX02), YP, VM, VZV, dengue 2,3,4 and 95% (95%CI 75–100) for BA (pX01) and dengue 1 using spiked clinical specimens. The specificity of both BioT multiplex assays on spiked specimens was 100% (95% CI 99–100). Compared to other available assays (culture, serology, PCR, etc.) both the BioT DNA mPCR-EHA and BioT RNA mRT-PCR-EHA are rapid, sensitive and specific assays for detecting many category “A” Bioterrorism agents using a standard thermocycler.

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Section: Discussionmentioning

confidence: 99%

Simultaneous Detection of CDC Category "A" DNA and RNA Bioterrorism Agents by Use of Multiplex PCR & RT-PCR Enzyme Hybridization Assays

Kraft

Fan

et al. 2009

Viruses

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“…Bacillus anthracis Sterne 7702, Ames, UT500 and UT538 (Drysdale et al, 2005) spores were prepared in PA media (Green et al, 1985) at 30°C while shaking at 200 r.p.m. for 5 days.…”

Section: Spore Preparationmentioning

confidence: 99%

Potential dissemination of Bacillus anthracis utilizing human lung epithelial cells

Russell¹,

Vasan²,

Keene³

et al. 2008

Cell Microbiol

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SummaryDissemination of Bacillus anthracis spores from the lung is a critical early event in the establishment of inhalational anthrax. We recently reported that B. anthracis could adhere to and be internalized by cultured intestinal epithelial and fibroblast cells. Here, using gentamicin protection assays and/or electron microscopy, we found that Sterne strain 7702 spores were able to adhere to and subsequently be internalized by polarized A549 cells and primary human small airway epithelial cells. We showed for the first time that internalized spores were able to survive and that spores could translocate across an A549 cell barrier from the apical side to the basolateral side without disrupting the barrier integrity, suggesting a transcellular route. In addition, dormant spores of fully virulent Ames and UT500 strains were able to adhere to A549 cells at a frequency similar to that of 7702, whereas the capsule in germinated Ames and UT500 spores prevented adherence. Fluorescence microscopy also revealed that dormant Ames spores were internalized at a frequency similar to that of 7702. These findings highlight the possibility of a novel route of dissemination in which B. anthracis utilizes epithelial cells of the lung. The implications of these results to B. anthracis pathogenesis are discussed.

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“…PCR-based methods can readily differentiate vaccine or fully virulent B. anthracis plasmid genotypes [22][23][24]26,35]. However, plasmid-cured B. anthracis [36][37][38][39], or near-neighbor species containing B. anthracis closely-related plasmids [40], are very difficult to distinguish from B. anthracis. In addition, plasmids or their virulence genes have been readily transferred within these groups by means of conjugation or transformation [41][42][43][44][45][46][47][48][49][50].…”

Section: Introductionmentioning

confidence: 99%

Rapid genotypic detection ofBacillus anthracisand theBacillus cereusgroup by multiplex real-time PCR melting curve analysis

Kim

Seo

Wheeler³

et al. 2005

FEMS Immunology &Amp; Medical Microbiology

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Bacillus anthracis has four plasmid possible virulence genotypes: pXO1+/pXO2+, pXO1+/pXO2-, pXO1-/pXO2+ or pXO1-/pXO2-. Due to the lack of a specific chromosomal marker for B. anthracis, differentiation of the pXO1-/pXO2- form of B. anthracis from closely related Bacillus cereus group species is difficult. In this study, we evaluate the ability of sspE, pXO1 and pXO2 primers to discriminate individual B. anthracis and the B. cereus group genotypes using multiplex real-time PCR and melting curve analysis. Optimal conditions for successful multiplex assays have been established. Purified DNAs from 38 bacterial strains including 11 strains of B. anthracis and 18 B. cereus group strains were analyzed. Nine of the B. cereus group near-neighbor strains were shown by multilocus sequence typing to be phylogenetically proximate to the B. anthracis clade. We have demonstrated that the four plasmid genotypes of B. anthracis and B. cereus group near-neighbors were differentially and simultaneously discriminated by this assay.

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Demonstration of a capsule plasmid in Bacillus anthracis

Cited by 459 publications

References 17 publications

Simultaneous Detection of CDC Category "A" DNA and RNA Bioterrorism Agents by Use of Multiplex PCR & RT-PCR Enzyme Hybridization Assays

Simultaneous Detection of CDC Category "A" DNA and RNA Bioterrorism Agents by Use of Multiplex PCR & RT-PCR Enzyme Hybridization Assays

Potential dissemination of Bacillus anthracis utilizing human lung epithelial cells

Rapid genotypic detection ofBacillus anthracisand theBacillus cereusgroup by multiplex real-time PCR melting curve analysis

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