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Multiplex PCR for species-level identification of Bacillus anthracis and detection of pXO1, pXO2, and related plasmids.

ABSTRACT: The Bacillus anthracis virulence plasmids pXO1 and pXO2 have critical implications for biosafety and select agent status. The proper identification and characterization of B. anthracis and its plasmid profile is important to the biodefense research community. Multiplex PCR was used to simultaneously detect a B. anthracis-specific chromosomal mutation, 4 targets distributed across pXO1, 3 targets distributed across pXO2, and highly conserved regions of the 16S gene, allowing an internal positive control for each sample. The multiplex PCR can produce as many as 9 easily separable and distinguishable amplicons, ranging in size from 188 to 555?bp. The PCR results were used to characterize DNA samples extracted from B. anthracis, other Bacillus species, and other bacterial species from many different genera. With the exception of 2 novel putative plasmids discovered, testing against inclusion and extensive exclusion panels showed 100% correlation to previously published and expected results. Upon testing 29 previously unpublished B. anthracis strains, 10 (34.5%) were pXO1(+)/pXO2(+), 9 (31.0%) were pXO1(+)/pXO2(-), 7 (24.1%) were pXO1(-)/pXO2(+), and 3 (10.3%) were pXO1(-)/pXO2(-). The present work presents a novel 9-target multiplex PCR assay capable of species-level identification of B. anthracis via a unique chromosomal marker and the detection of pXO1 and pXO2 via multiply redundant targets on each.

SUBMITTER: Riojas MA

PROVIDER: S-EPMC4394167 | biostudies-literature | 2015 Mar-Apr

REPOSITORIES: biostudies-literature

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Multiplex PCR for species-level identification of Bacillus anthracis and detection of pXO1, pXO2, and related plasmids.

Riojas Marco A MA Kiss Katalin K McKee Marian L ML Hazbón Manzour Hernando MH

Health security 20150320 2

The Bacillus anthracis virulence plasmids pXO1 and pXO2 have critical implications for biosafety and select agent status. The proper identification and characterization of B. anthracis and its plasmid profile is important to the biodefense research community. Multiplex PCR was used to simultaneously detect a B. anthracis-specific chromosomal mutation, 4 targets distributed across pXO1, 3 targets distributed across pXO2, and highly conserved regions of the 16S gene, allowing an internal positive ...[more]

PMID: 25813976

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Project description:Bacillus anthracis plasmids pXO1 and pXO2 carry the main virulence factors responsible for anthrax. However, the extent of copy number variation within the species and how the plasmids are related to pXO1/pXO2-like plasmids in other species of the Bacillus cereus sensu lato group remain unclear. To gain new insights into these issues, we sequenced 412 B. anthracis strains representing the total phylogenetic and ecological diversity of the species. Our results revealed that B. anthracis genomes carried, on average, 3.86 and 2.29 copies of pXO1 and pXO2, respectively, and also revealed a positive linear correlation between the copy numbers of pXO1 and pXO2. No correlation between the plasmid copy number and the phylogenetic relatedness of the strains was observed. However, genomes of strains isolated from animal tissues generally maintained a higher plasmid copy number than genomes of strains from environmental sources (P < 0.05 [Welch two-sample t test]). Comparisons against B. cereus genomes carrying complete or partial pXO1-like and pXO2-like plasmids showed that the plasmid-based phylogeny recapitulated that of the main chromosome, indicating limited plasmid horizontal transfer between or within these species. Comparisons of gene content revealed a closed pXO1 and pXO2 pangenome; e.g., plasmids encode <8 unique genes, on average, and a single large fragment deletion of pXO1 in one B. anthracis strain (2000031682) was detected. Collectively, our results provide a more complete view of the genomic diversity of B. anthracis plasmids, their copy number variation, and the virulence potential of other Bacillus species carrying pXO1/pXO2-like plasmids. IMPORTANCE Bacillus anthracis microorganisms are of historical and epidemiological importance and are among the most homogenous bacterial groups known, even though the B. anthracis genome is rich in mobile elements. Mobile elements can trigger the diversification of lineages; therefore, characterizing the extent of genomic variation in a large collection of strains is critical for a complete understanding of the diversity and evolution of the species. Here, we sequenced a large collection of B. anthracis strains (>400) that were recovered from human, animal, and environmental sources around the world. Our results confirmed the remarkable stability of gene content and synteny of the anthrax plasmids and revealed no signal of plasmid exchange between B. anthracis and pathogenic B. cereus isolates but rather predominantly vertical descent. These findings advance our understanding of the biology and pathogenomic evolution of B. anthracis and its plasmids.

Dataset Information

Multiplex PCR for species-level identification of Bacillus anthracis and detection of pXO1, pXO2, and related plasmids.

Publications

Multiplex PCR for species-level identification of Bacillus anthracis and detection of pXO1, pXO2, and related plasmids.

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