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Detailed genomic analysis of the Wbeta and gamma phages infecting Bacillus anthracis: implications for evolution of environmental fitness and antibiotic resistance.

ABSTRACT: Phage-mediated lysis has been an essential laboratory tool for rapidly identifying Bacillus anthracis for more than 40 years, relying on the gamma phage derivative of a Bacillus cereus prophage called W. The complete genomic sequences of the temperate W phage, referred to as Wbeta, and its lytic variant gamma were determined and found to encode 53 open reading frames each, spanning 40,864 bp and 37,373 bp, respectively. Direct comparison of the genomes showed that gamma evolved through mutations at key loci controlling host recognition, lysogenic growth, and possibly host phenotypic modification. Included are a cluster of point mutations at the gp14 tail fiber locus of gamma, encoding a protein that, when fused to green fluorescent protein, binds specifically to B. anthracis. A large 2,003-bp deletion was also identified at the gamma lysogeny module, explaining its shift from a temperate to a lytic lifestyle. Finally, evidence of recombination was observed at a dicistronic Wbeta locus, encoding putative bacterial cell surface-modifying proteins, replaced in gamma with a locus, likely obtained from a B. anthracis prophage, encoding demonstrable fosfomycin resistance. Reverse transcriptase PCR analysis confirmed strong induction at the dicistronic Wbeta locus and at four other phage loci in B. anthracis and/or B. cereus lysogens. In all, this study represents the first genomic and functional description of two historically important phages and is part of a broader investigation into contributions of phage to the B. anthracis life cycle. Initial findings suggest that lysogeny of B. anthracis promotes ecological adaptation, rather than virulence, as with other gram-positive pathogens.

SUBMITTER: Schuch R

PROVIDER: S-EPMC1446989 | biostudies-literature | 2006 Apr

REPOSITORIES: biostudies-literature

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Detailed genomic analysis of the Wbeta and gamma phages infecting Bacillus anthracis: implications for evolution of environmental fitness and antibiotic resistance.

Schuch Raymond R Fischetti Vincent A VA

Journal of bacteriology 20060401 8

Phage-mediated lysis has been an essential laboratory tool for rapidly identifying Bacillus anthracis for more than 40 years, relying on the gamma phage derivative of a Bacillus cereus prophage called W. The complete genomic sequences of the temperate W phage, referred to as Wbeta, and its lytic variant gamma were determined and found to encode 53 open reading frames each, spanning 40,864 bp and 37,373 bp, respectively. Direct comparison of the genomes showed that gamma evolved through mutations ...[more]

PMID: 16585764

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Project description:In 2015, a laboratory of the United States Department of Defense (DoD) inadvertently shipped preparations of gamma-irradiated spores of Bacillus anthracis that contained live spores. In response, a systematic evidence-based method for preparing, concentrating, irradiating, and verifying the inactivation of spore materials was developed. We demonstrate the consistency of spore preparations across multiple biological replicates and show that two different DoD institutions independently obtained comparable dose-inactivation curves for a monodisperse suspension of B. anthracis spores containing 3 × 1010 CFU. Spore preparations from three different institutions and three strain backgrounds yielded similar decimal reduction (D10) values and irradiation doses required to ensure sterility (DSAL) to the point at which the probability of detecting a viable spore is 10-6 Furthermore, spores of a genetically tagged strain of B. anthracis strain Sterne were used to show that high densities of dead spores suppress the recovery of viable spores. Together, we present an integrated method for preparing, irradiating, and verifying the inactivation of spores of B. anthracis for use as standard reagents for testing and evaluating detection and diagnostic devices and techniques.IMPORTANCE The inadvertent shipment by a U.S. Department of Defense (DoD) laboratory of live Bacillus anthracis (anthrax) spores to U.S. and international destinations revealed the need to standardize inactivation methods for materials derived from biological select agents and toxins (BSAT) and for the development of evidence-based methods to prevent the recurrence of such an event. Following a retrospective analysis of the procedures previously employed to generate inactivated B. anthracis spores, a study was commissioned by the DoD to provide data required to support the production of inactivated spores for the biodefense community. The results of this work are presented in this publication, which details the method by which spores can be prepared, irradiated, and tested, such that the chance of finding residual living spores in any given preparation is 1/1,000,000. These irradiated spores are used to test equipment and methods for the detection of agents of biological warfare and bioterrorism.

Project description:BackgroundBacillus anthracis, which causes anthrax in humans and animals, is enzootic in parts of the U.S. state of Texas where cases are typically reported in animals annually. The gamma phage lysis assay is a common diagnostic method for identification of B. anthracis and is based on the bacterium's susceptibility to lysis. This test has been shown to be 97% specific for B. anthracis, as a small number of strains of other Bacillus spp. are known to be susceptible. In this study, we evaluated the performance of a combination of B. anthracis diagnostic assays on 700 aerobic, spore-forming isolates recovered from soil collected in Texas. These assays include phenotypic descriptions, gamma phage susceptibility, and real-time polymerase chain reaction specific for B. anthracis. Gamma phage-susceptible isolates were also tested using cell wall and capsule direct fluorescent-antibody assays specific for B. anthracis. Gamma phage-susceptible isolates that were ruled out as B. anthracis were identified by 16S rRNA gene sequencing.FindingsWe identified 29 gamma phage-susceptible isolates. One was confirmed as B. anthracis, while the other 28 isolates were ruled out for B. anthracis by the other diagnostic tests. Using 16S rRNA gene sequencing results, we identified these isolates as members of the B. cereus group, Bacillus sp. (not within B. cereus group), Lysinibacillus spp., and Solibacillus silvestris. Based on these results, we report a specificity of 96% for gamma phage lysis as a diagnostic test for B. anthracis, and identified susceptible isolates outside the Bacillus genus.ConclusionsIn this study we found gamma phage susceptibility to be consistent with previously reported results. However, we identified non-B. anthracis environmental isolates (including isolates from genera other than Bacillus) that are susceptible to gamma phage lysis. To date, susceptibility to gamma phage lysis has not been reported in genera other than Bacillus. Though these isolates are not of clinical origin, description of unexpected positives is important, especially as new diagnostic assays for B. anthracis are being developed based on gamma phage lysis or gamma phage proteins.

Dataset Information

Detailed genomic analysis of the Wbeta and gamma phages infecting Bacillus anthracis: implications for evolution of environmental fitness and antibiotic resistance.

Publications

Detailed genomic analysis of the Wbeta and gamma phages infecting Bacillus anthracis: implications for evolution of environmental fitness and antibiotic resistance.

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