Bacillus anthracis gamma phage lysis among soil bacteria: an update on test specificity

Abstract

Background: Bacillus 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.

Findings: We 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.

Conclusions: In 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.

Keywords: Anthrax; Bacillus anthracis; Gamma phage.

Fig. 1

Neighbor-joining dendrogram of 16S rRNA gene sequences, showing the relationship of isolates and representative isolates from each grouping (shown in bold) to a panel of related bacteria. Bootstrap values (based on 1000 replications) are given as percentages at branch nodes. Brevibacillus brevis is used as an outgroup for this analysis

Fig. 2

Relationships between B. cereus group member isolates of this study and select reference isolates using concatenated sequences from seven housekeeping alleles. Sequence types (STs) with previously identified gamma phage susceptible isolates are shown in bold, and isolates from this study with new STs are marked (asterisk). The tree was constructed using the neighbor joining method and percent bootstrap confidence levels were calculated using 1000 resamplings of the original data