Identification of anthrax toxin genes in a Bacillus cereus associated with an illness resembling inhalation anthrax

Abstract

Bacillus anthracis is the etiologic agent of anthrax, an acute fatal disease among mammals. It was thought to differ from Bacillus cereus, an opportunistic pathogen and cause of food poisoning, by the presence of plasmids pXO1 and pXO2, which encode the lethal toxin complex and the poly-gamma-d-glutamic acid capsule, respectively. This work describes a non-B. anthracis isolate that possesses the anthrax toxin genes and is capable of causing a severe inhalation anthrax-like illness. Although initial phenotypic and 16S rRNA analysis identified this isolate as B. cereus, the rapid generation and analysis of a high-coverage draft genome sequence revealed the presence of a circular plasmid, named pBCXO1, with 99.6% similarity with the B. anthracis toxin-encoding plasmid, pXO1. Although homologues of the pXO2 encoded capsule genes were not found, a polysaccharide capsule cluster is encoded on a second, previously unidentified plasmid, pBC218. A/J mice challenged with B. cereus G9241 confirmed the virulence of this strain. These findings represent an example of how genomics could rapidly assist public health experts responding not only to clearly identified select agents but also to novel agents with similar pathogenic potentials. In this study, we combined a public health approach with genome analysis to provide insight into the correlation of phenotypic characteristics and their genetic basis.

Fig. 1.

(A-B) Circular representations of the plasmids of B. cereus G9241. The plasmids were compared by using blastp to the B. anthracis pXO1 and pXO2 plasmids and B. cereus ATCC10987 pBC10987 and Bacillus thuringiensis israeliensis pBtoxis. The inner two rings represent the plasmid coding regions in forward and reverse orientation; color scheme is as described (13). The outer-ring color scheme is as follows: red, B. anthracis plasmids; blue, B. cereus ATCC10987 plasmid pBC10987; cyan, proteins equally similar in both species; and green, proteins unique to that plasmid. The third ring represents the remaining physical gaps of the plasmids. (C) Synteny plots of the plasmids of B. cereus G9241 pBCXO1 versus B. anthracis Ames pXO1. Each triangle represents a single protein of G9241. They are color coded as follows: red, normalized score ≥0.95; orange, normalized scores ≥0.80 and <0.95; yellow, normalized scores ≥0.60 and <0.80; green, normalized scores ≥0.40 and <0.60; cyan, normalized scores ≥0.20 and <0.40; and blue, normalized scores≥0.0 and <0.20.

Fig. 2.

Unrooted, neighbor-joining tree derived from multiple-locus sequence typing of B. cereus G9241 and other Bacillus spp. (http://pubmlst.org/bcereus).

Fig. 3.

B. cereus G9241 capsule. India ink stain of B. cereus G9241 grown in ambient atmosphere (A) and B. anthracis grown in 5% CO₂ (B). (Scale bar, ≈10 μm.)

Fig. 4.

Survival of A/J mice i.p. challenged with B. cereus G9241 (green), B. anthracis Sterne (yellow), and B. cereus ATCC10987 (red). High-spore dose (1 × 10⁶) and low-spore dose (1 × 10⁴) are represented by triangles and circles, respectively. The experiment was monitored for 14 days, after which the mice inoculated with B. cereus ATCC10987 were still alive.