Anthrax, but Not Bacillus anthracis?

B acillus anthracis, the etiologic agent of anthrax, is a close relative of B. cereus, a soil organism and known opportunistic pathogen that causes a variety of human infections [1]. B. anthracis is very similar to B. cereus and B. thuringiensis except that all confirmed samples of B. anthracis suggest that it is a monophyletic clone derived from the B. cereus and B. thuringiensis clade. The major distinguishing feature of B. anthracis is the presence of two large virulence plasmids, pXO1 and pXO2, that harbor the tripartite toxin complex [2] and the genes responsible for the synthesis of a poly-c-D-glutamic acid capsule [3,4], respectively. Although virulence factors associated with pathogenic B. cereus isolates are not understood, large plasmids are known to be associated with many of the soil and pathogenic isolates [5] and are likely to impart advantageous phenotypes that promote opportunistic pathogenic properties and/or growth in soil. Recent studies now demonstrate that the ‘‘genetic backbones’’ for both the pXO1 and pXO2 plasmids are not restricted to B. anthracis but rather can be found in related B. cereus and B. thuringiensis isolates as well [6–9]. Importantly, several close relatives of B. anthracis were recently identified because they were associated with diseases that resembled anthrax [10–14]. Whole-genome sequencing of one of these isolates, B. cereus G9241, revealed a homolog of pXO1 that includes an expressed pagA gene and a complete pathogenicity island. This isolate did not harbor pXO2, but it did express a capsule under experimental conditions that is not poly-c-D-glutamic acid. Two other B. cereus isolates (03BB102 and 03BB108) with clinical properties similar to B. cereus G9241 have recently been characterized and shown by PCR to be positive for the pXO1 toxin complex and, in one case (03BB102), positive for the pXO2 cap genes [13]. Unlike B. anthracis, neither of these isolates was sensitive to the cphage, and both were penicillin-resistant. A third series of isolates was obtained from chimpanzees that died in Tai National Park, Côte d’Ivoire (CI), and from chimpanzees and a gorilla that died in Dja Reserve, Cameroon (CA), reportedly from an anthrax-like disease [12]. Isolates from CI were genetically indistinguishable, but different from those obtained from CA. The CI and CA isolates contained the pagA gene and capC genes as measured by real-time PCR assays, suggesting the presence of pXO1and pXO2-like sequences [11]. What, then, is B. anthracis? Should these new isolates be categorized as B. anthracis? Should the definition be based purely upon clinical disease definitions or based upon other phenotypes? Historically, conventional bacteriology has suggested that motility, hemolysis, and the production of capsule were the only useful markers that could distinguish B. anthracis from B. cereus [15]. Hoffmaster et al. have taken a similar tack in maintaining the B. cereus nomenclature for the B. cereus G9241 isolate by expanding the strict definition of B. anthracis at the United States Centers for Disease Control and Prevention Special Bacteriology Reference Laboratory to include the following phenotypic and biochemical properties: (a) capsule-producing, (b) nonmotile, (c) susceptible to cphage, (d) nonhemolytic, (e) susceptible to penicillin, and (f) having other cell-wall, capsule, and 16S RNA features [10]. More recently, a definitive molecular genotypic marker has been found in the B. anthracis plcR gene in the form of a nonsense mutation [16]. This mutation was present in all B. anthracis isolates (89) tested but not in any of an array of close and distant B. cereus relatives [17]. While this paper was in review, an additional study of the CI and CA isolates showed that they are motile bacilli and that their primary cultures are not susceptible to the c-phage [14]. These results, combined with several other features—including the lack of the nonsense mutation in the plcR gene and the absence of large B. anthracis–specific prophage regions in their chromosomes—indicate that the CI and CA isolates are not B. anthracis. All currently accepted isolates and strains of B. anthracis fall into a monophyletic clade, and only the combined use of rapidly evolving variable number tandem repeat markers and single nucleotide polymorphism analysis using whole-genome comparisons allowed for discrimination between individual isolates and construction of a highly accurate phylogeny with precise rooting for this species [18–20]. These results led to the conclusion that B. anthracis was derived from the clonal expansion of a single ancestral B. cereus that acquired the two virulence plasmids and the nonsense plcR mutation. Strains that diverged close to this species boundary are being discovered principally because they share many B. anthracis– like traits, but correct nomenclature is dependent on determining where isolates fall in relation to this boundary. B. cereus G9241 and the CI/CA chimpanzee isolates diverged from the B. anthracis ancestor before the species boundary and are not included in the B. anthracis clade. Amplified fragment length polymorphism analysis (AFLP) indicates that B. cereus G9241 falls into a large cluster that includes B. anthracis and a number of clinical isolates known as AFLP group F [21,22]. Included in this AFLP cluster are two of the closest confirmed relatives of B. anthracis: B. cereus E33L and B. thuringiensis 97–27 [21]. Neither of these genomes contains a