Characterization of Bacillus anthracis-like bacteria isolated from wild great apes from Cote d'Ivoire and Cameroon

Abstract

We present the microbiological and molecular characterization of bacteria isolated from four chimpanzees and one gorilla thought to have died of an anthrax-like disease in Côte d'Ivoire and Cameroon. These isolates differed significantly from classic Bacillus anthracis by the following criteria: motility, resistance to the gamma phage, and, for isolates from Cameroon, resistance to penicillin G. A capsule was expressed not only after induction by CO(2) and bicarbonate but also under normal growth conditions. Subcultivation resulted in beta-hemolytic activity and gamma phage susceptibility in some subclones, suggesting differences in gene regulation compared to classic B. anthracis. The isolates from Côte d'Ivoire and Cameroon showed slight differences in their biochemical characteristics and MICs of different antibiotics but were identical in all molecular features and sequences analyzed. PCR and Southern blot analyses confirmed the presence of both the toxin and the capsule plasmid, with sizes corresponding to the B. anthracis virulence plasmids pXO1 and pXO2. Protective antigen was expressed and secreted into the culture supernatant. The isolates possessed variants of the Ba813 marker and the SG-749 fragment differing from that of classic B. anthracis strains. Multilocus sequence typing revealed a close relationship of our atypical isolates with both classic B. anthracis strains and two uncommonly virulent Bacillus cereus and Bacillus thuringiensis isolates. We propose that the newly discovered atypical B. anthracis strains share a common ancestor with classic B. anthracis or that they emerged recently by transfer of the B. anthracis plasmids to a strain of the B. cereus group.

FIG. 1.

Colony morphology and capsule production studied by SEM and TEM. Bacteria isolated from great apes (A, C, E, and G) and classic B. anthracis strains (B, D, F, and H) presented the same morphological criteria. The same colony morphology on agar was seen by SEM (A to D). Cells from suspension cultures showed the same capsule and filament (piles) morphology by negative staining (E and F), and a capsule fringe at the outer wall of the bacteria in thin cross-sections by TEM (G and H). Similar results were obtained with all bacterial isolates tested.

FIG. 2.

Cell morphology studied by TEM and SEM. Bacteria isolated from great apes (A, C, and E) and classic B. anthracis strains (B, D, and F) presented different morphological criteria. Cells from suspension cultures studied by TEM had flagella (A), in contrast to what was seen in B. Cells from agar cultures studied by SEM showed capsule structures (arrows) on the bacteria, in C in contrast to D, and twisted bacteria (E) in contrast to very rare structures (arrows) shown in F. Similar results were obtained with all bacterial isolates tested. All bars represent 1 μm.

FIG. 3.

Neighbor-joining phylogenetic trees for the concatenated allele sequences of different strains of the B. cereus (Bc) group. (A) Tree based on the scheme described previously by Helgason et al. (17) comparing 2,977 bp. B. anthracis strain B19-39 possessed a new pyrE allele, and the other 16 classic B. anthracis strains had ST-1. (B) Tree based on the scheme described previously by Priest et al. (45) comparing 2,838 bp. B. anthracis strain 5261 was ST-2, and the other 16 B. anthracis strains were ST-1. Other STs that could be assigned according to the database at http://pubmlst.org/bcereus/ were as follows: ST-4 (B. cereus ATCC 14579), ST-32 (B. cereus ATCC 10987), ST-26 (B. cereus DSM 4312), ST-34 (B. cereus ATCC 11778), ST-10 (B. thuringiensis [Bt] DSM 2046 and DSM 350), ST-16 (B. thuringiensis DSM 5815), ST-116 (B. mycoides [Bm] DSM 2048), ST-38 (B. cereus ATCC 4342), and ST-113 (B. thuringiensis serovar konkukian strain 97-27). The trees were statistically evaluated with a bootstrap analysis with 1,000 bootstraps. Only relevant bootstrap values above 70% are shown.

FIG. 4.

Detection of large plasmids in B. anthracis isolates. Plasmids of B. anthracis strains CI (lane 1) and UDIII-7 (lane 2) were detected by agarose gel electrophoresis (A) or by Southern blot analysis with probes for the capC gene (B) or the pag gene (C).

FIG. 5.

Western blot analysis demonstrating the expression of protective antigen. Culture supernatants of B. anthracis strains CI (lane 2) and UDIII-7 (lane 3) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane, and the protein was detected with a monoclonal anti-PA antibody. Purified recombinant PA was included as a control (lane 1). The position of the 85-kDa band of the protein standard is indicated.