Polyphasic Approach for Identifying Bacillus spp

Abstract

According to a recent paper by Blackwood and colleagues (1), the Bacillus cereus and B. anthracis ATCC type strains share identical 16S rRNA gene sequences. From the study of a number of additional phylogenetic markers in 27 strains of the B. cereus group, they concluded that the rpoB gene sequence was the most discriminatory marker, as the B. cereus type strain revealed four mismatches at gene positions considered key positions for B. anthracis (5). We fully agree with the notion of the importance of a polyphasic approach to species identification among the Bacillus cereus group. However, in our experience rpoB may not be specific enough for the identification of B. anthracis and B. cereus to the species level. (This research was presented as an abstract at the 103rd General Meeting of the American Society for Microbiology in Washington, D.C., 2003.) We isolated a Ba813-positive member of the B. cereus group with the phenotypic markers of B. cereus. A 19-year-old male developed wound infection and septic shock after surgery because of femur osteosarcoma. A Bacillus sp. was isolated from blood cultures and wound swabs in pure culture. Grey, dry, beta-hemolytic colonies grew on 5% sheep blood agar. The gram-positive, rod-shaped bacteria showed ellipsoidal central and subterminal spores. The isolate ifik1001803 was motile, resistant to penicillin G, and catalase positive. Analysis of the cellular fatty acids (MIDI system; Microbial ID, Inc., Newark, Del.) was consistent with B. cereus (without 17:1 anteiso A; with ISO 17:1 w10c) (personal communication of R. Paisley, MIDI, Inc., Newark, Del.). Sequence analysis of the 16S rRNA full gene showed a 100% match with B. anthracis type 6 (6). PCR amplifications of two virulence factors of B. anthracis (2), the protective antigen gene pag and the capsule gene fragment capC, were negative. PCR amplification of the Ba813 chromosomal fragment (4) gave a band of the expected size; sequencing of the PCR product revealed a 5-bp mismatch to B. anthracis ({type:entrez-nucleotide,attrs:{text:U46157,term_id:1389560,term_text:U46157}}U46157). In addition, the sequence of the rpoB gene of ifik1001803 was identical to that of B. anthracis at three of the four key positions, but it differed from it by 2 bp (Table ​(Table1).1). The variability of the rpoB gene sequence among B. cereus strains and ifik1001803 was in the range of 3 to 5 bp. Interestingly, B. cereus ATCC 49064, the B. cereus strain most closely related to ifik1001803, had a match with B. anthracis at one of the four key positions. From the perspective of sequence information, ifik1001803 might be viewed as a transitional composite of B. cereus and B. anthracis, supporting the recent concept of B. anthracis being a lineage of B. cereus (3). In this respect, B. cereus ATCC 49064 might represent another transitional member of the B. cereus group. TABLE 1. Alignment of B. cereus and B. anthracis sequence information of the rpoB gene Our results suggest that position 84 of the rpoB gene may represent the only key position for the differentiation of B. cereus from B. anthracis. However, the number of strains examined as well the choice of strains studied may influence the outcome of phylogenetic studies. In addition, a true polyphasic approach would require the consideration of independent information, such as morphological and physiological characteristics, including pathogenicity.