PCR assay of the groEL gene for detection and differentiation of Bacillus cereus group cells

Abstract

Strains of species in the Bacillus cereus group are potentially enterotoxic. Thus, the detection of all B. cereus group strains is important. As 16S ribosomal DNA sequence analysis cannot adequately differentiate species of the B. cereus group, we explored the potential of the groEL gene as a phylogenetic marker. A phylogenetic analysis of the groEL sequences of 78 B. cereus group strains revealed that the B. cereus group strains were split into two major clusters, one including six B. mycoides and one B. pseudomycoides (cluster II) and the other including two B. mycoides and the rest of the B. cereus group strains (cluster I). Cluster I was further differentiated into two subclusters, Ia and Ib. The sodA gene sequences of representative strains from different clusters were also compared. The phylogenetic tree constructed from the sodA sequences showed substantial similarity to the tree constructed from the groEL sequences. Based on the groEL sequences, a PCR assay for detection and identification of B. cereus group strains was developed. Subsequent restriction fragment length polymorphism (RFLP) analysis verified the PCR amplicons and the differentiation of the B. cereus group strains. RFLP with MboI was identical for all the B. cereus group strains analyzed, while RFLP with MfeI or PstI classified all B. cereus and B. thuringiensis strains into two groups. All cluster II B. mycoides and B. pseudomycoides strains could be discriminated from other B. cereus group bacteria by restriction analysis with TspRI.

FIG. 1.

Phylogenetic trees of B. cereus group bacteria based on nucleotide sequences of groEL (a) and sodA (b). The percentage of 1,000 bootstrap replicates is indicated above the nodes. The scale bar represents 10% differences in nucleotide sequence. (a) DNA neighbor-joining phylogram inferred from the nucleotide sequences of groEL gene fragments originating from 78 B. cereus group strains, including 56 reference strains and 22 Taiwan B. cereus soil isolates. The 533-bp sequences were used. Nine groups of B. cereus group bacteria had identical sequences (Table 1). One to three representative strains in each group are represented in the figure. The tree was rooted with B. stearothermophilus as the outgroup. (b) DNA neighbor-joining phylogram inferred from the nucleotide sequences of sodA gene fragments originating from 21 reference B. cereus group strains. The 428-bp sequences were used. The tree was rooted with B. stearothermophilus as the outgroup. B. t, B. thuringiensis; B. m, B. mycoides; B. c, B. cereus; B. a, B. anthracis.

FIG. 1.

Phylogenetic trees of B. cereus group bacteria based on nucleotide sequences of groEL (a) and sodA (b). The percentage of 1,000 bootstrap replicates is indicated above the nodes. The scale bar represents 10% differences in nucleotide sequence. (a) DNA neighbor-joining phylogram inferred from the nucleotide sequences of groEL gene fragments originating from 78 B. cereus group strains, including 56 reference strains and 22 Taiwan B. cereus soil isolates. The 533-bp sequences were used. Nine groups of B. cereus group bacteria had identical sequences (Table 1). One to three representative strains in each group are represented in the figure. The tree was rooted with B. stearothermophilus as the outgroup. (b) DNA neighbor-joining phylogram inferred from the nucleotide sequences of sodA gene fragments originating from 21 reference B. cereus group strains. The 428-bp sequences were used. The tree was rooted with B. stearothermophilus as the outgroup. B. t, B. thuringiensis; B. m, B. mycoides; B. c, B. cereus; B. a, B. anthracis.