Genomic insights into the taxonomic status of the Bacillus cereus group

Abstract

The identification and phylogenetic relationships of bacteria within the Bacillus cereus group are controversial. This study aimed at determining the taxonomic affiliations of these strains using the whole-genome sequence-based Genome BLAST Distance Phylogeny (GBDP) approach. The GBDP analysis clearly separated 224 strains into 30 clusters, representing eleven known, partially merged species and accordingly 19-20 putative novel species. Additionally, 16S rRNA gene analysis, a novel variant of multi-locus sequence analysis (nMLSA) and screening of virulence genes were performed. The 16S rRNA gene sequence was not sufficient to differentiate the bacteria within this group due to its high conservation. The nMLSA results were consistent with GBDP. Moreover, a fast typing method was proposed using the pycA gene, and where necessary, the ccpA gene. The pXO plasmids and cry genes were widely distributed, suggesting little correlation with the phylogenetic positions of the host bacteria. This might explain why classifications based on virulence characteristics proved unsatisfactory in the past. In summary, this is the first large-scale and systematic study of the taxonomic status of the bacteria within the B. cereus group using whole-genome sequences, and is likely to contribute to further insights into their pathogenicity, phylogeny and adaptation to diverse environments.

Figure 1. Whole-genome phylogeny of 224 bacteria of the B. cereus group inferred using the latest GBDP version and rooted with Bacillus subtilis.

Numbers above branches are greedy-with-trimming pseudo-bootstrap support values from 100 replicates if larger than 50%. Leaves are colored according to their affiliation to clusters (i.e., Bacillus cereus groups, BCG). The three outer circles show whether or not the (i) cry locus, (ii) pXO1(-like) plasmid and/or the (iii) pXO2(-like) plasmid is found. Type strains are printed in bold font as well as marked by an asterisk (*). The tree was inferred using FastME and visualized using iTOL. The leaf labels correspond to the encoding as listed in Data S1.

Figure 2. Distribution of pairwise 16S rRNA gene similarities of the three levels: inter-species, intra-species and intra-strain.

A total of 1007 16S rRNA gene sequences were analyzed, sharing all sites from positions 352 to 1051 in the complete 16S rRNA gene; more sequences could not be considered due to the condition of some of the draft genome sequences. The x-axis indicated the pairwise similarity (in %) of the 16S rRNA gene sequences, whereas the y-axis represents the proportion of each respective similarity value.

Figure 3. Correlation analysis between dDDH values and nMLSA similarities.

The exponential equation (y = 91.99*e^0.0008756*x – 111.5*e^−0.1064*x, R² = 0.9832) was obtained using a nonlinear simulation analysis method with the default option of the Curve Fitting Tool implemented in MATLAB 8.1. The vertical line indicates the 70% dDDH threshold. The upper horizontal line (y1 = 97.74) indicates the estimated nMLSA similarity threshold (inter-species) corresponding to 70% dDDH threshold that was calculated on the basis of the above exponential equation. The lower horizontal line (y1 = 97.50) indicates the actual lowest similarity for nMLSA among strains of intra-species.

Figure 4. Phylogenetic trees inferred from the pycA gene of 224 bacteria of the B. cereus group.

Numbers on branches are bootstrap support values from 1,000 replicates given in percent. Branches are scaled in terms of the expected number of substitutions per site. Leaves are colored according to their affiliation to clusters (compare Fig. 1). The three outer circles show whether or not the (i) cry locus, (ii) pXO1(-like) plasmid and/or the (iii) pXO2(-like) plasmid are found. Type strains are printed in bold font and marked by an asterisk (*). Bacillus subtilis ATCC 6051^T (CP003329) was used as the outgroup.