Novel Effective Bacillus cereus Group Species " Bacillus clarus" Is Represented by Antibiotic-Producing Strain ATCC 21929 Isolated from Soil

Abstract

Gram-positive, spore-forming members of the Bacillus cereus group species complex are widespread in natural environments and display various degrees of pathogenicity. Recently, B. cereus group strain Bacillus mycoides Flugge ATCC 21929 was found to represent a novel lineage within the species complex, sharing a relatively low degree of genomic similarity with all B. cereus group genomes (average nucleotide identity [ANI] < 88). ATCC 21929 has been previously associated with the production of a patented antibiotic, antibiotic 60-6 (i.e., cerexin A); however, the virulence potential and growth characteristics of this lineage have never been assessed. Here, we provide an extensive genomic and phenotypic characterization of ATCC 21929, and we assess its pathogenic potential in vitro. ATCC 21929 most closely resembles Bacillus paramycoides NH24A2^T (ANI and in silico DNA-DNA hybridization values of 86.70 and 34.10%, respectively). Phenotypically, ATCC 21929 does not possess cytochrome c oxidase activity and is able to grow at a range of temperatures between 15 and 43°C and a range of pH between 6 and 9. At 32°C, ATCC 21929 shows weak production of diarrheal enterotoxin hemolysin BL (Hbl) but no production of nonhemolytic enterotoxin (Nhe); at 37°C, neither Hbl nor Nhe is produced. Additionally, at 37°C, ATCC 21929 does not exhibit cytotoxic effects toward HeLa cells. With regard to fatty acid composition, ATCC 21929 has iso-C17:0 present in highest abundance. Based on the characterization provided here, ATCC 21929^T (= PS00077A^T = PS00077B^T = PSU-0922^T = BHP^T) represents a novel effective B. cereus group species, which we propose as effective species "Bacillus clarus"IMPORTANCE The B. cereus group comprises numerous closely related lineages with various degrees of pathogenic potential and industrial relevance. Species-level taxonomic classification of B. cereus group strains is important for risk evaluation and communication but remains challenging. Biochemical and phenotypic assays are often used to assign B. cereus group strains to species but are insufficient for accurate taxonomic classification on a genomic scale. Here, we show that antibiotic-producing ATCC 21929 represents a novel lineage within the B. cereus group that, by all metrics used to delineate prokaryotic species, exemplifies a novel effective species. Furthermore, we show that ATCC 21929 is incapable of producing enterotoxins Hbl and Nhe or exhibiting cytotoxic effects on HeLa cells at human body temperature in vitro These results provide greater insight into the genomic and phenotypic diversity of the B. cereus group and may be leveraged to inform future public health and food safety efforts.

Keywords: Bacillus cereus group; Bacillus cereus sensu lato; Bacillus clarus; antibiotic production; cerexin A; cytotoxicity; enterotoxins; novel species; taxonomy; whole-genome sequencing.

FIG 1

Maximum likelihood phylogeny constructed using concatenated amino acid sequences derived from the type strain/representative genomes of the 22 published and effective B. cereus group species (gray font), outgroup genome Bacillus panaciterrae^T (itself not a member of the B. cereus group; gray font), and novel effective B. cereus group species “B. clarus” strain ATCC 21929^T (black font). B. panaciterrae^T was used to root the phylogeny, and branch lengths are reported in substitutions per site. Node labels correspond to branch support percentages obtained using 1,000 replicates of the ultrafast bootstrap approximation. OrthoFinder (19) was used to identify orthologues among all genomes and produce the amino acid sequence alignment, and IQ-TREE (20) was used to construct the phylogeny. s.s., sensu stricto.

FIG 2

Percent viability of HeLa cells when treated with supernatants of novel effective B. cereus group species “B. clarus” strain ATCC 21929^T or one of 17 published B. cereus group species type strains, as determined by the WST-1 assay. Viability was calculated as the ratio of corrected absorbance of suspension when HeLa cells were treated with supernatants to the ratio of corrected absorbance of suspension when HeLa cells were treated with BHI (i.e., negative control), converted to percentages. The columns represent the mean viabilities, while the error bars represent standard deviations for 12 technical replicates.