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 NH24A2T (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 21929T (= PS00077AT = PS00077BT = PSU-0922T = BHPT) 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.
Bacillus cereus group or B. cereus sensu lato (s.l.), is comprised of Gram-positive spore-forming, rod-like bacteria that are widespread in natural environments. Although the species in this group are known to be highly related in terms of phenotypic characteristics, they display different levels of pathogenicity. Biochemical assays are therefore considered to be insufficient for accurate taxonomic classification of B. cereus group species. To facilitate accurate taxonomic classification and associated prediction of pathogenic potential, we have conducted comparative genomic analyses of publicly available genome assemblies of B. cereus group isolates. Through that, we found that an isolate previously known as B. mycoides ATCC 21929 was sufficiently distant from valid and effective type strains to be considered a putative new species. We have conducted biochemical and bioinformatic characterization of strain ATCC 21929 that had been isolated from soil in Papua New Guinea. Strain ATCC 21929 most closely resembles B. paramycoides NH24A2T, producing ANIb and DDH values of 86.70% and 34.1%, respectively. Phenotypically, isolate ATCC 21929 does not possess cytochrome c oxidase activity, and is able to grow at a range of temperatures 15°C - 43°C and at a range of pH 6 - 9. With regards to fatty acid composition, this isolate has iso-C17:0 in highest abundance. We propose the strain ATCC 21929T (=PS00077AT = PS00077BT = PSU-0922T = BHPT) as a new species named Bacillus clarus sp. nov. to facilitate accurate taxonomic classification of B. cereus group isolates.
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