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 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.

Characterization of a Deep Sea Bacillus toyonensis Isolate: Genomic and Pathogenic Features

“…To date, little is known about the pathogenicity of B. toyonensis . Several studies have provided evidences that suggested a lack of or low cytotoxic potential for some B. toyonensis strains ( Guinebretiere et al., 2010 ; Johler et al., 2018 ; Abdulmawjood et al., 2019 ; Carroll et al., 2020 ; Mendez Acevedo et al., 2020 ). Two other reports showed that B. toyonensis could exhibit larvacidal effect against beet armyworm ( Eski et al., 2018 ) or induce HeLa cell membrane damage ( Miller et al., 2018 ).…”

Genomic Sequencing of Bacillus cereus Sensu Lato Strains Isolated from Meat and Poultry Products in South Africa Enables Inter- and Intranational Surveillance and Source Tracking

“…Prokka v1.14.6 ( 78 ) was used to annotate each of the 25 B. cereus sensu lato genomes sequenced in this study. Protein-coding sequences derived from the type strain genomes of each of the 23 validly published and effective B. cereus sensu lato species (accessed 28 August 2021) were downloaded from NCBI’s RefSeq Assembly database (see Table 1 of Méndez Acevedo et al for all type strain accession numbers) ( 79 ). OrthoFinder v2.5.2 ( 80 , 81 ) was used to identify orthologues among protein-coding sequences associated with all 47 genomes (25 B. cereus sensu lato genomes sequenced in this study plus 23 B. cereus sensu lato species type strain genomes) using MAFFT v7.475 ( 82 , 83 ) for sequence alignment and RAxML-NG v1.0.2 ( 84 ) for phylogeny construction.…”

“…All assembled genomes submitted to the National Center for Biotechnology Information RefSeq database ( 60 , 61 ) as one of 23 validly published or effective B. cereus sensu lato species (i.e., albus , anthracis , “ bingmayongensis ,” cereus , “ clarus ,” cytotoxicus , fungorum , “ gaemokensis ,” luti , “ manliponensis ,” mobilis , mycoides , nitratireducens , pacificus , paramycoides , paranthracis , proteolyticus , pseudomycoides , thuringiensis , toyonensis , tropicus , weihenstephanensis , and wiedmannii ) ( 1 , 29 , 57 , 79 , 91 , 93 – 105 ) were downloaded ( n = 2,733; accessed 20 March 2021). QUAST and CheckM were used to assess the quality of each assembled genome (see Data preprocessing and quality control), and BTyper3 (using default settings) and GTDB-Tk were used for typing and/or taxonomic assignment as described above (see In silico typing and taxonomic characterization).…”

Genomic surveillance of Bacillus cereus sensu lato strains isolated from meat and poultry products in South Africa enables inter- and intra-national surveillance and source tracking

“…Protein coding sequences derived from the type strain genomes of each of the 23 validly published and effective B. cereus s.l . species (accessed 28 August 2021) were downloaded from NCBI’s RefSeq Assembly database (see Table 1 of Méndez Acevedo, et al for all type strain accessions) (71). OrthoFinder v2.5.2 (72, 73) was used to identify orthologues among protein coding sequences associated with all 47 genomes (25 B. cereus s.l .…”

“…the National Center for Biotechnology Information (NCBI) RefSeq database (83,84) as one of 23 validly published or effective B. cereus s.l. species (i.e., albus, anthracis, "bingmayongensis", cereus, "clarus", cytotoxicus, fungorum, "gaemokensis", luti, "manliponensis", mobilis, mycoides, nitratireducens, pacificus, paramycoides, paranthracis, proteolyticus, pseudomycoides, thuringiensis, toyonensis, tropicus, weihenstephanensis, wiedmannii) (1,29,52,69,81,(85)(86)(87)(88)(89)(90)(91)(92)(93)(94)(95)(96)(97) were downloaded (n = 2,733; accessed 20 March 2021). QUAST and CheckM were used to assess the quality of each assembled genome (see section "Data pre-processing and quality control" above), and BTyper3 (using default settings) and GTDB-Tk were used for typing and/or taxonomic assignment as described above (see section "In silico typing and taxonomic characterization").…”