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.
OBSERVATION
The Bacillus cereus group is a complex of closely related, spore-forming, facultatively anaerobic bacterial species. Currently, the B. cereus group contains 19 published species: albus (1), anthracis (2), cereus sensu stricto (2), cytotoxicus (3), fungorum (4), luti (1), mobilis (1), mycoides (2), nitratireducens (1), pacificus (1), paramycoides (1), paranthracis (1), proteolyticus (1), pseudomycoides (5), thuringiensis (2), toyonensis (6), tropicus (1), weihenstephanensis (7), and wiedmannii (8). Additionally, the group currently contains three effective species: “bingmayongensis” (9), “gaemokensis” (10), and “manliponensis” (11).
We recently queried all publicly available B. cereus group genomes (12) and identified a singleton strain, B. mycoides Flugge ATCC 21929 (referred to here as ATCC 21929), that shared a relatively low degree of genomic similarity with all other genomes. Prior publications indicated that ATCC 21929 had been isolated from soil in Papua New Guinea and is able to produce a patented antibiotic compound, antibiotic 60-6 (also known as cerexin A), which is active against Gram-positive pathogens (13, 14). Here, a polyphasic approach that integrated genomic and phenotypic analyses was used to characterize ATCC 21929, a representative of novel effective species “Bacillus clarus.”
ATCC 21929 belongs to a novel B. cereus group genomospecies.The ATCC 21929 genome was resequenced to confirm its identity (NCBI accession no. QVOD00000000; see Text S1 in the supplemental material). The original genome (NCBI RefSeq accession no. GCF_000746925.1) (14) was used in subsequent analyses.
TEXT S1
Copyright © 2020 Méndez Acevedo et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
Despite sharing 99.8% and 100% 16S rRNA gene sequence similarity and coverage with Bacillus tropicus (Fig. S1 and Text S1), respectively, ATCC 21929 shared <88 average nucleotide identity (ANI) with all B. cereus group genomes (accessed 19 November 2018; calculated using FastANI version 1.0) (12). Based on ANI and in silico DNA-DNA hybridization (DDH) values calculated between all published and effective B. cereus group species type strain/representative genomes, as well as the whole-genome phylogeny, ATCC 21929 most closely resembled Bacillus paramycoides (Fig. 1, Table 1, and Table S1). ATCC 21929 shared 86.70 ANI with B. paramycoides (calculated using JSpeciesWS, http://jspecies.ribohost.com/jspeciesws/, accessed 15 July 2020; Table 1 and Table S1) (15), which is well below all proposed species thresholds for the B. cereus group (i.e., 92.5 to 96 ANI) (1, 3, 6, 8, 12). The in silico DDH value (calculated using the Genome-to-Genome Distance Calculator [GGDC], https://ggdc.dsmz.de/, accessed 15 July 2020) (16) shared by ATCC 21929 and B. paramycoides was 34.10% (95% confidence interval, 31.60 to 36.60%; Table 1 and Table S1), with 0.48% probability that the DDH value is greater than the 70% species threshold (Table S1) (16). Based on these results, ATCC 21929 is a member of the B. cereus group but is not a member of any published (valid) or effective species.
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 panaciterraeT (itself not a member of the B. cereus group; gray font), and novel effective B. cereus group species “B. clarus” strain ATCC 21929T (black font). B. panaciterraeT 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.
List of B. cereus group strains used in this studya
FIG S1
Copyright © 2020 Méndez Acevedo et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
TABLE S1
Copyright © 2020 Méndez Acevedo et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
ATCC 21929 does not produce Hbl or Nhe at human body temperature in vitro.Thirteen biosynthetic gene clusters (BGCs) were detected in the ATCC 21929 genome (Table 2) using antiSMASH (Text S1) (17). Genes encoding enterotoxins hemolysin BL (Hbl; hblABCD) and nonhemolytic enterotoxin (Nhe; nheABC) were additionally detected (Text S1). The Duopath Cereus Enterotoxins kit (Merck; Text S1) confirmed weak production of Hbl at 32°C but no production of Nhe. At 37°C, neither Hbl nor Nhe was produced. ATCC 21929 additionally demonstrated a lack of cytotoxic activity toward HeLa cells at 37°C (Fig. 2 and Text S1).
Biosynthetic gene clusters (BGCs) identified in the genome of “B. clarus” strain ATCC 21929a
Percent viability of HeLa cells when treated with supernatants of novel effective B. cereus group species “B. clarus” strain ATCC 21929T 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.
Unlike the B. mycoides and B. paramycoides type strains, ATCC 21929 is oxidase negative.ATCC 21929 cells stained Gram positive and were approximately 3 μm long. Morphology of ATCC 21929 was observed by transmission electron microscopy (Fig. S2 and Text S1). ATCC 21929 was hemolytic and oxidase negative. The strain was able to hydrolyze starch and casein at 32°C after 72 h of incubation, indicating that it possesses both amylase and caseinase activity. ATCC 21929 was motile and grew a visible biomass after 3 days of incubation under anaerobic conditions. It grew at temperatures of 15 to 43°C, pH of 6 to 9, and NaCl concentrations of 0 to 5% (Table 3). The spore-forming capabilities of ATCC 21929 were not specifically assessed.
Phenotypic characteristics of novel effective species “B. clarus” strain ATCC 21929T and published and effective Bacillus cereus group species type strainsi
FIG S2
Copyright © 2020 Méndez Acevedo et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
Fatty acid composition of ATCC 21929 (Text S1) revealed that iso-C15:0 was most abundant. Among the least abundant fatty acids were C15:1 ω5c and iso-11:0 3OH. The latter two fatty acids, along with iso-13:0 3OH, were not reported for any other B. cereus group species type strain (Table 4). API 20E and CH50 biochemical assays (bioMérieux; Text S1) indicated that ATCC 21929 has a metabolic capacity similar to that of other B. cereus group species type strains (Table 4).
Fatty acid composition of novel effective species “B. clarus” strain ATCC 21929T and other published and effective Bacillus cereus group species type strainsa
“B. clarus” is an effective B. cereus group species.By all contemporary metrics used to delineate prokaryotic species, singleton genome ATCC 21929 represents a novel B. cereus group species. However, in order to validly publish a novel species, its type strain cannot be patented, as is the case here (13, 18). Furthermore, ATCC 21929 had been deposited in the American Type Culture Collection (ATCC), which did not allow for its deposition in another international culture collection (a hard requirement for new species validation) (18). ATCC 21929 should be evaluated as a member of a novel effective species, for which we propose the name “B. clarus.”
Description of effective species “Bacillus clarus.”“Bacillus clarus” (cla′rus. L. masc. adj. clarus clear).
Cells stained Gram positive and displayed a long rod-like appearance, 3 μm in length. “B. clarus” ATCC 21929T is highly motile, oxidase negative, hemolytic, possesses amylase and caseinase activity, can reach stationary phase in 16 h when grown at 32°C in brain heart infusion (BHI), and can grow under aerobic and anaerobic conditions. “B. clarus” ATCC 21929T can grow at pH 6 to 9, temperatures of 15 to 43°C, and NaCl concentrations of 0 to 5%; optimal conditions for growth are 6 to 9, 37°C, and 0.5 to 3%, respectively. “B. clarus” ATCC 21929T shows weak Hbl production at 32°C, as indicated by faint bands in the Duopath Enterotoxins test, but does not reduce the metabolic activity of HeLa cells under the tested conditions. The most abundant fatty acid was iso-C15:0. Among the least abundant fatty acids were C15:1 ω5c and iso-11:0 3OH. The latter two fatty acids, along with iso-13:0 3OH, were not reported for any other B. cereus group type strain. Unique characteristics of “B. clarus” ATCC 21929T include a higher abundance of iso-C17:0, lower abundance of iso-C16:0, and the ability to grow optimally at 3% NaCl. “B. clarus” ATCC 21929T is oxidase negative, a trait shared only by B. cereus group members B. wiedmannii, “B. gaemokensis,” and “B. manliponensis.”
Availability of data.The “B. clarus” ATCC 21929 genome is available under NCBI RefSeq accession no. GCF_000746925.1 (original genome sequenced by Los Alamos National Laboratory) (14) and NCBI accession no. QVOD00000000 (the resequencing effort described here). “B. clarus” ATCC 21929 has been deposited in the PubMLST Isolates database under ID number 2468.
FIG S3
Copyright © 2020 Méndez Acevedo et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
ACKNOWLEDGMENTS
We thank Ryan Michael Gaboy and bioMérieux for donating API 20E and CH50 assay kits.
M.M.A. was supported by the USDA-funded REEU project “Bugs in my Food: Research and Professional Development in Food Safety for Undergraduates from Non-Land Grant Institutions” (USDA-NIFA 2017-67032-26022), and L.M.C. by the National Science Foundation Graduate Research Fellowship Program under grant no. DGE-1144153. J.K. and E.G.D. were supported by the USDA National Institute of Food and Agriculture Hatch Appropriations under projects #PEN04646 and #PEN04522 and accessions #1015787 and #0233376, respectively. L.X. and sequencing were supported by the U.S. Food and Drug Administration grant number 1U18FD006222-01 in support of GenomeTrakr in Pennsylvania.
FOOTNOTES
- Received August 29, 2020.
- Accepted October 20, 2020.
- Copyright © 2020 Méndez Acevedo et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
