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Cereulide Synthetase Acquisition and Loss Events within the Evolutionary History of Group III Bacillus cereus Sensu Lato Facilitate the Transition between Emetic and Diarrheal Foodborne Pathogens.

ABSTRACT: Cereulide-producing members of Bacillus cereus sensu lato group III (also known as emetic B. cereus) possess cereulide synthetase, a plasmid-encoded, nonribosomal peptide synthetase encoded by the ces gene cluster. Despite the documented risks that cereulide-producing strains pose to public health, the level of genomic diversity encompassed by emetic B. cereus has never been evaluated at a whole-genome scale. Here, we employ a phylogenomic approach to characterize group III B. cereus sensu lato genomes which possess ces (ces positive) alongside their closely related, ces-negative counterparts (i) to assess the genomic diversity encompassed by emetic B. cereus and (ii) to identify potential ces loss and/or gain events within the evolutionary history of the high-risk and medically relevant sequence type (ST) 26 lineage often associated with emetic foodborne illness. Using all publicly available ces-positive group III B. cereus sensu lato genomes and the ces-negative genomes interspersed among them (n?=?159), we show that emetic B. cereus is not clonal; rather, multiple lineages within group III harbor cereulide-producing strains, all of which share an ancestor incapable of producing cereulide (posterior probability = 0.86 to 0.89). Members of ST 26 share an ancestor that existed circa 1748 (95% highest posterior density [HPD] interval = 1246.89 to 1915.64) and first acquired the ability to produce cereulide before 1876 (95% HPD = 1641.43 to 1946.70). Within ST 26 alone, two subsequent ces gain events were observed, as well as three ces loss events, including among isolates responsible for B. cereus sensu lato toxicoinfection (i.e., "diarrheal" illness).IMPORTANCE B. cereus is responsible for thousands of cases of foodborne disease each year worldwide, causing two distinct forms of illness: (i) intoxication via cereulide (i.e., emetic syndrome) or (ii) toxicoinfection via multiple enterotoxins (i.e., diarrheal syndrome). Here, we show that emetic B. cereus is not a clonal, homogenous unit that resulted from a single cereulide synthetase gain event followed by subsequent proliferation; rather, cereulide synthetase acquisition and loss is a dynamic, ongoing process that occurs across lineages, allowing some group III B. cereus sensu lato populations to oscillate between diarrheal and emetic foodborne pathogens over the course of their evolutionary histories. We also highlight the care that must be taken when selecting a reference genome for whole-genome sequencing-based investigation of emetic B. cereus sensu lato outbreaks, since some reference genome selections can lead to a confounding loss of resolution and potentially hinder epidemiological investigations.

SUBMITTER: Carroll LM

PROVIDER: S-EPMC7448271 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Cereulide Synthetase Acquisition and Loss Events within the Evolutionary History of Group III Bacillus cereus Sensu Lato Facilitate the Transition between Emetic and Diarrheal Foodborne Pathogens.

Carroll Laura M LM Wiedmann Martin M

mBio 20200825 4

Cereulide-producing members of Bacillus cereussensu lato group III (also known as emetic B. cereus) possess cereulide synthetase, a plasmid-encoded, nonribosomal peptide synthetase encoded by the ces gene cluster. Despite the documented risks that cereulide-producing strains pose to public health, the level of genomic diversity encompassed by emetic B. cereus has never been evaluated at a whole-genome scale. Here, we employ a phylogenomic approach to characteri ...[more]

PMID: 32843545

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Project description:BackgroundCereulide, a depsipeptide structurally related to valinomycin, is responsible for the emetic type of gastrointestinal disease caused by Bacillus cereus. Recently, it has been shown that this toxin is produced by a nonribosomal peptide synthetase (NRPS), but its exact genetic organization and biochemical synthesis is unknown.ResultsThe complete sequence of the cereulide synthetase (ces) gene cluster, which encodes the enzymatic machinery required for the biosynthesis of cereulide, was dissected. The 24 kb ces gene cluster comprises 7 CDSs and includes, besides the typical NRPS genes like a phosphopantetheinyl transferase and two CDSs encoding enzyme modules for the activation and incorporation of monomers in the growing peptide chain, a CDS encoding a putative hydrolase in the upstream region and an ABC transporter in the downstream part. The enzyme modules responsible for incorporation of the hydroxyl acids showed an unusual structure while the modules responsible for the activation of the amino acids Ala and Val showed the typical domain organization of NRPS. The ces gene locus is flanked by genetic regions with high homology to virulence plasmids of B. cereus, Bacillus thuringiensis and Bacillus anthracis. PFGE and Southern hybridization showed that the ces genes are restricted to emetic B. cereus and indeed located on a 208 kb megaplasmid, which has high similarities to pXO1-like plasmids.ConclusionThe ces gene cluster that is located on a pXO1-like virulence plasmid represents, beside the insecticidal and the anthrax toxins, a third type of B. cereus group toxins encoded on megaplasmids. The ces genes are restricted to emetic toxin producers, but pXO1-like plasmids are also present in emetic-like strains. These data might indicate the presence of an ancient plasmid in B. cereus which has acquired different virulence genes over time. Due to the unusual structure of the hydroxyl acid incorporating enzyme modules of Ces, substantial biochemical efforts will be required to dissect the complete biochemical pathway of cereulide synthesis.

Project description:BackgroundBacillus cereus sensu lato (s. l.) is an ecologically diverse bacterial group of medical and agricultural significance. In this study, I use publicly available genomes and novel bioinformatic workflows to characterize the B. cereus s. l. pan-genome and perform the largest phylogenetic and population genetic analyses of this group to date in terms of the number of genes and taxa included. With these fundamental data in hand, I identify genes associated with particular phenotypic traits (i.e., "pan-GWAS" analysis), and quantify the degree to which taxa sharing common attributes are phylogenetically clustered.MethodsA rapid k-mer based approach (Mash) was used to create reduced representations of selected Bacillus genomes, and a fast distance-based phylogenetic analysis of this data (FastME) was performed to determine which species should be included in B. cereus s. l. The complete genomes of eight B. cereus s. l. species were annotated de novo with Prokka, and these annotations were used by Roary to produce the B. cereus s. l. pan-genome. Scoary was used to associate gene presence and absence patterns with various phenotypes. The orthologous protein sequence clusters produced by Roary were filtered and used to build HaMStR databases of gene models that were used in turn to construct phylogenetic data matrices. Phylogenetic analyses used RAxML, DendroPy, ClonalFrameML, PAUP*, and SplitsTree. Bayesian model-based population genetic analysis assigned taxa to clusters using hierBAPS. The genealogical sorting index was used to quantify the phylogenetic clustering of taxa sharing common attributes.ResultsThe B. cereus s. l. pan-genome currently consists of ≈60,000 genes, ≈600 of which are "core" (common to at least 99% of taxa sampled). Pan-GWAS analysis revealed genes associated with phenotypes such as isolation source, oxygen requirement, and ability to cause diseases such as anthrax or food poisoning. Extensive phylogenetic analyses using an unprecedented amount of data produced phylogenies that were largely concordant with each other and with previous studies. Phylogenetic support as measured by bootstrap probabilities increased markedly when all suitable pan-genome data was included in phylogenetic analyses, as opposed to when only core genes were used. Bayesian population genetic analysis recommended subdividing the three major clades of B. cereus s. l. into nine clusters. Taxa sharing common traits and species designations exhibited varying degrees of phylogenetic clustering.ConclusionsAll phylogenetic analyses recapitulated two previously used classification systems, and taxa were consistently assigned to the same major clade and group. By including accessory genes from the pan-genome in the phylogenetic analyses, I produced an exceptionally well-supported phylogeny of 114 complete B. cereus s. l. genomes. The best-performing methods were used to produce a phylogeny of all 498 publicly available B. cereus s. l. genomes, which was in turn used to compare three different classification systems and to test the monophyly status of various B. cereus s. l. species. The majority of the methodology used in this study is generic and could be leveraged to produce pan-genome estimates and similarly robust phylogenetic hypotheses for other bacterial groups.

Project description:The Bacillus cereus group comprises multiple species capable of causing emetic or diarrheal foodborne illness. Despite being responsible for tens of thousands of illnesses each year in the U.S. alone, whole-genome sequencing (WGS) is not yet routinely employed to characterize B. cereus group isolates from foodborne outbreaks. Here, we describe the first WGS-based characterization of isolates linked to an outbreak caused by members of the B. cereus group. In conjunction with a 2016 outbreak traced to a supplier of refried beans served by a fast food restaurant chain in upstate New York, a total of 33 B. cereus group isolates were obtained from human cases (n = 7) and food samples (n = 26). Emetic (n = 30) and diarrheal (n = 3) isolates were most closely related to B. paranthracis (group III) and B. cereus sensu stricto (group IV), respectively. WGS indicated that the 30 emetic isolates (24 and 6 from food and humans, respectively) were closely related and formed a well-supported clade distinct from publicly available emetic group III genomes with an identical sequence type (ST 26). The 30 emetic group III isolates from this outbreak differed from each other by a mean of 8.3 to 11.9 core single nucleotide polymorphisms (SNPs), while differing from publicly available emetic group III ST 26 B. cereus group genomes by a mean of 301.7-528.0 core SNPs, depending on the SNP calling methodology used. Using a WST-1 cell proliferation assay, the strains isolated from this outbreak had only mild detrimental effects on HeLa cell metabolic activity compared to reference diarrheal strain B. cereus ATCC 14579. We hypothesize that the outbreak was a single source outbreak caused by emetic group III B. cereus belonging to the B. paranthracis species, although food samples were not tested for presence of the emetic toxin cereulide. In addition to showcasing how WGS can be used to characterize B. cereus group strains linked to a foodborne outbreak, we also discuss potential microbiological and epidemiological challenges presented by B. cereus group outbreaks, and we offer recommendations for analyzing WGS data from the isolates associated with them.

Dataset Information

Cereulide Synthetase Acquisition and Loss Events within the Evolutionary History of Group III Bacillus cereus Sensu Lato Facilitate the Transition between Emetic and Diarrheal Foodborne Pathogens.

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Cereulide Synthetase Acquisition and Loss Events within the Evolutionary History of Group III <i>Bacillus cereus Sensu Lato</i> Facilitate the Transition between Emetic and Diarrheal Foodborne Pathogens.

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