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.

No Assembly Required: Using BTyper3 to Assess the Congruency of a Proposed Taxonomic Framework for the Bacillus cereus Group With Historical Typing Methods

“…species) were omitted. Carroll and Wiedmann, 2020) and regardless of whether the legacy seven-group or adjusted eight-group panC typing schemes were used (Figure 7 and Supplementary Table S1).…”

“…isolates that are closely related to emetic toxin (cereulide)-producing isolates are incapable of causing emetic intoxication themselves but can cause the diarrheal form of B. cereus s.l. illness (Ehling-Schulz et al, 2005;Jessberger et al, 2015;Riol et al, 2018;Carroll and Wiedmann, 2020). However, as there is no standardized name for these isolates, they have been referred to as "emetic-like B. cereus" (Ehling-Schulz et al, 2005), "B. paranthracis" (Liu et al, 2017;Bukharin et al, 2019), "B. cereus," "Group III B. cereus" (i.e., assigned to Group III using the sequence of panC and the seven-phylogenetic group framework proposed by Guinebretiere et al, 2010), and "B. cereus s.s." (although it should be noted that these strains do not fall within the genomospecies boundary of the B. cereus s.s. type strain and thus are not actually members of the B. cereus s.s. species) (Guinebretiere et al, 2010;Gdoura-Ben Amor et al, 2018;Glasset et al, 2018;Zhuang et al, 2019).…”

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

“…For reference, in a previous point source foodborne outbreak caused by B. cereus s.l . (37), outbreak isolates could differ by up to seven core SNPs (using the same SNP calling methodology used here) (38).…”

“…This strain, which had been isolated from processed beef mince from a processing plant in Mpumalanga, was a member of ST26 (Figure 5 and Table 2), the ST to which most cereulide-producing B. cereus s.l . strains belong (although it should be noted that ST26 strains may be capable of producing enterotoxins and causing diarrheal illness, regardless of whether they produce cereulide or not) (38, 40, 41). While members of ST26 are comparatively closely related (>99.52 pairwise ANI), WGS was able to distinguish the South African strain sequenced here from closely related ST26 strains (pairwise core SNP distance >913 relative to publicly available genomes; Figure 5 and Table 3).…”

“…strains from beef and poultry products, which were assigned to the “ B. paranthracis ” genomospecies via GTDB and similar ANI-based methods (Table 1). As noted previously, “ B. paranthracis ” was proposed as a “novel” species in 2017 (55) but was later found to encompass the well-known foodborne pathogen “emetic B. cereus ” within its genomospecies boundary (29, 37, 38). One of the “ B.paranthracis ” strains isolated here indeed possessed cereulide synthetase-encoding genes and belonged to ST26 (Table 2), the ST that encompasses most B. cereus s.l .…”