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

Cereulide-producing members of group III (also known as emetic ) possess cereulide synthetase, a plasmid-encoded, nonribosomal peptide synthetase encoded by the gene cluster. Despite the documented risks that cereulide-producing strains pose to public health, the level of genomic diversity encompassed by emetic has never been evaluated at a whole-genome scale. Here, we employ a phylogenomic approach to characterize group III genomes which possess ( positive) alongside their closely related, -negative counterparts (i) to assess the genomic diversity encompassed by emetic and (ii) to identify potential 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 -positive group III genomes and the -negative genomes interspersed among them ( = 159), we show that emetic 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 gain events were observed, as well as three loss events, including among isolates responsible for toxicoinfection (i.e., "diarrheal" illness). 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 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 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 outbreaks, since some reference genome selections can lead to a confounding loss of resolution and potentially hinder epidemiological investigations.