While the food-borne pathogen Listeria monocytogenes can persist in food associated environments, there are no whole-genome sequence (WGS) based methods to differentiate persistent from sporadic strains. Whole-genome sequencing of 188 isolates from a longitudinal study of L. monocytogenes in retail delis was used to (i) apply single-nucleotide polymorphism (SNP)-based phylogenetics for subtyping of L. monocytogenes, (ii) use SNP counts to differentiate persistent from repeatedly reintroduced strains, and (iii) identify genetic determinants of L. monocytogenes persistence. WGS analysis revealed three prophage regions that explained differences between three pairs of phylogenetically similar populations with pulsed-field gel electrophoresis types that differed by <3 bands. WGS-SNP-based phylogenetics found that putatively persistent L. monocytogenes represent SNP patterns (i) unique to a single retail deli, supporting persistence within the deli (11 clades), (ii) unique to a single state, supporting clonal spread within a state (7 clades), or (iii) spanning multiple states (5 clades). Isolates that formed one of 11 deli-specific clades differed by a median of 10 SNPs or fewer. Isolates from 12 putative persistence events had significantly fewer SNPs (median, 2 to 22 SNPs) than between isolates of the same subtype from other delis (median up to 77 SNPs), supporting persistence of the strain. In 13 events, nearly indistinguishable isolates (0 to 1 SNP) were found across multiple delis. No individual genes were enriched among persistent isolates compared to sporadic isolates. Our data show that WGS analysis improves food-borne pathogen subtyping and identification of persistent bacterial pathogens in food associated environments.
Listeria monocytogenes is an opportunistic food-borne pathogen responsible for approximately 250 deaths per year in the United States (1). The annual costs of listeriosis, including morbidity, mortality, and lost wages, are estimated at $2.8 billion (2). A 2003 risk assessment attributed 90% of listeriosis cases in the United States to consumption of contaminated ready-to-eat (RTE) deli meats (3), and most (Ͼ60%) of U.S. listeriosis cases linked to RTE deli meats were estimated to be due contamination during retail handling and slicing (4, 5). Consequently, the retail deli environment is a focal point for listeriosis reduction efforts.L. monocytogenes can persist in food-associated environments for months to years (6-8). Persistent strains have been linked to outbreaks of food-borne disease. For example, L. monocytogenes that was responsible for an outbreak linked to 29 cases and 4 deaths persisted in the source plant for at least 12 years (9). Therefore, the food processing industry has implemented the seek-anddestroy process to identify and eliminate point sources of persistence through enhanced environmental monitoring, sanitation, and equipment and process design (10).One challenge for persistent pathogen control is to differentiate true persistence from repeated reintroduction of a give...