Over the last 10 to 15 years, increasing evidence suggests that persistence of Listeria monocytogenes in food processing plants for years or even decades is an important factor in the transmission ...

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.

Sampling of agricultural and natural environments in two US states (Colorado and Florida) yielded 18 Listeria-like isolates that could not be assigned to previously described species using traditional methods. Using whole-genome sequencing and traditional phenotypic methods, we identified five novel species, each with a genome-wide average blast nucleotide identity (ANIb) of less than 85 % to currently described species. Phylogenetic analysis based on 16S rRNA gene sequences and amino acid sequences of 31 conserved loci showed the existence of four well-supported clades within the genus Listeria ; (i) a clade representing Listeria monocytogenes , L. marthii , L. innocua , L. welshimeri , L. seeligeri and L. ivanovii , which we refer to as Listeria sensu stricto, (ii) a clade consisting of Listeria fleischmannii and two newly described species, Listeria aquatica sp. nov. (type strain FSL S10-1188^T = DSM 26686^T = LMG 28120^T = BEI NR-42633^T) and Listeria floridensis sp. nov. (type strain FSL S10-1187^T = DSM 26687^T = LMG 28121^T = BEI NR-42632^T), (iii) a clade consisting of Listeria rocourtiae , L. weihenstephanensis and three novel species, Listeria cornellensis sp. nov. (type strain TTU A1-0210^T = FSL F6-0969^T = DSM 26689^T = LMG 28123^T = BEI NR-42630^T), Listeria grandensis sp. nov. (type strain TTU A1-0212^T = FSL F6-0971^T = DSM 26688^T = LMG 28122^T = BEI NR-42631^T) and Listeria riparia sp. nov. (type strain FSL S10-1204^T = DSM 26685^T = LMG 28119^T = BEI NR- 42634^T) and (iv) a clade containing Listeria grayi . Genomic and phenotypic data suggest that the novel species are non-pathogenic.

The persistence of Listeria monocytogenes in food-associated environments represents a key factor in transmission of this pathogen. To identify persistent and transient strains associated with production of fermented meat sausages in northern Portugal, 1,723 L. monocytogenes isolates from raw material and finished products from 11 processors were initially characterized by random amplification of polymorphic DNA (RAPD), PCR-based molecular serotyping, and epidemic clone characterization, as well as cadmium, arsenic, and tetracycline resistance typing. Pulsed-field gel electrophoresis (PFGE) typing of 240 representative isolates provided evidence for persistence of L. monocytogenes for periods of time ranging from 10 to 32 months for all seven processors for which isolates from different production dates were available. Among 50 L. monocytogenes isolates that included one representative for each PFGE pattern obtained from a given sample, 12 isolates showed reduced invasion efficiency in Caco-2 cells, including 8 isolates with premature stop codons in inlA . Among 41 isolates representing sporadic and persistent PFGE types, 22 isolates represented lysogens. Neither strains with reduced invasion nor lysogens were overrepresented among persistent isolates. While the susceptibility of isolates to lysogenic phages also did not correlate with persistence, it appeared to be associated with molecular serotype. Our data show the following. (i) RAPD may not be suitable for analysis of large sets of L. monocytogenes isolates. (ii) While a large diversity of L. monocytogenes subtypes is found in Portuguese fermented meat sausages, persistence of L. monocytogenes in this food chain is common. (iii) Persistent L. monocytogenes strains are diverse and do not appear to be characterized by unique genetic or phenotypic characteristics.