Prevalence and Clonal Diversity of over 1,200 Listeria monocytogenes Isolates Collected from Public Access Waters near Produce Production Areas on the Central California Coast during 2011 to 2016

Abstract

A 5-year survey of public access surface waters in an agricultural region of the Central California Coast was done to assess the prevalence of the foodborne pathogen Listeria monocytogenes. In nature, L. monocytogenes lives as a saprophyte in soil and water, which are reservoirs for contamination of preharvest produce. Moore swabs were deployed biweekly in lakes, ponds, streams, and rivers during 2011 to 2016. L. monocytogenes was recovered in 1,224 of 2,922 samples, resulting in 41.9% prevalence. Multiple subtypes were isolated from 97 samples, resulting in 1,323 L. monocytogenes isolates. Prevalence was higher in winter and spring and after rain events in some waterways. Over 84% of the isolates were serotype 4b. Whole-genome sequencing was done on 1,248 isolates, and in silico multilocus sequence typing revealed 74 different sequence types (STs) and 39 clonal complexes (CCs). The clones most isolated, CC639, CC183, and CC1, made up 27%, 19%, and 13%, respectively, of the sequenced isolates. Other types were CC663, CC6, CC842, CC4, CC2, CC5, and CC217. All sequenced isolates contained intact copies of core L. monocytogenes virulence genes, and pathogenicity islands LIPI-3 and LIPI-4 were identified in 73% and 63%, respectively, of the sequenced isolates. The virulence factor internalin A was predicted to be intact in all but four isolates, while genes important for sanitizer and heavy metal resistance were found in <5% of the isolates. These waters are not used for crop irrigation directly, but they are available to wildlife and can flood fields during heavy rains. IMPORTANCE Listeria monocytogenes serotype 4b and 1/2a strains are implicated in most listeriosis, and hypervirulent listeriosis stems from strains containing pathogenicity islands LIPI-3 and LIPI-4. The waters and sediments in the Central California Coast agricultural region contain widespread and diverse L. monocytogenes populations, and all the isolates contain intact virulence genes. Emerging clones CC183 and CC639 were the most abundant clones, and major clones CC1, CC4, and CC6 were well represented. CC183 was responsible for three produce-related outbreaks in the last 7 years. Most of the isolates in the survey differ from those of lesser virulence that are often isolated from foods and food processing plants because they contain genes encoding an intact virulence factor, internalin A, and most did not contain genes for sanitizer and heavy metal resistance. This isolate collection is important for understanding L. monocytogenes populations in agricultural and natural regions.

Keywords: Listeria monocytogenes; food safety; microbial ecology; sediment; water quality.

FIG 1

(A) Map of region indicating sampling sites along the Upper and Lower Salinas River. Sampling sites are labeled with a letter and number. The Upper Salinas area is indicated in the blue-shaded region. The Lower Salinas area is in the yellow-shaded region. Sites labeled X1 and X2 are not part of any of the designated waterways. (B) Map of the sampling region and the Alisal, Carr Lake, Gabilan, and Tembladero waterway areas. Waterways are indicated on the map by blue dotted lines. Sampling sites indicated with letter corresponding to the waterway to which they were assigned. A, Alisal (yellow-shaded region); C, Carr Lake (pink-shaded region); G, Gabilan (brown-shaded region); T, Tembladero (blue-shaded region); S, Salinas River. The map backgrounds are from The National Map courtesy of the U.S. Geological Survey and are in the public domain.

FIG 2

Phylogenetic trees of sequenced isolates. L. monocytogenes isolates were grouped into their clonal complex or sequence type. The tree was constructed by the neighbor-joining algorithm using pairwise allelic differences generated by cgMLST and rooted at midpoint. The scale bar at the bottom represents the percentage of dissimilarity. Shown is the relatedness of CCs and/or STs, the presence or absence of genes associated with virulence (including LIPI-1, -2, -3, and -4), stress tolerance, biofilm formation, detergent and heavy metal resistance, and motility. Dark blue boxes indicate all isolates in that branch carrying intact genes. Light blue boxes indicate there is variability among the isolates in the branch on specific gene content. Orange boxes indicate that isolates in the branch are missing the labeled gene. LIPI-1 contains genes necessary for L. monocytogenes virulence (prfA, plcA, hly, mpl, actA, and plcB). LIPI-3 (which contains llsABDGHPXY, with llsX encoding lysteriolysin S) is an additional pathogenicity island present in some strains. LIPI-4, which contains genes encoding a cellobiose phosphor-transferase system (LM9005581_70009, -70010, -70012, -70013, and -70014), is an additional pathogenicity island present in some strains). Internalin genes inlEFGHKL are involved in virulence with inlL playing a role in initial adhesion for biofilm development. The brcABC genes encode resistance determinants for benzalkonium chloride. ladR and mdlL are involved in a multidrug efflux pump. qacA/C, ermE, ermC, and Tn1688 are involved in resistance to quaternary ammonium compounds. bapL is a gene coding for a biofilm-associated protein. lmo0673 represents lmo0673, lmo2504, luxS, and recO, which are involved in biofilm development. LGI-2 (Listeria genomic island 2) encodes stress determinants for cadmium and arsenic resistance on a genomic island. cadA1A2, cadA2C2, and cadA3C3 encode extrachromosomal cadmium resistance determinants. SSI-1 (stress survival islet 1) contains lmo0444, lmo0445, pva, gadD1, and gadT1 and is involved in response to low pH and high salt, and SSI-2 (stress survival islet 2) contains lin0464, lin0465, LM6179_0748, and yoaZ and is involved in alkaline and oxidative stress responses. The remaining columns are labeled as to what processes they have been shown to influence.