Salmonella enterica Serovar Diversity, Distribution, and Prevalence in Public-Access Waters from a Central California Coastal Leafy Green-Growing Region from 2011 to 2016.

Prevalence and serovar diversity of Salmonella enterica were measured during a 5-year survey of surface waters in a 500-mi2 agricultural region of the Central California Coast. Rivers, streams, lakes, and ponds were sampled bimonthly resulting in 2,979 samples. Overall prevalence was 56.4% with higher levels detected in spring than in fall. Small, but significant, differences in prevalence were detected based on sample locations. Detection of Salmonella was correlated positively with both significant rain events and, in some environments, levels of generic Escherichia coli. Analysis of 1,936 isolates revealed significant serovar diversity, with 91 different serovars detected. The most common isolated serovars were S. enterica subsp. enterica serovars I 6,8:d:- (406 isolates, 21.0%, and potentially monophasic Salmonella Muenchen), Give (334 isolates, 17.3%), Muenchen (158 isolates, 8.2%), Typhimurium (227 isolates, 11.7%), Oranienburg (106 isolates, 5.5%), and Montevideo (78 isolates, 4%). Sixteen of the 24 most common serovars detected in the region are among the serovars reported to cause the most human salmonellosis in the United States. Some of the serovars were associated with location and seasonal bias. Analysis of XbaI pulsed field gel electrophoresis (PFGE) patterns of strains of serovars Typhimurium, Oranienburg, and Montevideo showed significant intraserovar diversity. PFGE pulsotypes were identified in the region for multiple years of the survey, indicating persistence or regular reintroduction to the region. IMPORTANCE Nontyphoidal Salmonella is among the leading causes of bacterial foodborne illness, and increasing numbers of outbreaks and recalls are due to contaminated produce. High prevalence and 91 different serovars were detected in this leafy green growing region. Seventeen serovars that cause most of the human salmonellosis in the United States were detected, with 16 of those serovars detected in multiple locations and multiple years of the 5-year survey. Understanding the widespread prevalence and diversity of Salmonella in the region will assist in promoting food safety practices and intervention methods for growers and regulators.

Growth and Survival of Attached Listeria on Lettuce and Stainless Steel Varies by Strain and Surface Type.

ABSTRACT: The foodborne pathogen Listeria monocytogenes lives as a saprophyte in nature and can adhere to and grow on surfaces as diverse as leaves, sediment, and stainless steel. To discern the mechanisms used by L. monocytogenes for attachment and growth on various surfaces, we studied interactions between the pathogen on lettuce and stainless steel. A panel of 24 strains (23 L. monocytogenes and 1 Listeria innocua) were screened for attachment and growth on lettuce at 4 and 25°C and on stainless steel at 10 and 37°C. Overnight growth of attached cells resulted in a 0- to 3-log increase on lettuce, depending on the strain and the temperature. Among the worst-performing strains on lettuce were two from a large cantaloupe outbreak, indicating that factors important for interactions with cantaloupe may be different from those required on lettuce tissue. Strains that grew the best on lettuce belonged to serotypes 1/2a, 1/2b, and 4b and were from cheese, potatoes, and water-sediment near produce fields. Confocal microscopy of L. monocytogenes tagged with constitutively expressed green fluorescent protein indicated associations with the cut edges and veins of lettuce leaves. On stainless steel coupons, there was a 5- to 7-log increase at 10°C after 7 days and a 4- to 7-log increase at 37°C after 40 h. Statistically, surface growth on stainless steel was better for serotype 1/2a than for serotype 4b strains, even though certain serotype 4b strains grew well on the coupons. The latter included strains that originated from produce and water-sediment. Some strains were fit in both environments, whereas others showed variability between the two different surfaces. Further analysis of these strains should reveal molecular factors needed for adherence and surface growth of L. monocytogenes on different biotic and abiotic surfaces.

The Majority of Genotypes of the Virulence Gene inlA Are Intact among Natural Watershed Isolates of Listeria monocytogenes from the Central California Coast.

Internalin A is an essential virulence gene involved in the uptake of the foodborne pathogen Listeria monocytogenes into host cells. It is intact in clinical strains and often truncated due to Premature Stop Codons (PMSCs) in isolates from processed foods and processing facilities. Less information is known about environmental isolates. We sequenced the inlA alleles and did Multi Locus Variable Number Tandem Repeat Analysis (MLVA) on 112 L. monocytogenes isolates from a 3-year period from naturally contaminated watersheds near a leafy green growing area in Central California. The collection contained 14 serotype 1/2a, 12 serotype 1/2b, and 86 serotype 4b strains. Twenty-seven different inlA alleles were found. Twenty-three of the alleles are predicted to encode intact copies of InlA, while three contain PMSCs. Another allele has a 9-nucleotide deletion, previously described for a clinical strain, indicating that it is still functional. Intact inlA genes were found in 101 isolates, and 8 isolates contained the allele predicted to contain the 3-amino acid deletion. Both allele types were found throughout the 3-year sampling period. Three strains contained inlA alleles with PMSCs, and these were found only during the first 3 months of the study. SNP analysis of the intact alleles indicated clustering of alleles based on serotype and lineage with serotypes 1/2b and 4b (lineage I strains) clustering together, and serotype 1/2a (lineage II strains) clustering separately. The combination of serotype, MLVA types, and inlA allele types indicate that the 112 isolates reflect at least 49 different strains of L. monocytogenes. The finding that 90% of environmental L. monocytogenes isolates contain intact inlA alleles varies significantly from isolates found in processing plants. This information is important to public health labs and growers as to the varieties of L. monocytogenes that could potentially contaminate fresh produce in the field by various means.

High genotypic and phenotypic similarity among Shiga toxin-producing Escherichia coli O111 environmental and outbreak strains.

Escherichia coli serogroup O111 is among the six most commonly reported non-O157:H7 Shiga toxin-producing E. coli (STEC), which are emerging as important foodborne pathogens. We have assembled a collection of environmental and clinical strains of E. coli O111 from diverse sources and investigated various genotypic and phenotypic characteristics of these strains to gain a better understanding of the epidemiology and biology of this serogroup. Sixty-three percent of the strains (24/38) were of H-type 8, which dominated the environmental- and outbreak-strains group, whereas the sporadic-case strains were more heterogeneous in H-type. All of the environmental and outbreak strains harbored the Shiga toxin 1 gene (stx1), eae, and ehx, and a subset of these also carried the Shiga toxin 2 gene (stx2). Only 9 of 16 sporadic-case strains produced stx1 and/or stx2, and these were mostly of H-type 8 and 10. Pulsed-field gel electrophoresis analysis revealed a cluster of environmental, outbreak, and sporadic illness strains with high phylogenetic similarity. Strains in this pulsogroup were all of the H8 type and STEC pathotype, and carried eae and ehx. Smaller clusters of highly similar STEC O111 strains included outbreak and sporadic illness strains isolated during different time periods or from different geographical locations. A distinct aggregative behavior was observed in the cultures of all environmental and outbreak STEC O111 strains, but not in those of sporadic-case strains. Among environmental and outbreaks strains, aggregation was positively correlated with production of curli fimbriae and RpoS function, and negatively with cellulose synthesis, while the nonaggregative behavior of sporadic-case strains correlated (positively) only with cellulose production. Our results indicate that STEC O111 strains sharing high genotypic similarity and important phenotypic traits with STEC O111 outbreak strains are present in the agricultural environment and may contribute to the burden of foodborne disease.

Comparison of subtypes of Listeria monocytogenes isolates from naturally contaminated watershed samples with and without a selective secondary enrichment.

Two enrichment methods for Listeria monocytogenes using Immuno Magnetic Separation (IMS) were tested to determine if they selected the same subtypes of isolates. Both methods used a non-selective primary enrichment and one included subculture in Fraser Broth, while the other involved direct plating of IMS beads. Sixty-two naturally contaminated watershed samples from the Central California Coast were used as a source of L. monocytogenes, and subtype diversity was measured by serotype and Multiple Number Variable Tandem Repeat Analysis (MLVA). Three different serotypes were detected from both methods with serotype 4b strains making up 87% of the isolates, serotype 1/2a making up 8%, and serotype 1/2b making up 5%. The data suggest that serotype 1/2a strains were more likely to be isolated from the Fraser Broth culture method. Sixty-two different MLVA types were detected and the more common MLVA types were detected by both culture methods. Forty-three MLVA types were detected only from one culture method or the other, while 19 types were detected from both culture methods. The most common MLVA type-12 was detected in 33 of the 62 water samples, and represented 31% of the isolates from both culture methods. This limited study provides evidence that using both enrichment culture methods allowed for detection of a greater diversity of isolates among the samples than the use of one method alone, and that a wide diversity of L. monocytogenes strains exist in this watershed.

Development of a robust method for isolation of shiga toxin-positive Escherichia coli (STEC) from fecal, plant, soil and water samples from a leafy greens production region in California.

During a 2.5-year survey of 33 farms and ranches in a major leafy greens production region in California, 13,650 produce, soil, livestock, wildlife, and water samples were tested for Shiga toxin (stx)-producing Escherichia coli (STEC). Overall, 357 and 1,912 samples were positive for E. coli O157:H7 (2.6%) or non-O157 STEC (14.0%), respectively. Isolates differentiated by O-typing ELISA and multilocus variable number tandem repeat analysis (MLVA) resulted in 697 O157:H7 and 3,256 non-O157 STEC isolates saved for further analysis. Cattle (7.1%), feral swine (4.7%), sediment (4.4%), and water (3.3%) samples were positive for E. coli O157:H7; 7/32 birds, 2/145 coyotes, 3/88 samples from elk also were positive. Non-O157 STEC were at approximately 5-fold higher incidence compared to O157 STEC: cattle (37.9%), feral swine (21.4%), birds (2.4%), small mammals (3.5%), deer or elk (8.3%), water (14.0%), sediment (12.3%), produce (0.3%) and soil adjacent to produce (0.6%). stx1, stx2 and stx1/stx2 genes were detected in 63%, 74% and 35% of STEC isolates, respectively. Subtilase, intimin and hemolysin genes were present in 28%, 25% and 79% of non-O157 STEC, respectively; 23% were of the "Top 6″ O-types. The initial method was modified twice during the study revealing evidence of culture bias based on differences in virulence and O-antigen profiles. MLVA typing revealed a diverse collection of O157 and non-O157 STEC strains isolated from multiple locations and sources and O157 STEC strains matching outbreak strains. These results emphasize the importance of multiple approaches for isolation of non-O157 STEC, that livestock and wildlife are common sources of potentially virulent STEC, and evidence of STEC persistence and movement in a leafy greens production environment.

Diversity of pulsed-field gel electrophoresis pulsotypes, serovars, and antibiotic resistance among Salmonella isolates from wild amphibians and reptiles in the California Central Coast.

A survey of cold-blooded vertebrates and associated surface waters in a produce-growing region on the Central California Coast was done between May and September 2011 to determine the diversity of Salmonella. Samples from 460 amphibians and reptiles and 119 water samples were collected and cultured for Salmonella. Animals sampled were frogs (n=331), lizards (n=59), newts (n=5), salamanders (n=6), snakes (n=39), and toads (n=20). Salmonella was isolated from 37 individual animals, including frogs, lizards, snakes, and toads. Snakes were the most likely to contain Salmonella, with 59% testing positive followed by 15.3% of lizards, 5% of toads, and 1.2% of frogs. Fifteen water samples (12.6%) were positive. Twenty-two different serovars were identified, and the majority of isolates were S. enterica subsp. IIIb, with subsp. I, II, and IIIa also found. The serovar isolated most frequently was S. enterica subsp. IIIb 16:z10:e,n,x,z15, from snakes and frogs in five different locations. S. enterica subsp. I serovar Typhimurium and the monophasic I 6,8:d:- were isolated from water, and subspecies I Duisburg and its variants were found in animals and water. Some samples contained more than one type of Salmonella. Analysis of pulsed-field gel electrophoresis pulsotypes indicated that some strains persisted in animals and water collected from the same location. Sixty-six isolates displayed antibiotic resistance, with 27 isolates resistant to more than one antibiotic, including a subspecies IIIb isolate from snake having resistance to five different antibiotics. Twenty-three isolates were resistant to more than one class of antibiotic, and six isolates were resistant to three classes. While these subspecies of IIIa and IIIb cause fewer instances of human illness, they may serve as reservoirs of antibiotic resistance, determinants in the environment, and be sources of contamination of leafy greens associated with product recalls.