Produce contaminated with enteric pathogens is a major source of foodborne illness in the United States. Lakes, streams, rivers, and ponds were sampled with Moore swabs bi-monthly for over two years at 30 locations in the vicinity of a leafy green growing region on the Central California Coast and screened for Shiga toxin producing Escherichia coli (STEC), Salmonella enterica, and Listeria monocytogenes to evaluate the prevalence and persistence of pathogen subtypes. The prevalence of STEC from 1,386 samples was 11%; 110 samples (8%) contained E. coli O157:H7 with the highest prevalence occurring close to cattle operations. Non-O157 STEC isolates represented major clinical O-types and 57% contained both shiga toxin types 1 and 2 and intimin. Multiple Locus Variable Number Tandem Repeat Analysis of STEC isolates indicated prevalent strains during the period of study. Notably, Salmonella was present at high levels throughout the sampling region with 65% prevalence in 1,405 samples resulting in 996 isolates with slightly lower prevalence in late autumn. There were 2, 8 and 14 sites that were Salmonella-positive over 90%, 80% and 70% of the time, respectively. The serotypes identified most often were 6,8:d:-, Typhimurium, and Give. Interestingly, analysis by Pulsed Field Gel Electrophoresis indicated persistence and transport of pulsotypes in the region over several years. In this original study of L. monocytogenes in the region prevalence was 43% of 1,405 samples resulting in 635 individual isolates. Over 85% of the isolates belonged to serotype 4b with serotypes 1/2a, 1/2b, 3a, 4d with 4e representing the rest, and there were 12 and 2 sites that were positive over 50% and 80% of the time, respectively. Although surface water is not directly used for irrigation in this region, transport to the produce can occur by other means. This environmental survey assesses initial contamination levels towards an understanding of transport leading to produce recalls or outbreaks.

Background: Although serotype O157:H7 is the predominant enterohemorrhagic Escherichia coli (EHEC), outbreaks of non-O157 EHEC that cause severe foodborne illness, including hemolytic uremic syndrome have increased worldwide. In fact, non-O157 serotypes are now estimated to cause over half of all the Shiga toxin-producing Escherichia coli (STEC) cases, and outbreaks of non-O157 EHEC infections are frequently associated with serotypes O26, O45, O103, O111, O121, and O145. Currently, there are no complete genomes for O145 in public databases. Results: We determined the complete genome sequences of two O145 strains (EcO145), one linked to a US lettuce-associated outbreak (RM13514) and one to a Belgium ice-cream-associated outbreak (RM13516). Both strains contain one chromosome and two large plasmids, with genome sizes of 5,737,294 bp for RM13514 and 5,559,008 bp for RM13516. Comparative analysis of the two EcO145 genomes revealed a large core (5,173 genes) and a considerable amount of strain-specific genes. Additionally, the two EcO145 genomes display distinct chromosomal architecture, virulence gene profile, phylogenetic origin of Stx2a prophage, and methylation profile (methylome). Comparative analysis of EcO145 genomes to other completely sequenced STEC and other E. coli and Shigella genomes revealed that, unlike any other known non-O157 EHEC strain, EcO145 ascended from a common lineage with EcO157/EcO55. This evolutionary relationship was further supported by the pangenome analysis of the 10 EHEC str ains. Of the 4,192 EHEC core genes, EcO145 shares more genes with EcO157 than with the any other non-O157 EHEC strains. Conclusions: Our data provide evidence that EcO145 and EcO157 evolved from a common lineage, but ultimately each serotype evolves via a lineage-independent nature to EHEC by acquisition of the core set of EHEC virulence factors, including the genes encoding Shiga toxin and the large virulence plasmid. The large variation between the two EcO145 genomes suggests a distinctive evolutionary path between the two outbreak strains. The distinct methylome between the two EcO145 strains is likely due to the presence of a Bsu BI/Pst I methyltransferase gene cassette in the Stx2a prophage of the strain RM13514, suggesting a role of horizontal gene transfer-mediated epigenetic alteration in the evolution of individual EHEC strains.

Airborne dust from feedlots is a potential mechanism of contamination of nearby vegetable crops with Escherichia coli O157:H7 (EcO157). We compared the fitness of clinical and environmental strains of EcO157 in <45 µm soil from a spinach farm. Differences in survival were observed among the 35 strains with D-values (days for 90% decreases) ranging from 1–12 days. Strains that survived longer, generally, were from environmental sources and lacked expression of curli, a protein associated with attachment and virulence. Furthermore, the proportion of curli-positive (C⁺) variants of EcO157 strains decreased with repeated soil exposure and the strains that were curli-negative (C⁻) remained C⁻ post-soil exposure. Soil exposure altered expression of stress-response genes linked to fitness of EcO157, but significant clonal variation in expression was measured. Mutations were detected in the stress-related sigma factor, rpoS, with a greater percentage occurring in parental strains of clinical origin prior to soil exposure. We speculate that these mutations in rpoS may confer a differential expression of genes, associated with mechanisms of survival and/or virulence, and thus may influence the fitness of EcO157.