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

Abstract

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.

Comanaging fresh produce for nature conservation and food safety

“…Although established methods for detecting EHEC and Salmonella are readily available (11,12,21), the specific methods used by the third-party company were not disclosed. Generally, however, the company's pathogen testing procedure consists of DNA preparation, amplification, and detection.…”

“…Both pathogens are carried by domestic animals (e.g., cattle) and wildlife; however, whereas S. enterica is readily isolated from many wildlife hosts (10,11), EHEC is generally more prevalent in cattle than in wildlife. In one study, for example, 37.9% of cattle vs. only 7.4% of wildlife samples obtained from California's Central Coast tested positive for EHEC (12). Prevalence can vary by location, however; for example, ∼12% of cattle shed the EHEC strain E. coli O157:H7 in the midwestern United States (13), whereas only 2.6-7.1% did so in California's Central Coast (12,14).…”

“…In one study, for example, 37.9% of cattle vs. only 7.4% of wildlife samples obtained from California's Central Coast tested positive for EHEC (12). Prevalence can vary by location, however; for example, ∼12% of cattle shed the EHEC strain E. coli O157:H7 in the midwestern United States (13), whereas only 2.6-7.1% did so in California's Central Coast (12,14). Similarly, whereas E. coli O157:H7 was detected in <2% of bird, deer/elk, and feral pig samples across 50 studies worldwide (15), detection rates were higher in the Central Coast region (22%, 3.4%, and 4.7%, respectively) (12).…”

Top-Down Proteomic Identification of Shiga Toxin 2 Subtypes from Shiga Toxin-Producing Escherichia coli by Matrix-Assisted Laser Desorption Ionization–Tandem Time of Flight Mass Spectrometry

“…Table 1 summarizes the reference and environmental E. coli strains analyzed in this work (1,(42)(43)(44)(45)(46)(47). Stx2 induction and sample preparation have been described in detail previously (38,39).…”

Shiga Toxin 2 Subtypes of Enterohemorrhagic E. coli O157:H- E32511 Analyzed by RT-qPCR and Top-Down Proteomics Using MALDI-TOF-TOF-MS

“…E. coli O157:H-strain E32511 (RM7004) is a STEC that has two stx2 genes, one designated as a stx2a subtype and the other as a stx2c subtype [13][14][15][16][17]. E. coli strain RM7787 was isolated from a feral pig in Northern California, and has a single stx2 gene that is designated as a stx2c subtype [33].…”