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

• DOI: 10.1371/journal.pone.0065716
• Publication Date: 2013-06-06T21:06:10Z
• Authors: Michael B. Cooley; Michele Jay-Russell; Edward R. Atwill; Diana Carychao; Kimberly Nguyen; Beatriz Quiñones; Ronak Patel; Samarpita Walker; Michelle Swimley; Edith Pierre-Jerome; Andrew G. Gordus; Robert E. Mandrell
• Keywords: 0301 basic medicine; 570; General Science & Technology; Swine; Science; Clinical Sciences; Wastewater; Escherichia coli O157; Microbiology; 630; California; Feces; 03 medical and health sciences; Vegetables; 2.2 Factors relating to the physical environment; Animals; Aetiology; Phylogeny; Soil Microbiology; 2. Zero hunger; Biomedical and Clinical Sciences; Shiga-Toxigenic Escherichia coli; 9. Industry and infrastructure; Drinking Water; Escherichia coli Proteins; Q; R; O Antigens; Biological Sciences; 6. Clean water; 3. Good health; Food Microbiology; Medicine; Cattle; Digestive Diseases; Infection; Multiplex Polymerase Chain Reaction; Biotechnology; Research Article; Multilocus Sequence Typing

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.