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United States Department of Agriculture

Agricultural Research Service

Research Project: Biology and Control of Human Pathogens on Fresh Produce

Location: Produce Safety and Microbiology Research

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

Authors
item Cooley, Michael
item Jay-Russell, Michele -
item Atwill, Edward -
item Carychao, Diana
item Nguyen, Kimberly
item Quiñones, Beatriz
item Patel, Ronak -
item Walker, Samarpita
item Swimley, Michelle -
item Pierre-Jerome, Edith -
item Gordus, Andrew -
item Mandrell, Robert -

Submitted to: PLoS One
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 16, 2013
Publication Date: June 6, 2013
Citation: Cooley, M.B., Jay-Russell, M., Atwill, E.R., Carychao, D.K., Nguyen, K.M., Quinones, B., Patel, R., Walker, S., Swimley, M., Pierre-Jerome, E., Gordus, A., Mandrell, R.E. 2013. Development of a robust methos for isolation of shiga toxin positive Escherichia coli (STEC) from fecal, plant, soil and water samples from leafy greens production region in California. PLoS One. 8(6):e65716. doi:10.1371/journal.pone.0065716.

Interpretive Summary: Contamination of produce with pathogenic bacteria occurs where it is grown, from a variety of sources in the surrounding environment, including livestock, wildlife and water. We developed a method to detect and culture bacteria which contain the genes necessary to produce shigatoxin. Production of shigatoxin is important for disease in humans. We used this method in a 2.5 year survey of ranches and farms in the Salinas region, which is responsible for growth of large amounts of produce for human consumption. We isolated 3256 different shigatoxin producing bacteria. Some of these bacteria are identical to other bacteria from the Salinas region and cultured from a variety of sources (cattle, wildlife, water, sediment and produce) and from a variety of locations (ranches, farms, waterways). This work demonstrates the movement of these bacteria in the environment. Such movement is a risk because it could lead to contamination of produce and human illness.

Technical Abstract: Pre-harvest contamination of produce occurs by the transport of pathogenic bacteria from point sources in the surrounding environment. To identify vertebrate sources, we surveyed livestock and wildlife over a 2.5 year period on 33 farms and ranches in the Salinas valley region of California for the incidence of Shiga Toxin-producing E. coli (STEC). We developed a culture method for potential recovery of any non-O157 STEC strain from multiple types of animal and environmental samples (produce, soil, water) for the purpose of robust source tracking. The method involves two parallel isolation schemes using both immunomagnetic separation (IMS) with anti-O157 beads and real-time multiplex PCR, as well as three different indicator media (O157 Rainbow agar, O157 Chromagar and sheep’s blood agar). The multiplex PCR was designed to amplify all know variants of shiga toxin (stx) genes. From more than 13,000 samples, we isolated 3256 non-O157 STECs, representing 1599 different non-O157 STEC strains, as defined by any difference in Multi-Locus Variable number tandem repeat Analysis (MLVA) and DNA sequence of the hypervariable loop region of outer membrane protein A (ompA). Recovery of non-O157 STECs was dependent on the indicator medium, source of the sample, month/season of collection, sample quality (delay between collection and culture) and the presence of IMS and PCR inhibitors. Of the 3,256 non-O157 STECs, 2,879 (88%) were isolated from cattle and wild animal feces; 377 non-O157 STECs were isolated from water, soil, sediment and produce. Stx genes, stx1, stx2 and stx1/stx2, were present in 63%, 74%, 35% of the strains, respectively. Subtilase, intimin and hemolysin genes were present in 28%, 25% and 79% of the non-O157 STECs, respectively, indicating potentially virulent non-O157 STECs. Phylogenetic analysis identified matching non-O157 STEC strains isolated from as many as 4 different sources (e.g. cattle, wildlife, soil and produce at multiple locations) and greater than 12 months apart. We conclude that livestock and wild animals are common sources of non-O157 STECs in this production environment, matching strains are present also in watersheds, especially subsequent to heavy precipitation events. Non-O157 STECs may be variably culturable by different methods and associated with varying virulence gene profiles and serotypes.

Last Modified: 10/25/2014
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