Summary
Though exceptionally rare events, relative to the scale of production and consumption, there is ample evidence to know that produce samples sometimes contain pathogens of serious potential human health consequences. A group of bacterial pathogens, Shiga toxin‐producing E. coli (STEC) from diverse fresh produce were recovered from multi‐year sampling programs conducted by the UDSA, largely at wholesale distribution centers. Leafy greens, herbs, and specifically spinach were singled out for concern due to a STEC prevalence rate exceeding 50% of the total isolates recovered. Product testing is used by many but not all producers to pre‐screen leafy greens for bacterial pathogens, including STEC. Unfortunately, not all testing platforms rapidly distinguish STEC likely to cause human illness from those that lack the genetic traits necessary for infection. Due to the high perishability of these commodities, testing can lead to destruction of a field due to false association with dangerous STEC. The combined objectives of protecting consumers, reducing food waste, and improving sustainability can be enhanced by applying new advancements proposed in this research in specific detection of clinically relevant Shiga toxin‐ producing E. coli to risk management decisions and better defining the role of wildlife as vectors of preharvest contamination.
Technical Abstract
Though exceptionally rare events, relative to the scale of production and consumption, there is ample evidence to know that produce samples sometimes contain pathogens of serious potential human health consequences. After the E. coli O157:H7 outbreak on spinach in 2006, the programmatic testing of leafy greens at preharvest for raw commodity and fields intended for processing, incoming raw material, and/or finished packaged product greatly increased. A group of bacterial pathogens, Shiga toxin‐producing E. coli (STEC) from diverse fresh produce were recovered from multi‐year sampling programs conducted by the USDA, largely at wholesale distribution centers. Leafy greens, herbs, and specifically spinach were singled out for concern due to a STEC prevalence rate exceeding 50% of the total isolates recovered. Product testing is used by many but not all producers to pre‐screen leafy greens for bacterial pathogens, including STEC. Unfortunately, not all testing platforms rapidly distinguish STEC likely to cause human illness from those that lack the genetic traits necessary for infection. There has been a rapid expansion of platforms, kits, pathogen targets, and diversity of approaches to lot acceptance criteria. There has been a gradual but accelerating shift in product testing criteria and policies for the group of pathogens that includes EHEC and STEC. Some commercial kit test systems have been recently used that screen for the top‐seven EHEC (O157, O26, O45, O103, O111, O121, O145) based on the premise that these sub‐types are responsible for over 85% of clinical cases. However, due to the increasing recognition of diverse STEC in clinical cases, many commercial service labs have more recently been using detection and lot acceptance systems that employ the least diagnostic genetic markers for this group, presence of eae (intimin; attaching and effacing) and stx (either of two key forms of shigatoxin) in an enrichment culture. Presence of these two markers, alone or being contributed individually by independent cell lines, has resulted in frequent crop destruction involving many acres and substantial economic loss, at the individual grower level. Due to the high perishability of these commodities, testing can lead to destruction of a field due to false association with dangerous STEC. The combined objectives of protecting consumers, reducing food waste, and improving sustainability can be enhanced by applying new advancements proposed in this research in specific detection of clinically relevant Shiga toxin‐ producing E. coli to risk management decisions and better defining the role of wildlife as vectors of preharvest contamination. The anticipated outcome of this proposal is the development of a set of recommendations relative to rapid virulence profiling and its application to routine compliance and lot acceptance testing for fresh produce. This project is a starting point for a longer‐term effort to consolidate and clarify the available information on risk associated with the diverse STEC group and present this information in a guidance format that can help form industry‐based standards of practice. The outcomes would lead to both immediate transferable actions in pathogen testing protocols and serve as a first step towards fulfilling our collective responsibility to stewardship of the ag‐environment and associated regional landscape.
Research Objectives
1. Test retained E. coli O157:H7 negative leafy greens samples from CPS Project 2011‐136 for evidence of clinically significant STEC.
2. Test E. coli O157:H7 negative but STEC positive enrichments from commercial preharvest, raw material, and packaged leafy greens and culinary herb products for evidence of clinically significant STEC.
3. Characterize cultures identified as positive and negative by the ROKA Atlas EHEC screen for clinically relevant virulence markers by diagnostic multiplex PCR.
4. Characterize the clinical significance of EHEC/STEC isolates from nuisance bird populations exhibiting flocking and foraging behavior in leafy green vegetable fields. We will culture EHEC/STEC strains from bird cloacal samples following live‐capture and release at two enrolled farms in proximity to confined and range beef cattle operations.
Findings & Recommendations
Over the course of the project period, more than 600 samples from diverse agricultural production and farmscape environments were collected and analyzed by several test methods for the presence of STEC contamination. In addition, 120 retained samples from pre-project commercial and research lab isolations were screened. All confirmed E. coli were further genetically characterized with 12 virulence-associated genetic markers. The activities required multiple trips to commercial production farms for collection of crop samples, irrigation water, and soil samples. During the project period, 177 purified samples from produce/environment, and 120 retained enrichments were tested for STEC single marker (Atlas® System, Roka Biosciences), STEC marker profiles (stx1/2 and eae) by real-time PCR, and cultural assessment. In addition, 164 retained isolates were examined for STEC single marker and three-target STEC marker profiles; also, 25 strains recovered from commercial labs from positive enrichments were characterized by PCR. Of the 400 purified STEC colonies recovered from all environmental project sources, 32 variants of the combined twelve identity-confirming or virulence-associated genetic markers were observed (Marker Profile Groups). Of these, the Roka system positively identified 97% of those that met U.S. FDA criteria as STEC of presumptive clinical significance for human illness. Early in the project performance period, only 25 presumptive positive enrichment samples were obtained from commercial testing labs. Of these, eight Marker Profile Groups were observed, including those aligning with environmental isolates, as above. Twelve samples yielded colonies confirmed as presumptive clinical STEC and were positive by Roka, while 13 samples were determined to be false positive in the original platform detection screening, based on our 12 Marker Profile patterns and a negative reaction by Roka. In a separate, related objective to generate additional isolates for genetic characterization, birds were captured and sampled at two study sites in 2015 and 2016. One of the study sites is a commercial produce ranch in San Benito County, on the Central Coast foothills. The other site is a National Wildlife Refuge in Yolo County on the delta of the California Central Valley, where land use is predominantly agriculture. In both sites, mist nets and traps were placed in proximity to a water body (reservoir or river). Of the 1,369 birdassociated samples, including 54 bird species from the study areas, 0.7% were positive for a presumptive clinically significant STEC. In addition, 12.5% of composited samples of Canada goose fecal mass were positive for a presumptive clinically significant STEC. Since the end of the first performance period in 2015, commercial producers in CA alone have tested tens of thousands of samples of diverse leafy greens, culinary herbs, and other fresh and fresh-cut produce. The concerns for economic losses due to false-positive reports have essentially ceased to be raised to our extension program or at industry meetings. The research team holds this to be the major success of the project. In addition, the project outcomes further supported prior CPS research that showed, while birds can be a source of clinically significant STEC contamination, the prevalence is very low. Current data do not support implementing environmentally damaging measures or immediate crop destruct or broad-scale buffering in response to minor bird presence or intrusion to leafy greens fields. Science-based Best Practices regarding bird intrusion is a major successful outcome. In summary, without comment to the need or advisability of pathogen testing of fresh produce in food safety programs, the team achieved its performance goal to expand and confirm the scientific basis for a rapid twostep detection protocol for routine compliance and lot acceptance testing of the presence or absence of clinically significant EHEC/STEC for fresh produce. Major service labs that are utilized by the fresh and freshcut industries rapidly adopted the study outcomes, in collaboration with and complimentary studies conducted by Roka Biosciences, early in the project performance period. The application of this two-step protocol remains in use today, and the original issue of potential false-positive test results has been managed to a minimal occurrence across the industry (personal communication with several large handlers).