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The FDA conducted an assignment in 2021 to collect romaine lettuce samples from commercial coolers in Yuma County, Arizona to test for Shiga toxin-producing Escherichia coli (STEC), specifically enterohemorrhagic Escherichia coli (EHEC), and Salmonella spp. This assignment is part of the FDA’s ongoing surveillance of the commodity following multistate E. coli O157:H7 outbreaks of foodborne illness in recent years linked to or potentially linked to romaine lettuce and is a key prevention action identified by FDA in the Leafy Greens STEC Action Plan , .
The FDA began the assignment on February 8, 2021. Since 2009, the FDA and Centers for Disease Control and Prevention (CDC) identified 40 foodborne outbreaks of STEC infections in the U.S. with a confirmed or suspected link to leafy greens. Most recently, in 2018 and 2020 multistate outbreak investigations of E. coli O157:H7 infections were associated with or potentially associated with the consumption of romaine lettuce from the Yuma agricultural region. Contamination of romaine with Salmonella is also of concern as the U.S. experienced a multistate outbreak of Salmonella Newport infections linked to romaine lettuce consumption in 2013.
The agency’s goals in conducting this assignment were to determine whether the target pathogens and specific strains may be present in romaine lettuce from the Yuma agricultural region and to prevent contaminated romaine lettuce from entering commerce, when possible.
Yuma is referred to as the nation’s “winter salad bowl” because most of the leafy greens and other vegetables eaten by U.S. consumers in the winter and early spring, including romaine lettuce, are grown in the region. California’s Imperial Valley growing region also produces a large portion of the leafy greens eaten in the U.S. during the same period.
Romaine lettuce is grown low to the ground making it susceptible to contamination from irrigation water splashing off the soil, run-off from adjacent land, animal intrusion, and windblown dust, among other routes. In addition, this food is typically eaten without having undergone a ‘kill step,’ such as cooking, to reduce or eliminate bacteria.
The FDA assignment called for the collection and testing of 500 samples of romaine lettuce for STEC’s and Salmonella spp, with the testing performed by an independent contract laboratory. The agency collected and tested 504 samples for STECs and Salmonella spp. Each sample consisted of 10 subsamples, and each subsample was made up of at least 300 grams of romaine lettuce (whole heads, hearts or individual leaves). This approach – the collection and testing of samples composed of multiple subsamples – increases the probability of detecting pathogens if present, given that microbial hazards may not be uniformly present.
The FDA collected the samples from commercial cooler and cold storage facilities, where field heat is removed from harvested romaine lettuce and product is cold stored before shipment and processing. The samples were collected at cooling operations in Yuma County, AZ during the second half of the harvest season (February to March). The focus on commercial coolers for sample collection enabled the agency to collect samples from multiple farms at centralized locations, an efficiency previously employed by the FDA for a similar assignment in FY19.
Agency field staff collected all samples aseptically in accordance with the FDA’s Investigations Operation Manual (IOM) to prevent contamination. The FDA’s aseptic sampling methods, which entail the use of sterile implements and containers, and prescribed collection procedures, are published in IOM Chapter 4.3.
The FDA took additional precautions to ensure the safety of personnel and the integrity of its sample collection given the COVID-19 pandemic. Agency field staff received training to help ensure their safety and the safety of firm employees. Investigators preannounced their visits to the firms and were outfitted with personal protective equipment (PPE) consistent with local, state, and applicable CDC guidance.
Testing by an Independent Laboratory
The FDA engaged an independent laboratory located near the collection sites to eliminate the need to ship samples to FDA’s own laboratories, reducing the time between sample collection and results reporting, and facilitating swift action by the agency in the event that a sample were to yield a pathogen. The use of the independent laboratory was a pilot project by the FDA, the objective of which was to quickly notify firms of negatives and ‘cannot rule out’ (CRO) initial test results, generally within a benchmark of 24 hours of the laboratory’s receipt of the sample.
All 504 samples collected and tested in this assignment were negative for Salmonella spp. The FDA detected one STEC, an E. coli O130:H11. Further characterization indicated that this strain of STEC was found to be moderate to high risk and could be capable of causing severe illness in humans. Based on Whole Genome Sequencing (WGS) and a bioinformatics analysis, this particular strain of O130:H11 was not a genetic match to any known human illness.
Upon identifying the E. coli O130:H11, the agency notified the management of the cooling operation where the positive sample had been collected. Most of the romaine lettuce associated with the positive had been voluntarily held at the cooling operation. A number of pallets of the lettuce were released but were called back, and the product never reached consumers. Notified of the finding, the owner of the farm that grew the lettuce chose not to harvest the crop from the field where it was grown.
The FDA opened an investigation at the farm in question to identify possible sources and routes of contamination. At the time of the investigation, romaine lettuce remained in the field, which allowed the agency to collect romaine from various locations within the field. The FDA also collected multiple samples of soil, water, sediment, and animal fecal material, and assessed surface contamination using drag swabs.,  The investigation occurred during the last two weeks of March. Only one sample collected during the investigation, which came from the outer leaves of romaine lettuce, yielded STEC (specifically, E. coli O116:H-). The strain was further characterized as low risk to human health and was not associated with any clinical illnesses upon WGS analysis. This type of follow-up action allows the agency to build our understanding of how contamination events might occur.
The findings of this assignment may suggest that STEC and Salmonella spp. were not widespread in romaine lettuce grown in the Yuma agricultural region during this time period, even though such contamination continues to pose a public health concern given the multistate outbreaks of foodborne illness that have been linked to or potentially linked to romaine lettuce in recent years. Importantly, even a low level of either pathogen can result in an outbreak of foodborne illness. As stated in the “Findings” section of this report, the FDA detected STEC in one sample out of the 504 collected and tested with one additional STEC found during the follow up investigation.
Ensuring the microbiological safety of leafy greens continues to be a priority of the FDA. Romaine lettuce and other leafy greens are among the most widely consumed vegetables in the U.S., and are an important part of a healthy diet. The agency is working on several fronts to help prevent microbial contamination of leafy greens and to forestall outbreaks of foodborne illness. Chief among these efforts is the FDA’s Leafy Greens STEC Action Plan, which features public health and regulatory approaches related to response, prevention and the addressing of knowledge gaps.
As part of the action plan, the FDA has launched two multi-year research studies that seek to shed light on the cause(s) of foodborne illness outbreaks linked to romaine lettuce produced in California and Arizona. The studies are designed to increase understanding of how human pathogens survive, move and possibly contaminate produce prior to harvest. Research teams collect and examine environmental samples, including from adjacent land, well and surface waters, and soil inputs that may contain compost, dust and animal fecal material. Research teams also collect scat samples to assess the impact that animal intrusion and native wildlife may have on the growing environment. The studies are being conducted in partnership with state and local governments and universities. We anticipate that study results will lead to improved practices to prevent or mitigate food safety risks, and ultimately enhance the safety of produce grown in the region.
Consumers can reduce the risk of foodborne illness by thoroughly washing romaine lettuce and other leafy greens under running water before eating them. Washing leafy greens is not a lethality step (i.e., it is not guaranteed to eliminate pathogens) but may well remove some pathogens, if present. Additional information on leafy greens and food safety is available at the website of the U.S. Centers for Disease Control and Prevention.
 The drag swab method that FDA uses in leafy greens investigations is an adaptation of the one used for poultry operations.