Assessing Romaine lettuce “Forward Processing” for potential impacts on EHEC growth, antimicrobial susceptibility, and infectivity

Consumption of Romaine lettuce has been linked to multiple foodborne illness outbreaks due to contamination of pathogenic Escherichia coli strains. Recently, these outbreaks have occurred in the United States every year since 2016, causing great damage to consumer health and economic wellbeing of the fresh produce industry. The pathogen strains isolated from these outbreaks showed great similarity by genomic analyses. These outbreaks showed a pattern of heavy concentration especially in northeastern USA, prompting questions from the leafy green industry that the practice of “forward processing” could be linked to the outbreaks. “Forward processing” is a practice that the raw lettuce commodity is transported in trucks to facilities far away from the production area for washing and packaging, and regional marketing. We propose to work closely with the leafy green industry to comprehensively assess the forward processing for its effects on the integrity and safety of the raw commodity and the packaged products. In addition, the forward processing conditions will be simulated in the laboratory with the pathogenic E. coli strains. Emphases will be on how these conditions would affect the physiology of the pathogenic strains as well as other microorganisms on the raw commodity and packaged products. The findings in the research could provide important information that can be used by the leafy green industry for improving the forward processing practice and reducing the risks of fresh produce such as Romaine lettuce.

Fresh-cut Romaine lettuce has been implicated in multiple outbreaks of enterohemorrhagic Escherichia coli (EHEC) O157:H7, including recently in 2016, 2017, 2018, 2019, and 2020, with nearly all implicated products traced back to ranches in the Salinas Valley in California. Whole genome sequencing showed that the EHEC strains in several of the outbreaks in different years constituted a cluster which was closely related to strains isolated from cattle fecal samples in the same area. After harvest, Romaine lettuce is processed at facilities in the production region (source processing), or transported in refrigerated trucks to facilities thousands of miles away for processing (forward processing). Although some of the recent EHEC outbreaks associated with Romaine lettuce affected multiple states across the country, the outbreaks often showed clusters of cases. Associative evidences from FDA traceback investigations for 2018 and 2019 outbreaks implicated regionally marketed freshcut products from forward processing plants. Research outlined in this proposal is aimed at addressing CPS 2021 Research Priority #2 Harvest, b. Pathogen risk increase during pre-processing time delay. In Objective 1 we propose to comprehensively assess forward processing for potential impacts on raw material integrity, and on microbial activity. Objective 2 will focus on microbiome dynamics on raw commodity over the entire process from harvest to fresh-cut, and on commercial fresh-cut product during the distribution and retail display. In Objective 3, we will conduct comparative studies to better define the underlining fitness of the outbreak EHEC strains, and to better understand why these closely related outbreak strains reemerge every year. We will use the outbreak EHEC strains to conduct laboratory studies to simulate forward and source processing, and to examine the impacts of the practices on the growth and physiology of the outbreak EHEC strains. Although the causative effect of temperature volatility and E. coli O157:H7 growth is an important aspect to examine for forward processing, it will not be the focus of this proposal. The goal is to assess the entire process, and to understand the effects of different processing practices on the produce microbiome dynamics, and on the physiology of EHEC, as represented by the dominant outbreak strains. The proposed research needs close collaboration with commercial leafy green processors. The results will be used for further improvement of these practices.

1. Comprehensive assessment of forward processing practice under routine operation conditions for product integrity and microbiological quality. 

2. Assessment of microbiome dynamics on Romaine lettuce from harvest to retail for products being forward and source processed. 

3. Comparative assessment of E. coli O157:H7 outbreak strains and laboratory strains, and the impacts of different practices on EHEC outbreak strains, on cell physiology that may affect their growth potential, susceptibility to antimicrobial treatment, or virulence. 

4. Improvement of forward processing management by applying findings from comprehensive assessment and simulation.

Forward processing of romaine lettuce or other leafy greens is an effective strategy for ensuring freshness and access to fresh-cut products for consumers far away from the commercial leafy green growing regions. This project demonstrated that the practice of forward processing, with its significant pre-processing delays due to long distance transportation and inventory control logistics, permitted significant microbial proliferation even without recorded evidence of temperature abuse, and negatively impacted product post-processing shelf life. In laboratory simulation of romaine lettuce source and forward processing transportation and inventory holding events with EHEC outbreaks strains, no evidence was obtained to support significantly increased risk of EHEC growth or EHEC cell physiology changes leading to decreased antimicrobial susceptibility or increased expression of virulence related genes under conditions of forward processing, except slightly high rates of EHEC persister and theoretical VBNC cell induction were observed after exposure to such conditions. Upregulation of EHEC detoxification genes, efflux pumps, and antimicrobial resistance genes were detected in both source and forward processed lettuce samples, compared to EHEC not exposed to lettuce surface. The prevalent EHEC strains in recent outbreaks also showed stronger potential for biofilm formation, indicating increased fitness for survival in fresh produce production environments. Based on the above summary findings, the following are proposed for the fresh produce industry and research community in considering their current and future operational recommendations: 

• The “pre-processing delay” (as exemplified by romaine lettuce forward processing) is unlikely a significant cause for “pathogen risk increase”, in the absence of temperature abuse. 

• The pre-processing time delay such as that in forward processing can be reflected as produce quality loss. 

• Foodborne pathogen environmental fitness and the underlying genomic and transcriptomic mechanisms, along with development of persister cells and the VBNC state detected under processing conditions require further research. 

• The finding that significant bacterial proliferation is not reflected in EHEC growth under forward processing conditions suggests a significant role of the lettuce microbiome in balancing the growth or survival of foodborne pathogens, and the need for deeper microbiome analyses to better understand the microbial dynamics.