Summary of Awards to Date

Quantifying risk associated with changes in EHEC physiology during post-harvest pre-processing stages of leafy green production

Date

Jan. 1, 2022 - Dec. 31, 2023

Amount Awarded

$328,442.00

Investigator

Teresa Bergholz, Ph.D.
Michigan State University

Co-Investigator(s)

Jade Mitchell, Ph.D.

Summary

The goal of this project is to determine if the time between harvest and end use of romaine lettuce impacts E. coli O157:H7 pathogenicity and detectability resulting in increased health risk. Laboratory scale experiments with inoculated lettuce undergoing simulated harvest and cooling will be used to measure changes in E. coli O157:H7 stress tolerance and virulence. Input from industry partners including temperature data from commercial romaine harvesting and cooling, and details on supply chain logistics, will be combined with the laboratory scale experimental data and used to model risk associated with specific harvest and handling practices. The resulting quantitative tool will be publicly available and allow for growers and producers to determine any practices that should be implemented to reduce the potential for O157 transmission on romaine lettuce.

Technical Abstract

If leafy greens are contaminated in the field, human pathogens such as E. coli O157:H7 are capable of surviving throughout the distribution chain. E. coli O157:H7 survival on lettuce in the field and during post-harvest washing has been quantified, but not during harvest and transport prior to processing. Leafy greens transported from the West coast to the East coast may be held for days at refrigeration temperature during transport prior to processing, likely impacting pathogen physiology. Physiological changes such as entering a dormant state can protect cells from environmental stress and decrease detectability. Recent studies have demonstrated that E. coli O157:H7 cells may enter a persister state, a type of dormancy characterized by resistance environmental stresses. Entering the viable but non culturable (VBNC) state impedes detection of E. coli O157:H7 by standard molecular detection methods, though cells remain virulent. While E. coli O157:H7 can enter the persister or VBNC state on lettuce, the extent to which this occurs during harvest, cooling, and refrigerated transport is unknown. Changes in the physiological state of E. coli O157:H7 have the potential to impact risk of illness associated with contaminated lettuce due to alterations in tolerance to sanitizers in wash water, virulence properties, and detectability. This study aims to 1) characterize physiological changes in E. coli O157:H7 cells on romaine lettuce during post-harvest pre-processing handling and refrigerated storage, 2) determine if changes in physiology impact detectability of E. coli O157:H7 on romaine lettuce, and 3) evaluate how changes in E. coli O157:H7 physiology, virulence, and stress tolerance impact potential health risks through a quantitative microbial risk assessment (QMRA). Romaine lettuce plants will be inoculated with E. coli O157:H7 in the laboratory and undergo simulated harvest and refrigerated storage. The proportion of E. coli O157:H7 cells that are culturable, persisters, or VBNC will be quantified before and after harvest and every day for 5 days during refrigerated storage. Changes in acid and chlorine tolerance will be assessed at each sampling point. Changes in virulence potential will be determined by measuring Shiga toxin gene expression and pathogenicity of E. coli O157:H7 in Galleria mellonella. Correlations between proportion of cells in the persister or VBNC state and changes in detectability with a standard molecular detection method will be determined. A QMRA model will be developed to evaluate the risk of illness after consumption of contaminated lettuce using the expected prevalence and concentration of E. coli O157:H7 across the farm-to-fork pathway including processing, production and supply chain stages. Experimental data will be used to determine changes in risks corresponding to the physiological changes in E. coli O157:H7 as the result of in-field cut-to-cool temperatures and durations. The models will also be populated with existing data from peer-reviewed literature on survival and decay rates of E. coli O157:H7 on romaine lettuce, transfer rates and other data sets required to parameterize the conditions across the supply chain. A tool will be developed to identify practices that are associated with reduced risk of E. coli O157:H7 illnesses transmitted via romaine lettuce.