A high-throughput, culture-independent approach to identify index and indicator species for E. coli O157:H7 contamination.

A high-throughput, culture-independent approach to identify index and indicator species for E. coli O157:H7 contamination.

Apr. 1, 2009 - Mar. 31, 2010

2009-39

$182,205.00

Gitta L. Coaker, Ph.D.
University of California, Davis

Johan Leveau, Ph.D., Trevor Suslow, Ph.D.

Final Report

Recurrent outbreaks of E. coli O157:H7 linked to lettuce and leafy greens produced in the California Central Coast region are of high concern to both the industry and public health regulators. The absence of recognized sporadic illness or outbreaks from products originating from winter-production regions in southern deserts has engendered broad speculation as to causes. One commonly held hypothesis is that although parallel risk factors for pathogen contamination exist in both regions, climatic conditions and other factors result in an exclusionary leaf surface microflora in the desert regions. In this proposal, we seek to identify bacterial species that have potential as indicators of contamination for E. coli O157:H7 on lettuce. Non-pathogenic microorganisms on the surface of fresh fruits and vegetables, including lettuce, can behave in a manner highly similar to those causing foodborne illness. Alternatively, some harmless bacteria are most abundant when conditions are not favorable for survival or growth of food borne pathogens. We will use a high-throughput DNA sequencing approach to identify bacterial species present on Romaine lettuce leaves grown in the Salinas, Imperial, and Yuma Districts. These indicator bacteria will be selected for positive or negative association with periods of E. coli O157:H7 detection or growth. The verification of both positive and negative risk indicators will result in optimized testing strategies and lower overall costs for monitoring of product for E. coli O157:H7. The development of novel rapid, DNA-based tests will help focus economic resources for pathogen testing on fields and lots of significant risk.

Technical Abstract

The contamination of lettuce and other leafy greens with enterohemorrhagic Escherichia coli (EHEC) O157:H7 has become a serious concern in the major regions of domestic production (California and Arizona) due to several recent outbreaks associated with consumption of both whole and minimally processed product. Between harvest and consumption, the abundance of E. coli O157:H7 on leaves may increase by as much as 1-2 orders of magnitude [1,2], while its infectious oral dose is 10-100 cells [3,4]. Hypothetically, this means that abundances as low as 1 cell on a serving size of pre-harvest lettuce leaves can already signify a potential health risk. While routine pre-harvest testing of lettuce and leafy greens for the presence of E. coli O157:H7 and related EHEC has greatly increased over the past two years, there is both the desire and increasing data-based justification to move to a more seasonally focused and predictive screening system. Based on post-contamination survival and growth potential under ideal conditions, it is untenable that early warning systems for E. coli O157:H7 outbreaks will be based on direct detection of EHEC. Instead, development of such systems must rely on other, indirect, indicators for the establishment of E. coli O157:H7 on plants in the field. An index organism acts as a benchmark for conditions that favor survival or growth of a
pathogen; an indicator is a benchmark for evidence of recent fecal contamination and shares key attributes of persistence and growth with the target pathogen [5]. The identification of both positive and negative index and indicator species for E. coli O157:H7 contamination will be a valuable resource to mitigate the downstream consequences of contamination by highlighting when conditions favor E. coli O157:H7 dispersal and growth on lettuce and other leafy greens. This information can be used to develop a practical, scientifically-based strategy for when to intensively sample fields or incoming raw product for enrichment-enhanced direct detection of E. coli O157:H7 and non-O157 EHEC. In this proposal, we seek to use a high-throughput, culture-independent approach to identify robust index and indicator species for E. coli O157:H7 contamination and survival on lettuce. High-throughput DNA sequencing technologies will be used to identify microbial communities associated with Romaine lettuce produced in the Salinas, Imperial, and Yuma districts across two growing seasons and over two production cycles. This approach will enable us to establish a baseline of microbial communities associated with lettuce for both immediate-term and long-term goals. This will be the first step towards taking a culture-independent approach to: (1) Identify which microbial species warrant additional investigation as potential index or indicator species. (2) Investigate how microbial communities and/or microbial ecology contribute to explaining the recurring pattern of outbreaks of E. coli O157:H7 on lettuce and leafy greens in the Central Coast region and the apparent absence of outbreak events from the Imperial and Yuma districts. Potential positive and negative indicator species will then be selected and analyzed for their utility in the field.