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This project seeks to understand the behavior of Escherichia coli O157:H7 on lettuce and to identify the biotic and abiotic factors that influence the persistence of this pathogen on these plants. E. coli serotype O157:H7 is a significant foodborne pathogen, owing to its low infectious dose, severity of infection, and high association with leafy green outbreaks. Preharvest contamination of the plants in the field is considered the main point of entry of the pathogen. Although E. coli O157:H7 is not considered to be a good colonist of plants, it is able to survive and, under some conditions, grow on lettuce leaf surfaces. The phyllosphere or aerial-portion of plants, is generally regarded as a harsh environment for microorganisms containing both abiotic and biotic (microbial) stresses. <P>
The objectives driving this project are to determine the effects of abiotic environmental conditions, specifically moisture and temperature, and composition of the phyllosphere microbiota (biotic conditions) on the survival of virulent and avirulent (attenuated) E. coli O157:H7 strains on Romaine lettuce plants. This project integrates results from field-trials with an attenuated E. coli O157:H7 strain with controlled growth-chamber studies using virulent isolates of the pathogen. We are currently collecting data that describe the fitness characteristics of attenuated E. coli O157:H7 applied as a surrogate to evaluate pathogen persistence in field trials. In the current study we will validate these data by comparing the growth and survival of virulent and attenuated E. coli O157:H7 isolates in the growth chamber on lettuce exposed to environmental regimes that mimic those found in the field. Quantification of E. coli and indigenous bacterial cell amounts will be measured by culture-dependent and independent methods including high-throughput DNA profiling analyses which will provide more complete data on cell viability, diversity, and abundance. <P>Identification of the factors influencing the survival of E. coli O157:H7 on lettuce is expected to inform development of agricultural practices that may reduce outbreaks associated with these economically valuable and otherwise healthy crops.
NON-TECHNICAL SUMMARY: Fresh fruits and vegetables have been increasingly implicated in outbreaks of foodborne illness, and in particular, outbreaks of enterohemorrhagic Escherichia coli O157:H7 have been associated with consumption of leafy green vegetables. Lettuce is the main item within this group responsible for produce-associated outbreaks and pathogen contamination of the lettuce plants likely occurred in the field prior to harvest. This project will investigate biotic (microbial) and abiotic factors which might affect persistence levels of E. coli O157:H7 on field-grown lettuce. Specifically, the indigenous microorganisms (collectively termed microbiota) residing on the leaf surfaces, or phyllosphere, of lettuce plants will be identified using high-throughput culture-independent approaches. Field-grown lettuce with and without prior exposure to an attenuated (non-pathogenic) E. coli O157:H7 strain will be examined to identify members of the indigenous microbiota positively or negatively correlated with the persistence of this strain in the field. Environmental conditions, and in particular extremes and fluctuations in moisture and temperature on plants, will also be investigated in growth chamber experiments designed to measure the contributions of these factors to E. coli persistence. This project will advance knowledge of the factors affecting E. coli O157:H7 survival on lettuce which can be applied to the development of improved control measures aimed at mitigating the risk of the organism surviving after a contamination event.
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APPROACH: Abiotic and biotic (microbial) factors affecting Escherichia coli O157:H7 persistence on lettuce plants will be determined in the growth chamber experiments and field studies during different seasons and irrigation regimes. Potential effects of the indigenous phyllosphere (leaf-surface associated) microbiota in supporting or diminishing E. coli O157:H7 survival will be determined after application of an attenuated E. coli O157:H7 strain onto research plots of Romaine lettuce in the Salinas Valley, California, a major lettuce growing region of the United States. Two field trials (early and late summer) from the 2009 season employing two irrigation regimes (sprinkler and drip) will inform field trials set for the same location and seasons in 2010. The phyllosphere will be assessed on inoculated and control plants over a period of 4 weeks using high-throughput 16S rRNA culture-independent approaches to capture the microbial diversity and abundance in the phyllosphere and to determine whether species and/or communities of bacteria on lettuce are directly and/or indirectly correlated to E. coli O157:H7 persistence on the same plants. Results from these experiments will be applied to design controlled growth-chamber experiments with lettuce plants designed to take into account the controlled moisture levels and temperatures present in the field. Such controlled laboratory studies afford comparisons between virulent and avirulent strains of E. coli O157:H7 and specific environmental conditions which modulate pathogen survival. Both flow-cytometry and real-time RT-PCR will be applied to quantify E. coli O157:H7 viability on lettuce plants. Flow cytometry offers the potential to rapidly and simultaneously perform several measurements on individual cells contained in complex systems; whereas real-time (RT) PCR procedures offer sensitive and specific detection of metabolically active bacteria targeting either genomic DNA or gene transcripts. These methods will be evaluated for plants in the growth chamber and then selectively applied to study E. coli O157:H7 on Romaine lettuce in field-trials.