Contribution of phyllosphere microbiota to the persistence of Escherichia coli O157:H7 ATCC 700728 on field-grown lettuce.

Contribution of phyllosphere microbiota to the persistence of Escherichia coli O157:H7 ATCC 700728 on field-grown lettuce.

Apr. 1, 2009 - Mar. 31, 2010

2009-34

$50,800.00

Maria Marco, Ph.D
University of California, Davis

Linda J. Harris, Ph.D.

Final Report

Plant surfaces (also known as the phyllosphere) are colonized by indigenous communities of microorganisms (here termed microbiota). The composition of the phyllosphere microbiota can differ depending on environmental factors including the time of year and moisture levels. Harris et ai (unpublished) found in previous California Lettuce Research Board (CLRB)-funded projects that these environmental factors might also influence the persistence of E. coli O157:H7 on Romaine lettuce plants in the field. However, a current gap in our knowledge is whether the indigenous phyllosphere microbiota also contributes to E. coli O157:H7 persistence. We propose to identify the bacterial communities present in the phyllosphere of overhead and drip irrigated Romaine lettuce prior to and following inoculation with attenuated E. coli O157:H7. Bacterial diversity will be assessed using culture-independent methods with high-density 16S rRNA phylogenetic DNA microarrays. The culturable fraction of bacteria will also be quantified on laboratory culture media. Bacterial plant isolates with the ability to inhibit the growth of E. coli O157:H7 will be identified. The project will provide much needed information aimed at reducing the risk of E. coli O157:H7 survival on leafy greens.

Technical Abstract

The communities of microorganisms residing in the lettuce phyllosphere are currently unexplored for their ability to control levels of E. coli O157:H7. In this project, we will determine if the diversity and abundance of the Romaine phyllosphere microbiota contributes to the persistence of attenuated E. coli O157:H7 ATCC 700728. The phyllosphere microbiota will be analyzed by culture-dependent and -independent methods for Romaine lettuce plants grown under different irrigation regimes (overhead and drip) within a single split-plot design field trial. This trial is described in a connected
CFP/CLRB proposal (Harris et al., current RFP) to compare the effects of irrigation on attenuated E. coli O157:H7 ATCC 700728 survival on Romaine lettuce planted in the spring and summer in the Salinas Valley. Previous field trials by Harris et al. in 2007 and 2008 have shown that population sizes of this organism rapidly decline within the first hours and days after inoculation, but low amounts of viable cells can persist until the time of harvest. E. coli O157:H7 ATCC 700728 survival differed between plants that were drip and overhead irrigated in the same field during the early summer season of 2008; such that overhead irrigation resulted in significantly higher amounts of plants with detectable, persistent levels of E. coli O157:H7 ATCC 700728. In collaboration with Harris et al., ten lettuce plants will be harvested from both overhead and drip irrigated plots immediately prior to the inoculation of attenuated E. coli O157:H7 ATCC 700728 (t= 0) and then after 2 and 7 days. Amounts of culturable bacteria will be determined by colony counting on standard bacteriological media. For a selection of 50 plant isolates, inhibition screening will be performed to identify any strains that are able to reduce the growth of E. coli O157:H7 and therefore interfere with the persistence of this pathogen on lettuce in the field and during post-harvest procedures. Because it is expected that only a fraction of the viable bacterial cells present on the plants are able to form colonies on laboratory culture media, a culture-independent bacterial profiling technique with high-density
16S rRNA microarrays will also be applied. This approach is needed to quantify the total bacterial diversity present on lettuce which might influence E. coli O157:H7 survival. Bacterial community diversity, abundance, and the presence of specific (potentially antagonistic) bacterial isolates from individual plants exposed to different irrigation treatments (overhead and drip) will be compared to the persistence of E. coli O157:H7 ATCC 700728 on the plants. This project will provide much needed information on whether there is a link between the phyllosphere microbiota and the long term
survival of E. coli O157:H7 on lettuce exposed to different irrigation regimes in the field. These data will be useful for risk assessments of E. coli O157:H7 survival on field grown lettuce and modifying agricultural practices to avoid future outbreaks of leafy-green associated foodborne illness