Bacterial and plant factors that influence adhesion of enterohaemorrhagic E. coli and Salmonella enterica to salad leaves

Lead Research Organisation: Harper Adams University
Department Name: Crops and Environment Sciences

Abstract

Until now, research into transmission of food-borne pathogens has largely focused on farm animal reservoirs. Although transmission of enteric pathogens via salad leaves is on the rise, only a few studies have addressed the molecular mechanisms used by human enteric bacterial pathogens to attach to plants. Likewise there have been few studies investigating variation between plant genotypes on bacterial adhesion and/or survival and those that have not addressed the genetic basis of any differences. In this project we aim to investigate how human bacterial pathogens attach to salad leaves (in particular lettuce). We will determine the effect of bacterial growth condition on the ability of the organism to attach to leaves. Similarly, we will determine the effect of plant growth conditions on susceptibility to bacterial attachment. Using genetic approaches, we will determine which bacterial genes are involved in leaf attachment and we will utilise a lettuce mapping population of recombinant inbred lines to initiate studies aimed at gaining an understanding of the genetic basis of a plant's susceptibility to bacterial adhesion. We will also determine survival and replication rates of Salmonella and E. coli on the different lettuce lines. The ultimate goal of our studies is to provide the underpinning knowledge of the interaction between the plant and enteric bacteria in the phyllosphere to allow the development of strategies to reduce the risk of crop contamination and to increase food safety.

Technical Summary

Traditionally, research into food borne pathogens in the UK has focused on zoonosis. However, recent surveys have shown that while there is a reduction in transmission of non-typhoidal serovars of Salmonella enterica via contaminated poultry products, there is a steep rise in transmission via salad leaves, particularly, but not exclusively, lettuce. Similarly, although Infections with enterohaemorrhagic E. coli (EHEC) are usually linked to consumption of contaminated beef products, sever outbreaks in Sakai city in 1996 and the US in 2006 were traced to consumption of contaminated radish sprouts and pre-packed lettuce, respectively. Only a few studies have addressed the molecular mechanisms used by human enteric bacterial pathogens to attach to plants. Likewise there have been few studies investigating variation between plant genotypes on bacterial adhesion and survival and those that have been carried out do not address the genetic basis of any differences. In this project we will determine the mechanisms used by S. enterica & EHEC (O157 and non-O157) to attach to salad leaves. We will determine the effect of bacterial growth conditions on leaf attachment potential. We will use immunofluoresence microscopy, scanning electron microscopy and viable counts to determine the level and pattern of adhesion and to visualize potential adhesion factors. We will determine survival and replication rates in planta. Combining with site directed mutagenesis, this will reveal common and species-specific leaf adhesion and survival strategies. We will also investigate host factors that contribute to leaf susceptibility to colonisation. We will determine the effect of plant growth conditions and age on colonisation by S. enterica & EHEC. Using genetic and genomic approaches we will initiate studies to determine the genetic basis of lettuce to susceptibility to colonisation. These studies will underpin development of new risk assessments and improvements in food safety.

Related Projects

Project Reference Relationship Related To Start End Award Value
BB/G014175/1 01/04/2009 30/11/2010 £416,422
BB/G014175/2 Transfer BB/G014175/1 01/12/2010 31/03/2012 £197,623
 
Description There were 3 significant discoveries arising from this grant as follows:
1. The identification of plant traits which affect pathogen attachment, especially specific compounds such as phenolics which are involved in plant defence responses.
2. The significant levels of internal (endophytic) colonisation of lettuce leaves by pathogens which has implications for post-harvest processing in the salads industry.
3. The discovery of bacteria in the natural leaf microbial community which are antagonistic to human pathogens raises the possibility of natural control methods.
Exploitation Route Our discovery that specific physical and biochemical components of lettuce leaves affect pathogen attachment and the fact that there is a genetic component to this could be taken forward by further research to further define the underlying genetics and understand the mechanism(s) involved. The information could be used in plant breeding to develop cultivars which are less susceptible to colonisation by human pathogens.
The significant degree of internal colonisation of leaves by pathogenic bacteria should be of interest to the salad processing and retail industry as it could reduce the effectiveness of current washing protocols for bagged salads. Washing and monitoring procedures should be reviewed and modified if neccessary.
We discovered that some bacteria found in the leaf microbial community were able to inhibit or prevent the growth of human pathogens in vitro. Further research would be needed to confirm this and to determine the nature of the interaction, but it raises the possibility of natural control methods to reduce or prevent contamination of leaves.
Sectors Agriculture, Food and Drink,Education,Healthcare,Retail

 
Description DACP member of BBC Rural Affairs Connittee
Geographic Reach Multiple continents/international 
Policy Influence Type Membership of a guidance committee
 
Description DACP member of Food Ethics Council
Geographic Reach National 
Policy Influence Type Membership of a guidance committee
 
Description DACP parliamentary discussion on global food crisis
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Title Bacterial 16S rRNA sequences 
Description A total of 215 equences from 16S rRNA genes of bacteria isolated from salad leaves have been submitted to GenBank (http://www.ncbi.nlm.nih.gov/ ) and are publically available under accession numbers JQ727437 - JQ727652 
Type Of Material Improvements to research infrastructure 
Year Produced 2012 
Provided To Others? Yes  
Impact Improved coverage of 16S rRNA database for better understanding of bacterial population community structure in the crop environment 
URL http://www.ncbi.nlm.nih.gov/
 
Title Lettuce phyllosphere bacterial culture collection 
Description A culture collection of bacteria (approx. 300 isolates) associated with epiphytic and endophytic colonisation of salad leaves was produced and is stored at Harper Adams University College. A number of these have been identified and further characterised by 16S rRNA sequence analysis, 215 sequences submitted to Genbank & publicly available 
Type Of Material Database/Collection of data 
Year Produced 2012 
Provided To Others? Yes  
Impact Clearer understanding of the microbial community structure on (and within) leaves. 
 
Description DACP BBC Countryfile interview 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact not known
Year(s) Of Engagement Activity 2009
 
Description DACP BBC Radio 4 interview 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact invitation to attend RHS conference on Horticulture careers

not known
Year(s) Of Engagement Activity 2012
 
Description DACP attended RHS Conference on Horticulture Careers 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact stimulated discussion of ways to encourage young people to consider careers in horticulture

not known
Year(s) Of Engagement Activity 2012
 
Description P Hand University Open Days 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact lively Q&A about careers in Agriculture and research

Increase in student applications to Harper Adams University
Year(s) Of Engagement Activity 2013,2014
 
Description P Hunter University Open Days 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact discussions with school pupils about degree choices and careers in Agriculture & land-based studies

increase in student applications to Harper Adams
Year(s) Of Engagement Activity 2011