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The past few years have seen an increasing number of human pathogenic E. coli and Salmonella outbreaks that have been associated with fresh produce. While this may be attributed to an increased consumption of fresh fruits and vegetables or, alternatively, to contamination during harvest and/or processing, it may also reflect an increasing ability of human pathogens to grow and persist on plant hosts. Knowledge concerning the nature of the interactions between these human pathogens and plant hosts is needed to develop strategies for preventing outbreaks of enteric disease due to consumption of contaminated produce. <P>
The long-term goal of this project is to understand how human pathogenic bacteria manage to persist on plants. There are three objectives: <OL> <LI> Determine what bacterial organisms make up the "community" associated with fresh produce and how frequently this includes human pathogens or their relatives. <LI> Develop an understanding of the interactions that take place between the bacteria and the plant. This will involve identifying specific plant genes involved in response as well as genes required by the bacteria for growth and persistence in the plant. <LI> Determine if bacteria that are better able to survive and persist on plants are selected in natural environments. This might involve selection of naturally occurring mutations or potentially the horizontal transfer of genes from organisms better adapted to a plant-based lifestyle. </ol>The first objective is designed to give us an idea of what bacterial species are commonly found on fresh produce using a method that does not require culturing or DNA cloning and is therefore relatively unbiased. This will not only tell us whether or not human pathogens are present, but also what closely related species might be present that could potentially act as DNA donors in horizontal gene transfer events. <P>
The goal of the second objective is to gain some idea of how well human pathogenic E. coli can grow and persist in plants as well as how this is influenced by plant defense responses and bacterial virulence factors. E. coli O157:H7 carries a type III secretion system that is well known to play an important role in both animal and plant pathogenesis. These experiments will tell us whether that system is important for E. coli to grow on a plant host as well as whether the plant perceives the bacteria and tries to defend itself against them. <P>The third objective is designed to test whether human pathogenic E. coli growing on a plant host are undergoing natural selection for strains that are better plant pathogens. If such strains are selected the DNA sequencing experiments will identify what genetic changes have taken place to allow the bacteria to better exploit their plant hosts. Since E. coli already has a type III secretion system, acquisition of one or more plant effector proteins from another bacterial species could enhance its ability to grow/persist in a plant environment.
NON-TECHNICAL SUMMARY: An increasing number of cases of human disease are associated with bacterial pathogens that are acquired from eating fresh produce. Between 1973 and 1997 foodborne disease outbreaks in the United States resulted in 16,058 illnesses, 598 hospitalizations, and 8 deaths. In 2009 the USDA estimated the total economic cost of foodborne disease caused by Salmonella and E. coli O157 in the United States at over $3 billion annually. Current efforts to curb these infections focus on the prevention of contamination of agricultural fields with animal wastes from adjacent areas and/or post-harvest technologies to sanitize the crops. If these bacterial species are capable of being facultative plant pathogens (or even endophytes) this would suggest that alternative approaches such as breeding resistant plant varieties or more effective means of eliminating the bacteria from plant tissues after harvest may be required to effectively deal with this threat. DNA sequencing, bioinformatics and genetic analyses will be used to identify species of bacteria found associated with fresh produce as well as to identify the types of genes required for them to grow on plant hosts. <P>The data generated by this project will help us understand (1) how well these human pathogens grow and persist on plants, (2) how commonly they or their relatives are associated with plants naturally, (3) if plants attempt to mount a defensive response against these bacteria, and (4) how quickly/easily genetic changes are acquired allowing human pathogens to utilize plant hosts more effectively. This knowledge will be critical in developing better strategies to deal with this growing problem. <P>
APPROACH: Determine what bacterial organisms make up the "community" associated with fresh produce and how frequently this includes pathogenic E. coli and Salmonella. Total DNA will be extracted from fresh produce samples and 16S ribosomal DNA primers will be used to amplify either the entire 16S rDNA or hypervariable subregions. Amplified DNA molecules will be sequenced using Roche 454 sequencing technology to produce a library of bacterial sequences obtained from each produce sample. These sequences will be compared with databases of known sequences to determine the most closely related species to each sequence. Samples that contain E. coli sequences will be tested by conventional PCR for the presence of sequences specific to human pathogenic strains. Different types of produce and produce obtained from different geographic regions of the United States will be compared. Develop an understanding of the interactions that take place between the bacteria and the plant. Initial experiments will be carried out in Arabidopsis due to the availability of both genomic resources and large number of plant-defense mutants, although subsequent experiments could be carried out in tomato or other genetically well-characterized species that are more relevant to food production. Interactions between specific strains of bacteria and accessions of Arabidopsis that affect the growth of the bacteria will be quantified. Quantitative PCR using known plant pathogen induced genes (PR1, PDF1.2, etc.) will be used to assess the plant's response to E. coli applied either by leaf-infiltration or spray inoculation. The importance of plant defense genes will be assessed by comparing bacterial growth rates following inoculation of either wild-type plants or plants that are homozygous for mutations that are known to impair plant defenses. In a similar manner the importance of bacterial virulence genes will be assessed by examining whether or not bacterial growth in the plant is reduced in bacteria that carry mutations in the type III secretion system or other genes related to bacterial pathogenesis. Determine if bacteria that are better able to survive and persist on plants are selected in natural environments. Seeds will be exposed to E. coli and germinating seedlings will be examined for visible lesions and total number of surviving bacteria. Individual bacterial colonies from those seedlings showing greatest lesion formation and/or greatest bacterial survival will be purified and used to repeat the experiment for several more generations. Bacterial strains that are isolated that show significant increases in lesion formation or survival will be compared to the parental strain by whole genome sequencing to determine the nature of any genetic differences between them. Bacterial genome sequencing will also be used to characterize E. coli strains that have been isolated from plant associated disease outbreaks and compared with those from animal associated outbreaks.