1a.Objectives (from AD-416):
The overall goal of the proposed project is to conduct research that will provide a science-based approach that will reduce or eliminate bacterial contamination of several produce commodities by Listeria monocytogenes. This will be accomplished by determining molecular mechanisms for the interactions between L. monocytogenes and leafy greens, sprouts, and celery. This will be done in four objectives:
1. Assessing 30-50 strains of L. monocytogenes for fitness in colonization of produce to identify strains for mutant library construction.
2. Identification of genes necessary for L. monocytogenes colonization of produce.
3. Identification of genes induced in L. monocytogenes during produce colonization.
4. Characterization of biofilms produced by L. monocytogenes on produce surfaces.
1b.Approach (from AD-416):
A panel of L. monocytogenes strains will be screened for fitness in colonization to several produce commoditites. Those bacterial strains that are most fit will be selected for construction of a mutant library. This library will be screened for resistance to various stresses incurred while colonizing a plant surface (e.g., oxidative stress), and the library will also be screened on various produce surfaces to identify genes necessary in L. monocytogenes for produce colonization. In addition a good colonizing strain of L. monocytogenes will be tagged with a chromosomally localized Green Fluorescent Protein tag in order to conduct real-time, microscopic observation of L. monocytogenes in bacterial biofilms present on produce surfaces.
Cantaloupe attachment experiments were done with 4 L. monocytogenes strains. Mariner transposon libraries were constructed in two strains, and these strains were used to make mutant pools enriched in strains that have defective attachment to cantaloupe. Additionally 8 lettuce cultivars were screened for their ability to support growth of L. monocytogenes in order to construct mutant pools enriched in strains that are defective in lettuce attachment. L. monocytogenes attach to cantaloupe flesh and rind very well, but shows cultivar differences in attachment to 28-day old lettuce plants. This research relates to Objective 1: Identify and characterize the microbial genes that are involved in the attachment, colonization and survivial of enteric pathogens on produce.