2012 Annual Report 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 commodities. 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. 3.Progress Report: The agreement was finalized May 31, 2012, so there is limited progress to report. The PI at North Carolina State University is constructing transposon libraries currently in two strains of L. monocytogenes (Lm). We have obtained four Lm strains isolated during the major 2011 cantaloupe outbreak as relevant strains for testing on pre-harvest lettuce. The lettuce cultivar used previously for quantification of Lm colonization is no longer available. Therefore, we have been testing the cantaloupe Lm strains and a mint-isolated Lm strain using green oakleaf baby lettuce as a model system. This cultivar does not support vibrant Lm colonization, so it will not be amenable to the research plan. We are testing other lettuce cultivars in pre-harvest models currently to find one suitable for selection of non-attaching mutants of Lm from the transposon libraries to be supplied. This work relates directly to the project objectives of parent project 5325-42000-046-00D to “Identify and characterize the microbial genes that are involved in the attachment, colonization and survival of enteric pathogens on produce (Objective 1)” and to “Determine the genetic and biochemical factors in plants that affect the attachment, growth, and survival of human pathogens in/on plants (Objective 2).”