Identifying Genes that Contribute to the Survival of Campylobacter Jejuni in Refrigerated and Frozen Foods

NON-TECHNICAL SUMMARY: Campylobacter jejuni is one of the leading causes of food-borne bacterial gastroenteritis in humans. Poultry is the major reservoir host where C. jejuni can be found at concentrations as high as 10E9 colony forming unts (cfu) per gram of feces. Epidemiological studies have frequently implicated raw and undercooked poultry meat as a major source human campylobacteriosis. In contrast to other food-borne pathogens, C. jejuni does not normally multiply on food products because of its fastidious nature and narrow range of growth temperatures (31C to 42C). C. jejuni does not grow below 30C and freezing or refrigeration results in cfu reductions of three orders of magnitude. As a result, refrigeration or freezing are the most commonly used intervention techniques to reduce the numbers of C. jejuni in poultry meat. Nevertheless, a substantial portion (as much as 60% to 80%) of frozen and fresh retail chicken in the U.S.A. is contaminated with C. jejuni with counts approaching 10E4 and 10E6 cfu, respectively. Given the very low infectious dose (100 to 500 cells) for humans, the ability of C. jejuni to survive in refrigerated and frozen meat is of obvious relevance to food safety and public health. As a species, C. jejuni exhibits considerable genetic and phenotypic diversity and unlike other bacteria, it does not harbor well recognized stress related genes (eg. Csps and rpoS). Consequently, the genetic factors required for the survival of C. jejuni during cold storage are not known. Thus, we peopose to identify the genetic factors (genes) that govern the survival of C. jejuni in refrigerated and frozen chicken meat. Transposon mutagenesis will be used to identify critical genes and complementation will be used to verify function. Identification of genes required for survival under cold conditions will provide novel targets for intervention or mitigation of colonization. We also expect to identify genes encoding novel cold shock proteins (Csps) and cold inducible proteins (Cips) of C. jejuni. These findings can have commercial implications in recombinant protein expression research. For instance, the newly identified Csps and Cips can be a potential target for design of cold inducible recombinant expression system for heterologous proteins that are heat sensitive or vulnerable to proteolysis.

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APPROACH: 1. Identify the genes required for survival of C. jejuni in chicken meat stored at refrigerated (4C) and frozen (-20C) conditions: We will construct a library of 6,000 mutants of C. jejuni NCTC11168 (a sequenced strain) by random mutagenesis using the EZ-Tn5-KAN transposon. To determine survivorship, a known cfu of each mutant strain will be mixed with a sterile chicken soup and subject to storage under refrigerated (4C) and frozen (-20C) condition. The cfu of each mutant strain before and after these treatments will be determined and compared with the wild-type parent. Subsequently, mutants with reduced survivorship will be selected for identification of EZ-Tn5-KAN insertion sites. The DNA sequences flanking transposon insertion sites will be identified by rescue cloning and sequencing. Once the gene sequences are identified, mutant strains will be complemented in trans by cloning the coding region of identified gene in vector pRY111 or pRY112. Our study is unique in this regard because we propose to use chicken juice as a food based model system to screen C. jejuni mutants for their survival under conditions that mimic the environment that C. jejuni experiences on raw chicken. 2. Determine if the genes identified in specific aim 1 are conserved among C. jejuni isolates recovered from different sources including human, swine, bovine, poultry and poultry products: PCR primers will be designed to amplify the genes identified in specific aim 1. Genomic DNA extracted from 50 C. jejuni isolates recovered from different sources will be subjected to PCR amplification of the genes identified in specific aim 1 (strains available from WSU-FDIU and Dr. Michael Konkel). We expect that the genes required for the survival of C. jejuni under cold storage conditions will be conserved in C. jejuni isolates. With this list of genes, we will pursue further functional characterization with the intent to identify targets for new antimicrobial agents or to devise intervention strategies to prevent or limit C. jejuni contamination of chicken meat and other food products.