#10-086

Complete

Date Full Report Received

01/05/2012

Date Abstract Report Received

01/05/2012

Investigation

Institution:
Primary Investigator:

Enterohemorrhagic and exotoxigenic E. coli (EHEC and ETEC) pose a variety of animal health and food safety challenges for the food animal industry. E. coli F4 is an ETEC pathogen that is an important cause of post-weaning diarrhea and death in piglets. E. coli O157:H7 is an EHEC strain that may cause illness in young animals, but is more widely recognized as a food adulterant (primarily from cattle). Successful control measures for both EHEC and ETEC strains would yield significant animal and public health benefits as well as financial benefits to producers and consumers.

We previously identified several strains of E. coli from cattle that are capable of inhibiting growth of EHEC and ETEC strains. This inhibition phenotype was called “proximity-dependent inhibition” (PDI) because of the apparent necessity for the inhibitor strain to be in close proximity or direct contact with the susceptible strain for the mechanism to function. The goal of the current project was to identify candidate genes that are responsible for this phenotype and to determine their functional contribution to the PDI phenotype.

For this project we identified two strains of E. coli, one that produces the PDI phenotype and one that does not, but that were otherwise genetically identical using a conventional typing system (PFGE). We then generated whole-genome sequence data for both strains and compared them with the goal of identifying unique regions of DNA sequence that might be responsible for the PDI phenotype. This effort identified a region of DNA sequence that was unique to the inhibitor strain.

This PDI region appears to encode a novel microcin. Microcins are a subclass of bacteriocin antimicrobial peptides. They tend to be small in molecular mass and secreted in very low concentrations. Bacteria use bacteriocins to limit competition from other bacteria, but these peptides are usually specific to the same species of bacteria and do not affect other species or genera. In our case specificity includes both EHEC and ETEC strains. We completed a series of gene knockouts and other experiments that demonstrated that the putative microcin is indeed responsible for the PDI phenotype. We also demonstrated that the microcin limits E. coli O157:H7 by an unidentified killing mechanism.

This discovery is important to the industry because microcins could be used to inhibit growth of EHEC and ETEC bacteria in food animals as they are already being applied in the food industry. For example, a probiotic strain that induces PDI could be fed to food animals either to prevent colonization or to limit the population size of pathogenic strains. It might also be possible to deliver the antimicrobial peptide directly to the animal. The first step in development of this type of tool is to identify the genetic mechanisms responsible for PDI and funding from the National Pork Checkoff made this possible. The next steps include identifying the receptor that is required for bacteria to be inhibited by the microcin and determining if our PDI positive strains can be used as probiotics to control EHEC and ETEC in food animals.