The Objectives of the project were (i) to characterize genetic and molecular mechanisms mediating resistance of Listeria monocytogenes to quaternary ammonium disinfectants (ii) to characterize genetic and molecular mechanisms responsible for temperature-dependent resistance of L. monocytogenes epidemic clone II strains to phage and (iii) to assess growth of different strains of L. monocytogenes and of Listeria spp. on ready-to-eat meat of different origin (chicken, turkey, beef).
Genetic analysis revealed that a gene cassette (bcrABC) first identified on a large plasmid (pLM80) harbored by strains implicated in the 1998-1999 hot dog outbreak conferred resistance to benzalkonium chloride and other quaternary ammonium disinfectants to L. monocytogenes. Transcription of the genes was induced by BC and expression was significantly higher at low temperature (4, 8, 25°C) than at 37°C. This cassette has become disseminated among different strains and serotypes of L. monocytogenes. A gene cassette unique to L. monocytogenes ECII was found to be responsible for the remarkable ability of these strains to resist phage infection when grown at low temperatures. The mechanism appears to involve the digestion of phage DNA upon injection into ECII cells via a temperature-regulated enzyme, encoded by one of the ECII-unique genes. Growth of Listeria on ready to eat deli meat (bologna) was not impacted by resistance to BC and was similar for L. monocytogenes and L. innocua. However, growth was more pronounced on chicken bologna than on turkey or beef.
Project findings contribute to elucidating the remarkable adaptations and underlying mechanisms that Listeria employs to colonize meat processing plants and persist in the meat industry environment. Strains have acquired dedicated, specialized mechanisms for resistance to disinfectants commonly used in the meat industry, and problematic lineages are resistant to phages when grown in the cold; genes conferring such adaptations are induced at low temperatures such as those that prevail in the meat industry. Understanding such capacity of Listeria for adaptation and growth will lead to novel, science-based interventions and control strategies. The project has identified genes and probes that can be used to monitor the presence of strains with natural resistance to quaternary ammonium compounds. Mutants of L. monocytogenes ECII that can methylate DNA, but lack the restriction enzyme responsible for phage resistance can be valuable propagating strains, along with non-pathogenic Listeria spp. that express the methyltransferase gene. Such strains have been constructed in this project. Phage from such strains can infect ECII regardless of temperature of growth, and would thus provide improved phage-based tools for enhanced biocontrol of Listeria in the meat industry.