Investigating the genetic determinants of L. monocytogenes stress tolerance to food-industry relevant stressors through adaptive laboratory evolution
Authors: Tyler Bechtel 1; John Gibbons 1,2,3
Affiliations: 1) Department of Food Science, University of Massachusetts, Amherst, MA; 2) Molecular and Cellular Biology Graduate Program, University of Massachusetts, Amherst, MA; 3) Organismic and Evolutionary Biology Graduate Program, University of Massachusetts, Amherst, MA
Keywords: Comparative genomics & genome evolution
Listeria monocytogenes is a foodborne bacterial pathogen that causes thousands of illness and billions in medical and food recall costs annually in the United States. L. monocytogenes can survive, replicate, and persist in a variety of harsh environments, including conditions encountered in food processing facilities and equipment. This presents a major problem for public health and the food industry as stress resistant strains of L. monocytogenes are more likely to persist in the food environment, and subsequently contaminate food products. Here, we devised an “evolve and resequence” experiment to shed light on the frequency, temporal patterns, and genetic determinants of stress resistance evolution in L. monocytogenes. Specifically, we grew three replicates of three phylogenetically and phenotypically distinct strains of L. monocytogenes for 500 generations in sub-lethal concentrations of (i) salt-induced osmotic stress, (ii) benzalkonium chloride (BAC), a sanitizer commonly used in the food industry, and (iii) tryptic soy broth (control). We have sequenced and annotated the genomes of each of the ancestral strains. Every 50 generations, we have (i) frozen and stored cultures, (ii) measured the minimum inhibitory concentration (MIC) of each stressor to track temporal changes in fitness, (iii) conducted a quantitative virulence assay to investigate the potential relationship between stress response and pathogenicity, and (iv) resequenced the genomes of each lineage to track changes in allele frequency and to identify candidate mutations underlying adaptive phenotypes. Lastly, we will create gene deletion mutants using the suicide plasmid pHoss1 to validate the function of candidate genes. Our results indicate that BAC-sensitive isolates, ALE_10_0415 and ALE_20_0415, exhibited a two-fold increase in MIC of BAC after ~300 generations. In comparison, BAC-tolerant isolate ALE_16_0415 showed minimal improvement at ~400 generations, which suggests that there is a physiological limit to L. monocytogenes BAC resistance. Our results will provide insight into the evolution and genetic mechanisms underlying L. monocytogenes stress-tolerance. In addition, the results of this study will identify candidate genes that will enable bacterial surveillance systems to detect and characterize persistent L. monocytogenes strains isolated from food production facilities.