Use of Experimental Evolution to Select for Listeria Phages in Food Environments and Assessing the Prevalence of Listeria spp. in Soil

The foodborne pathogen Listeria monocytogenes poses significant challenges for the food industry due to its ability for growth across diverse temperature ranges and in high-salt conditions. The research within this thesis evaluated whether experimental evolution can be used to select for Listeria phages that are more efficient at infecting L. monocytogenes in food specific environments. Listeria phages can be used as a biocontrol to target L. monocytogenes in food. Phage transfers were completed with Listeria phage LP-125 in ultra-pasteurized oat and whole milk, and bioinformatic tools were used to identify mutations present in the selected phages. We identified two nonsynonymous mutations in LP-125_108 and LP-125_112 genes encoding tail fiber glycerophosphodiester phosphodiesterase and baseplate wedge proteins, respectively. We then showed that the mutation in LP-125_112 allowed LP-125 to bind to L. monocytogenes more efficiently in high fat environments. Protein structural modeling found that the substituted amino acids in the affected phage proteins may alter the protein’s surface charge, which could explain the fat-dependent adsorption phenotype we observed. Our findings support that experimental evolution can be used to select for phages that are more efficient in specific environments than the ancestral phage. Furthermore, understanding the diversity and prevalence of Listeria spp. is important for preventing outbreaks. The work completed within this thesis also evaluated the prevalence of Listeria spp. in soil obtained from Nantahala National Forest. Twelve soil samples were obtained across 8 miles and 3,000 feet of elevation gain. These samples were enriched for Listeria spp. and L. monocytogenes. Bacterial DNA extractions were completed and whole genome sequencing (WGS) was utilized to identify Listeria isolates. Several of the isolates were identified as ‘L. swaminathanii’ a newly described species of Listeria. The highest prevalence of Listeria spp. was in soil samples at relatively lower altitudes. This research will assist in rapid and accurate detection of Listeria in natural environments, which is important for preventing potential outbreaks of Listeria.