The Development of a Plant-Growth Promoting Biocontrol Cocktail Using Microbiological Methods and Functional Genomics to Mitigate Salmonella enterica Typhimurium 4/74 Contamination in Alfalfa Sprouts

Abstract

Salmonella enterica is a serious threat to public health that is responsible for the vast majority of reported Salmonella outbreaks annually. Despite interventions from private industry and governmental agencies, Salmonella’s main mode of transmission continues to be foodborne, thus eliminating Salmonella reservoirs in the food supply chain is critical to controlling this pathogen. Fresh sprouts present a unique food safety challenge due to minimal processing and ideal growth conditions created during sprouting. Currently, sprout producers have implemented interventions such as seed decontamination, regular pathogen testing of irrigation water and harvested sprouts, and post-harvest treatments. However, an intervention has yet to be introduced during the sprout germination process when conditions are the most optimal for Salmonella growth and proliferation. To address the current gap in pathogen reduction, great interest has been put towards the use of bacterial biocontrol agents to reduce Salmonella contamination during sprout germination.In this thesis, two research goals were established. The first goal was focussed on isolating/characterizing plant-associated bacteria from alfalfa sprouts and subsequently formulating a cocktail of plant-associated bacteria that would function as a biocontrol agent to reduce Salmonella contamination on alfalfa sprouts. This biocontrol cocktail was then assayed for its ability to positively impact alfalfa biomass production and reduce Salmonella cell density on alfalfa sprouts. A plant-associated bacterial cocktail was composed of Pantoea agglomerans, Priestia megaterium, Pseudomonas koreensis, and Pseudomonas putida KT-2440. With a complex community of plant-associated bacteria, the cocktail was found to increase alfalfa biomass production by 25% when compared to uninoculated seeds while not causing a detriment to the overall shelf-life of harvested alfalfa sprouts. Furthermore, the cocktail led to a 3.1 log reduction of Salmonella Typhimurium 4/74 on the sprout surface after six days of germination. The second goal was centered on utilizing a functional genomics approach through transposon insertion sequencing to determine Salmonella conditional gene essentiality to the colonization and infection of alfalfa sprouts. From identified conditionally essential metabolic genes, metabolites produced by alfalfa or metabolites inherent to the sprout germination environment can be targeted for consumption by biocontrol agents. Therefore, the results of the transposon insertion sequencing study could then be used to guide the process of formulating a biocontrol cocktail specifically tailored towards the consumption of metabolite targets to ultimately decrease Salmonella cell density on alfalfa sprouts. Due to time limitations, the results of the transposon insertion sequencing study were not available prior to the submission of this thesis for review, and the results were not included.