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Abstract

Nontyphoidal Salmonella is a major global concern for food safety, with Salmonella enterica subsp. enterica serovar Enteritidis being particularly prevalent, often causing salmonellosis and resulting in gastroenteritis. The use of bacteriophages, a natural predator of bacteria, is a potential strategy to combat food-borne pathogens in the rise of anti-microbial resistance. However, multiple studies have observed the development of phage resistance in Salmonella over time, which can pose a challenge with phage therapies and phage biocontrol in the long run. In preliminary studies, a few genes that were differentially expressed or mutated in Salmonella adaptations against bacteriophage SF1 were identified. In this project, we deleted six of these genes in Salmonella Enteritidis to investigate their roles in bacterial growth activity, virulence, and disinfectant susceptibility. Suicide plasmid-assisted homologous recombination was utilized to achieve specific knockout strains of Salmonella Enteritidis. Subsequently, growth and phage characteristic assays were done to analyze differences between these strains and the wildtype. Additionally, various disinfectants were added to bacterial and phage growth assays to be able to observe the interaction between various disinfectants and phage. Finally, phenotypic differences in virulence were assessed using a macrophage uptake assay. The results demonstrated that the deletion of rfbC – a gene involved in the synthesis of O-antigen of the lipopolysaccharide (LPS) – rendered the bacterium insensitive to phage SF1 and compromised bacterial growth, consistent with previous findings. The ΔrfbC mutant also increased bacterial uptake by RAW 264.7 macrophages, highlighting the role of O-antigen in evading phagocytic uptake. Additionally, our study found that the deletion of rfaB – a gene that expresses a galactosyltransferase for the outer core LPS – increased bacterial sensitivity to benzalkonium chloride by 4-fold. Finally, our work revealed a promising potential for combined treatment between phage SF1 and benzalkonium chloride or hydrogen peroxide. This work contributes to understanding the roles of genes related to bacteriophage resistance in bacterial virulence and defenses against phage and disinfectants. Furthermore, the findings highlight the potential of combining treatment between phage and disinfectants as an effective strategy for controlling bacterial pathogens in the food industry and agricultural settings.