Date of Graduation
12-2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy in Cell & Molecular Biology (PhD)
Degree Level
Graduate
Department
Cell & Molecular Biology
Advisor/Mentor
Kwon, Young Min
Committee Member
Gibson, Kristen E.
Second Committee Member
Alrubaye, Adnan A.
Third Committee Member
Sun, Xiaolun
Keywords
Pathway enrichment; Peracetic acid; Reactive oxygen species; TCA; Tn-seq
Abstract
Peracetic acid (PAA) is extensively used as a bactericidal agent in poultry processing to reduce foodborne pathogens, including Salmonella enterica serotype Typhimurium (S. Typhimurium). Despite its widespread application, the exact mechanisms underlying its bactericidal effects, particularly the role of reactive oxygen species (ROS), remain incompletely understood. This dissertation examines the role of ROS in PAA-mediated bacterial killing and explores both genetic and metabolic factors that influence bacterial susceptibility to PAA. Findings from these studies have implications for enhancing the efficacy of PAA, thereby improving food safety practices in the industry.
The first study investigates ROS as a central component in PAA's bactericidal action against S. Typhimurium 14028. Exposure to PAA (20 ppm) resulted in significant ROS production, particularly hydroxyl radicals, which were shown to play a critical role in cell death. To test the involvement of ROS, S. Typhimurium was co-treated with either the ROS scavenger thiourea or the iron chelator 2,2’-dipyridyl (Dip), both of which substantially reduced PAA’s bactericidal activity. Additionally, single gene-deleted mutants predicted to have increased ROS production demonstrated a significantly lower survival rate compared to the wild-type strain, highlighting the potential for ROS enhancement to boost antimicrobial effectiveness. Furthermore, exposure to sublethal PAA concentrations led to a 28-fold increase in mutation rate, suggesting that bacterial survival under oxidative stress could drive the development of antibiotic resistance.
Expanding upon these findings, the second study explores the role of tricarboxylic acid (TCA) cycle metabolites in potentiating PAA's bactericidal effects. TCA metabolites, including oxalate, oxaloacetate, and pyruvate, were tested in combination with PAA. Results showed that oxalate, in particular, enhanced PAA efficacy and exhibited potent bactericidal activity when used independently. Among multiple S. Typhimurium strains tested, oxalate demonstrated consistent broad-spectrum activity, while oxaloacetate and pyruvate exhibited strain-specific effects, suggesting the potential for targeted applications of these metabolites in pathogen control. By combining PAA with select TCA metabolites, this study offers a promising strategy to enhance PAA's bactericidal efficiency.
The final study employs transposon sequencing (Tn-seq) to identify genetic determinants critical for S. Typhimurium tolerance to PAA. A genome-saturated Tn5 mutant library was exposed to PAA in 6% Chicken Meat Extract (CME) and 11% Luria-Bertani (LB) media, yielding conditionally essential genes required for survival under these conditions. Pathway enrichment analysis revealed that these genes were associated with pyruvate metabolism, oxidative phosphorylation, the TCA cycle, and stress response pathways, including genes previously identified as increasing PAA sensitivity when inactivated (e.g., sdhC, zwf, pta, and icdA). These findings validate the role of ROS-related metabolic pathways in bacterial tolerance to PAA, providing insight into potential molecular targets for improved pathogen control.
In conclusion, this dissertation advances our understanding of PAA’s bactericidal mechanism against S. Typhimurium, with an emphasis on ROS production and metabolic interactions. The results suggest that manipulating ROS levels within bacterial cells and integrating TCA cycle metabolites can enhance PAA efficacy, offering a basis for optimized disinfection protocols in food processing environments.
Citation
Al-Doury, M. K. (2024). Bactericidal Mechanism of Peracetic Acid Against Salmonella Mediated by Reactive Oxygen Species. Graduate Theses and Dissertations Retrieved from https://scholarworks.uark.edu/etd/5556
