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Guy, Thomas

University of British Columbia

Non-typhoidal Salmonella is a leading cause of foodborne diarrheal disease. The prevalence of antimicrobial resistant (AMR) isolates is rising, and the overexpression of the AcrAB–TolC efflux pump is one factor often linked to multidrug resistance. We propose a targeted strategy to elicit structural changes in the TolC protein via bacteriophage (phage) infection. We hypothesize that these alterations in TolC structure will restore antimicrobial sensitivity of the host Salmonella. The purpose of this thesis was to assess changes in AMR of bacteriophage-insensitive mutants (BIMs) of Salmonella Typhimurium resistant to chloramphenicol and tetracycline, following infection with a TolC-binding phage. Using a collection of newly-isolated phages (n=72), TolC dependence was determined via relative efficiency of plating using a ΔtolC mutant of S. Typhimurium ATCC 14028. BIMs were generated by co-inoculating S. Typhimurium with TolC binding phage in tryptic soy broth (TSB) in triplicate. Every day for three days, 5 µL of co-culture and an additional 5 µL of phage lysate (~10⁹ PFU/mL) was transferred to fresh TSB. Cultures were isolated on streak plates and ten colonies selected at random; which were confirmed as phage resistant over five rounds of subculture via spot test. Minimum inhibitory concentrations (MICs) of BIMs to chloramphenicol and tetracycline were determined via broth microdilution. Additionally, the ability of this phage to alter the frequency of AMR within populations of S. Typhimurium was investigated by a serial co-culture method. One phage, SeKF 80, showed reliance on TolC. Host resistance to this phage arose after three days. Of the ten randomly selected BIMs, #1, #5, and #9 displayed twofold increases in tetracycline resistance compared to the parental strain. In contrast, nine of the ten BIMs had unchanged MICs of chloramphenicol, with only BIM 5 showing a twofold reduction in chloramphenicol resistance. Phage SeKF 80 had minimal effect on the frequency of AMR when used to infect populations of S. Typhimurium. This study demonstrates that forced evolution of the TolC receptor through phage infection can lead to evolutionary trade-offs related to antimicrobial resistance; however, the phenotypes of bacteriophage-insensitive Salmonella mutants are complex and potentially specific to the host-phage pair.

Text

2024-01-26

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/87384

Food Science

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/