Date of Award
Spring 2025
Document Type
Thesis
Degree Name
Bachelor of Arts
Department
Biology
First Advisor
Joan Slonczewski
Abstract
Resistance breakers are drugs that decrease intrinsic antibiotic resistance of bacteria. While some resistance breakers (e.g. clavulanic acid) have been effective clinically, no drugs have been developed that target multidrug efflux pumps. These pumps are the frontline defense mechanisms of gram-negative bacteria to diverse antibiotics. To develop resistance breakers against efflux pumps, we must know the conditions that contribute to pump selection. Almost all bacterial efflux pumps are powered by PMF. Therefore, we are particularly interested in how conditions that alter PMF might contribute to pump selection, such as transient low pH exposure or the presence of antimicrobial food molecules (e.g. salicylate). We performed competition assays in which co-cultured Escherichia coli K-12 strains possessing or lacking a given pump complex were monitored using flow cytometry. We show how the PMF-dependent pumps AcrAB-TolC, MdtEF-TolC, and EmrAB-TolC undergo selection at low pH and in the presence of membrane-permeant phytochemicals. All three pumps showed negative selection under conditions that deplete PMF (pH 5.5 with CCCP, or at pH 8.0). The lipophilic weak acid salicylate selected against AcrA at pH 5.5. Nonetheless, selection against AcrAB-TolC did not correlate with the capacity of molecules to deplete ΔpH. Surprisingly, the lipophilic non-acidic molecules methyl salicylate and salicylamide selected against AcrA. Molecule lipophilicity (as indicated by logP) showed a strong negative correlation with AcrA selection. Molecule lipophilicity could be important because efflux pumps typically bind substrates embedded within the inner membrane. We hypothesize that lipophilic molecules may interact with the substrate-binding pocket of efflux pumps and inhibit efflux. Salicylate has often been considered a promising resistance breaker candidate because it selects against AcrA. However, other work has shown that salicylate can induce drug resistance regulons. Thus, we considered possible roles for the pumps MdtEF-TolC and EmrAB-TolC, both of which function to alleviate cytoplasmic acid stress. When treated with salicylate or benzoate, the fitness contribution for each pump was positive. However, all lipophilic non-acids showed negative or neutral selection effects. Thus, lipophilic non-acids appear to avoid off-target positive selection for efflux pumps. Finally, we demonstrated that salicylamide and methyl salicylate do not increase the minimum inhibitory concentration of E. coli to various antibiotics. Our results suggest that lipophilic non-acidic molecules could be promising resistance breaker candidates to select against a major efflux pump without inducing antibiotic resistance regulons.
Recommended Citation
Van Horn, Andrew, "Identification of Novel Multidrug Resistance Breakers in Escherichia coli K-12" (2025). Honors Theses. 983.
https://digital.kenyon.edu/honorstheses/983
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