Characterizing Environmental and Genetic Factors That Impact β-Lactamase-Mediated Antibiotic Resistance
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Authors
Zhao, Dylan Weisman
Date
2025-06-24
Type
thesis
Language
eng
Keyword
Antibiotic resistance , Outer membrane permeability , Urinary tract infections , β-lactamase-mediated antibiotic resistance , β-lactams , Diabetes , Hypertension
Alternative Title
Abstract
Antibiotic resistance, particularly among Gram-negative bacteria, poses a critical global health challenge. Pathogens including Escherichia coli and Pseudomonas aeruginosa can produce β-lactamases that inactivate β-lactam antibiotics as a resistance mechanism. Given the ability of bacteria to adapt to environmental stressors, understanding how environmental and genetic factors impact their physiology and β-lactamase-mediated resistance remains critical for developing more effective, individualized treatments.
E. coli is the most common uropathogen that causes urinary tract infections (UTIs). Hypothesizing that changes to the urinary environment would influence E. coli physiology and β-lactamase-mediated resistance, we demonstrated that growth in an artificial urine medium (AUM) significantly increased β-lactam hydrolysis by β-lactamases through an increase in outer membrane permeability to β-lactams via OmpF upregulation. Mimicking pathological urinary environments (glycosuria, hypernatriuria) caused a concentration-dependent decrease in outer membrane permeability to β-lactams through OmpF downregulation, impacting antibiotic resistance. Our results showcase that E. coli are particularly sensitive to gentamicin in AUM, but this sensitivity decreases in the presence of glucose, suggesting that glucose may impair the efficacy of gentamicin in vivo and potentially contribute to changes in morbidity in diabetic UTIs.
In parallel, we examined the impact of genetic mutations on an inducible β-lactamase system in P. aeruginosa, where β-lactam-mediated PBP4 inhibition triggers AmpC β-lactamase expression. An E. coli-based biosensor was engineered to luminesce upon PBP4 inhibition by co-transforming a luminescence-producing plasmid alongside a plasmid producing PBP4 from P. aeruginosa 2_1_26. Subsequently, we demonstrated that targeted mutations to dacB encoding PBP4 can significantly alter β-lactam binding affinity, where the L369A variant may exhibit an increased affinity for cephalosporins through enhanced luminescence following induction. These findings can help examine how clinically observed dacB mutations influence β-lactam-mediated PBP4 inhibition, AmpC induction, and cephalosporin susceptibility to hydrolysis.
Together, these findings highlight how environmental and genetic factors modulate β-lactamase-mediated resistance in clinically relevant pathogens. By elucidating the interplay between urinary environments, outer membrane permeability, and β-lactamase activity in E. coli, as well as the mechanistic consequences of dacB mutations in P. aeruginosa, this work contributes to a deeper understanding of antibiotic resistance and may inform more effective treatment strategies for bacterial infections.
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This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.
Attribution-NonCommercial-NoDerivatives 4.0 International