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Sutherland, Darcy

University of British Columbia

The World Health Organization has reported that the lack of new antibiotics is leading to a ‘post-antibiotic’ era, in which bacterial pathogens are resistant to most prevailing forms of antibiotic therapy. This thesis presents the laboratory evaluation of antimicrobial peptides (AMPs) for their capacity to combat multi-drug resistant bacteria, colloquially referred to as “superbugs”. The approach employed an artificial-intelligence classifier to discover novel AMPs from publicly-available datasets, and subsequently to generate mutations and truncations for improved potency. Top-scoring AMP candidates identified by the bioinformatic tools were assessed through antimicrobial susceptibility testing and cytotoxicity experiments in vitro. AMPs were assessed for antimicrobial activity based on measurement of their ability to interfere with microbial growth using standard minimum inhibitory concentration (MIC) assessments. AMP cytotoxicity was estimated by measuring the concentration of AMP that results in lysis of 50% of red blood cells (HC50) or 50% killing of human embryonic cell lines (CC50). This work identified 20 novel AMPs with high activity (MIC < 8μg/ml) against clinical isolates of multi-drug resistant bacteria, including carbapenem-resistant Escherichia coli and Acinetobacter baumannii. Many of these were also observed to be non-toxic (HC50 & CC50 > 128 μg/ml). Eight AMPs were examined for their propensity to stimulate resistance in E. coli using a 10-day sub-lethal passage experiment. Clinical strains of E. coli were observed to become 100X more-tolerant (MIC = 1-2μg/ml shifted to >256μg/ml) following repeated sub-lethal exposure with an AMP. Importantly, this demonstrated that certain AMPs can rapidly induce antimicrobial resistance in vitro. This was unique to one of the eight AMPs tested, whereas the other AMPs did not induce resistance over the 10-day experiment. These findings suggest that certain AMPs can induce resistance whereas others remain microbiologically active following repeated sub-lethal exposure to target microbes, and highlights that further understanding of distinct AMP mechanisms is required before translating them into antibiotics.

Text

2025-07-10

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/91545

Pathology and Laboratory Medicine

University of British Columbia

UBCV

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