Combatting antimicrobial resistance: small-molecule inhibitors as precision anti-virulence agents

Ansell-Downey, Holly (2024) Combatting antimicrobial resistance: small-molecule inhibitors as precision anti-virulence agents. Masters by Research thesis (MSc), Manchester Metropolitan University.

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Abstract

Background: Methicillin-resistant Staphylococcus aureus (MRSA) remains a major cause of acute and chronic infections in both community and healthcare associated settings, and novel treatments are urgently needed. Small molecule inhibitor (SMI) therapies, which have previously been employed in the treatment of eukaryotic cell conditions, present a promising alternative to current antimicrobial treatments. Methods: Preliminary MIC/MBC screening of eleven MRSA strains and synergy with traditional antimicrobials was assessed using checkerboard assays. Anti-biofilm activity of SMIs was then assessed using eradication and inhibition of biofilm formation using crystal violet assays and CDC bioreactor modelling. Cytotoxicity of one lead SMI was assessed using haemolytic and mammalian cell culture assays, and finally qRT-PCR was employed to evaluate changes in key gene expression in treated bacterial cells. Results: One lead SMI was chosen following MIC/MBC and checkerboard assays, revealing an additive interaction in combination with a traditional antibiotic. Time-kill kinetics assays revealed a 4-log reduction in cell viability when cells were exposed to 12.5 μM. Assessment of anti-biofilm activity revealed a 94.61% eradication of established biofilms at 200 μM, in addition to a 75.57% reduction in biofilm formation at 1.5625 μM. The lead SMI exhibited no cytotoxicity at <12.5 μM on NHDF cell lines and <2% haemolysis at 25 μM. Regulatory changes in sasG, sstD, perR, katA and msrA were observed in bacterial cells exposed to 6.25 μM. Conclusion: The lead SMI has anti-biofilm activity and may represent a novel treatment solution against MRSA infections. Future research should be targeted towards exploring global regulatory changes in SMI treated bacterial cells to fully understand the mechanisms of activity. Other related SMIs should also be explored to generate a library of compounds which may exhibit wider antimicrobial efficacy against both Gram-positive and negative bacterial pathogens.