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Belanger, Corrie Richel

University of British Columbia

This research assessed the ability and mechanism of azithromycin (AZM) alone, and in synergy, to treat Pseudomonas aeruginosa grown in physiologically relevant media and in vivo when compared to standard, nutrient rich conditions. When compared to treatment in Mueller Hinton broth (MHB), AZM has a substantially lower minimal inhibitory concentration (MIC) in RPMI ± human serum (RPMI/serum) and demonstrates increased synergy in combination with synthetic host defence peptides. Global transcriptional analysis revealed that genes mediating lipopolysaccharides (LPS) modification were downregulated in host-like media compared to MHB. Inactivation of these genes led to increased susceptibility to AZM and synergy between AZM and other antimicrobial agents. Thus indicating that dysregulation of LPS modification might result in increased AZM uptake in the host environment. Using Tn-Seq, mutants with severe growth defects under physiologically relevant conditions were determined. Genes involved in membrane integrity, iron acquisition, and nucleotide and cobalamin biosynthesis were implicated as required for survival under physiologically relevant conditions. The factors influencing growth of P. aeruginosa in physiologically relevant media were also essential in vivo, and may be used to explore alternative treatments that might take advantage or target these systems in the clinic. Finally, unique sets of genes important for susceptibility of P. aeruginosa to AZM under physiologically relevant media conditions and in vivo were identified using Tn-Seq in medium treated with AZM. Increased AZM susceptibility in murine abscess and human skin organoid models was observed for mutations in integral cell envelope genes, stress signaling and biofilm formation genes, type III secretion system genes, and transcriptional regulators. Most of the AZM susceptibility mutants had predicted interactions with iron acquisition components and increased susceptibility to AZM was induced as a function of iron limitation. Evidently, multiple factors in host-like environments are responsible for the observed changes in susceptibility of P. aeruginosa, including altered membrane permeability, altered iron availability, and stress responses. Understanding the mechanisms that underly bacterial susceptibility to antimicrobials in varying conditions could help change standard approaches to drug testing and lead to more complete and functional assays enabling more predictive drug discovery routes.

Text

2021-10-22

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/80033

Microbiology and Immunology

University of British Columbia

UBCV

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