Investigating R-pyocins as a therapeutic approach to treat high-risk, multidrug-resistant Pseudomonas aeruginosa wound infections

Project Summary Pseudomonas aeruginosa (Pa) is a versatile human pathogen responsible for a range of acute and chronic infections, notably those stemming from traumatic injuries, burns, chronic wounds and respiratory diseases such as Cystic Fibrosis. The prevalence of high-risk multidrug-resistant (MDR) and extensively drug-resistant (XDR) Pa has been rising over the past decade, causing significant concern due to limited treatment options and increased morbidity and mortality. Current therapeutic development approaches often rely on studies using laboratory strains, which may not accurately represent the efficacy against high-risk clinical isolates. The goal of the proposed work is to investigate the use of R-pyocins, narrow-spectrum antimicrobial proteins produced by Pa, as a novel therapeutic approach to treat wound infections caused by high-risk, antibiotic- resistant Pa strains by: (i) determining R-pyocin diversity and antimicrobial efficacy against high-risk MDR/XDR Pa strains, and (ii) investigating the reversal of antibiotic resistance in MDR/XDR Pa strains using R-pyocins as a selective pressure. The hypotheses are that (i) certain R-pyocin variants will demonstrate superior bactericidal activity against high-risk Pa strains in both in vitro and in vivo settings, potentially offering a novel therapeutic approach for treating resistant Pa infections, (ii) R-pyocin exposure will promote mutations or deletions in the galU gene, which is essential for the synthesis of a complete lipopolysaccharide (LPS) core and O-antigen, and that (iii) these mutations may also affect nearby genes, including the mexXY efflux pump genes, leading to increased susceptibility to conventional antibiotics. The first aim will involve identifying R-pyocin types in a biobank of 100 high-risk Pa strains using a bioinformatics tool called PyocinTyper, extracting and purifying R-pyocins from selected strains, and assessing their bactericidal activity through in vitro assays and in a murine chronic wound model. The second aim will involve exposing MDR/XDR Pa strains to increasing concentrations of R-pyocins, screening for surviving brown phenotype strains, characterizing the genetic changes in these mutants, and evaluating their antibiotic susceptibility profiles and efflux activity in vitro and in vivo. The findings of this project will provide new insights into the therapeutic applications of R-pyocins and potential strategies to combat antibiotic-resistant pathogens in human infections, addressing a critical healthcare challenge and informing the development of novel antimicrobial therapies.