Abstract
Pseudomonas aeruginosa and Staphylococcus aureus are leading causes of biofilm-associated chronic infections. These pathogens are commonly co-isolated from lung infections of cystic fibrosis patients, wound infections, and otitis media. The relationship between P. aeruginosa and S. aureus is complex, and their interactions are not fully understood. These studies aimed to further our understandings of how P. aeruginosa and S. aureus interact together within a polymicrobial biofilm, under physiologically relevant conditions. I used RNA-sequencing to study transcriptomic changes in both pathogens during coexistence and used Transposon-sequencing to identify genes that are essential to coexistence between P. aeruginosa and S. aureus in polymicrobial biofilms. To identify genes involved with competitive interactions between P. aeruginosa and S. aureus I designed a high throughput screen of transposon arrayed mutant libraries for growth competition assays. The last part of this study investigated the polymicrobial biofilm environment and how this potentiates the activity of a commonly used antibiotic, colistin in the presence of an anti-biofilm peptide, DJK-5.
I identified that S. aureus induced phosphate and potassium starvation in P. aeruginosa in polymicrobial biofilms. This starvation results in the high affinity potassium pump kdp being essential to P. aeruginosa in polymicrobial biofilms. S. aureus exhibited antimicrobial tolerance in polymicrobial biofilms to chloramphenicol, azithromycin and clindamycin, which is due to the significant upregulation of efflux pump encoding genes emrB and lmrP. Competitive interactions between P. aeruginosa and S. aureus were investigated using P. aeruginosa strains known to outcompete S. aureus, where the pqsABC genes were responsible for the production of secreted exoproducts that lead to S. aureus inhibition. Interestingly the production of 2-methyl-4- hydroxyquinoline (MHQ) by the pqsABC system in P. aeruginosa promoted the survival of S. aureus with P. aeruginosa strains known to inhibit S. aureus growth. In S. aureus, mutants that led to small colony variants promoted survival with P. aeruginosa in nutrient rich conditions. I used a combination of colistin a last line antibiotic with anti-biofilm peptide DJK-5 which showed synergistic activity against P. aeruginosa-S. aureus polymicrobial biofilms in vitro and within a murine subcutaneous abscess model.
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Overall, this collection of studies identifies important novel interspecies interactions that further our understanding of P. aeruginosa and S. aureus when they coexist in polymicrobial biofilms and during competitive interactions in planktonic co-cultures.