Isabell Pitigoi Courtney Ostromecki Madelyn Fischer Mitchell Shorgan


Introduction: Despite antimicrobial resistance topping the list of global health concerns, the development of novel antibiotics has been nearly abandoned due to strict regulations and dwindling economic incentives in the pharmaceutical industry. There is a critical need for alternative strategies to treat multidrug resistant pathogens like Pseudomonas aeruginosa (P. aeruginosa), a pestilent cause of nosocomial infections. Here, we aim to target adaptive resistance in P. aeruginosa biofilms by inducing hypersensitivity to existing antibiotics through phage-delivery of a particular gene cassette. Previous studies have suggested that the rpoS gene is repressed in P. aeruginosa biofilms and that its deletion is correlated with hypervirulence, increased biofilm thickness and antibiotic resistance.

Methods: In this protocol, we aim to explore the effect of inducing rpoS overexpression in P. aeruginosa colonies as a potential method to disrupt biofilm structure and increase sensitivity to tobramycin. Phagemids containing rpoS, an accompanying promoter, and a tellurite resistance gene are delivered by P1 bacteriophages to the biofilm to be shared through horizontal gene transfer (HGT). Tellurite is then administered to induce selective pressure for HGT, by favouring uptake of the phagemids due to the presence of the tellurite resistance gene. Consequently, we can assess the effect of rpoS overexpression on biofilm organization and tobramycin sensitivity using measures from confocal laser scanning microscopy (CLSM).

Anticipated Results: Given the hypervirulent effects of rpoS deletion, we expect that forcing rpoS overexpression in P. aeruginosa would result in decreased biofilm thickness compared to controls. Furthermore, the colonies are also expected to have lower cell viability following tobramycin administration.

Discussion: Overall, our experiment characterizes the effects of rpoS overexpression on biofilm thickness, cell viability and tobramycin resistance. As such, this protocol may have practical implications for re-sensitization of P.aeroginosa to antibiotics.

Conclusion: This would demonstrate a potential for phage-mediated hypersensitization of P. aeruginosa that is adaptable to more practical settings, such as in situ on hospital surfaces.

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Research Protocol