166 | Attenuation of Pseudomonas aeruginosa virulence by engineered phage

Domenico Franco¹, Salvatore Papasergi², Francesco Mediati¹, Salvatore P.P. Guglielmino¹, Laura Maria De Plano¹ | ¹Department of Chemical, Biological, Pharmaceutical and Environmental Sciences ChiBioFarAm, University of Messina, Italy; ²Institute of Translational Pharmacology of the National Research Council; ³Department of Occupational and Environmental Medicine, Epidemiology and Hygiene, Italian Workers’Compensation Authority INAIL, Italy.

Pseudomonas aeruginosa is a clinically relevant opportunistic pathogen whose virulence and persistence are largely controlled by quorum sensing and biofilm formation. In this study, we investigated the antivirulence activity of the engineered filamentous phage P9b, developed using phage display to present a peptide insert on its capsid that specifically recognizing P. aeruginosa [1-2]. We found that P9b induces a significant but transient inhibition of planktonic growth at early time points, with no effect on long-term bacterial viability. In contrast, P9b strongly and persistently reduced biofilm formation and pyocyanin production up to 72 hours - effects not observed with an insert-less control phagemid. At the molecular level, P9b exposure significantly modulated quorum-sensing pathways, with downregulation of key regulators (lasR, rhlI, and rhlR) and phenazine biosynthesis genes (phzM and phzS). Importantly, P9b efficiently bound to clinical P. aeruginosa isolates, which were characterized by distinct antibiotic resistance profiles [3], and inhibited their biofilm formation. Overall, our results show that P9b acts as a non-lytic, species-specific agent that interferes with quorum-sensing–regulated pathogenic pathways, highlighting engineered filamentous phages as promising tools to control P. aeruginosa virulence.