Characterization of Pseudomonas lytic phages and their application as a cocktail with antibiotics in controlling Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that causes nosocomial disease among immunocompromised and chronic cystic fibrosis (CF) patients. We characterized two newly isolated Pseudomonas phages, ϕPA01 and ϕPA02, with different host spectra, and examined their effect as a cocktail with antibiotics against P. aeruginosa, to indicate the possibility of combining a phage cocktail and antibiotics in treating pseudomonal infection. Phages ϕPA01 (66,220 bp) and ϕPA02 (279,095 bp) belong to the genus Pbunalikevirus and Phikzlikevirus, respectively. No virulence or lysogenic associated gene was found in their genomes, thus they are potentially safe for phage therapy. We generated respective phage-resistant strains to investigate cross-resistance between two phages. Slight cross-resistance to ϕPA02 in ϕPA01-resistant strain was observed, while ϕPA02-resistant strain remained susceptible to ϕPA01. A ϕPA01 resistant strain that was cross-resistant to ϕPA02 appeared in round 5 (R5-PA01R), revealed frameshift mutation in phosphoglucomutase (algC), which is important for the synthesis of core lipopolysaccharide (LPS). Knockout of algC was resistant to both phages. Complementation of ΔalgC restored phages' infectivity, suggesting that LPS as host receptor. Phage cocktail suppressed the growth of P. aeruginosa for longer (20 h) hour compared with single phage (8-9 h), further suggesting their potential to be used as a phage cocktail. Furthermore, application of the phage cocktail with ciprofloxacin (0.25 μg/ml) and meropenem (2 μg/ml), managed to suppress the growth of P. aeruginosa up to 96 h. Our results show the potential application of ϕPA01 and ϕPA02 as phage cocktail together with antibiotics for treatment of P. aeruginosa.