Phage cocktails containing a dual-receptor <i>Phikzvirus</i> suppress resistance evolution in <i>Pseudomonas aeruginosa</i>

While phage therapy is one of the promising strategies against antimicrobial resistant infections by <i>Pseudomonas aeruginosa</i>, the rapid emergence of phage-resistant variants remains a significant barrier to its long-term clinical efficacy, reflecting the constant evolutionary arms race between phages and their hosts. Here, we first characterized ΦBrmt, a <i>Phikzvirus</i> phage previously isolated from an LPS-defective <i>P. aeruginosa</i> mutant of the Pa12 strain. Whole-genome sequencing of ΦBrmt-resistant variants derived from the Pa12 strain (Pa12 mt^ΦBrmt) revealed mutations in genes for type IV pili and flagellar biosynthesis, resulting in decreased motility. To identify its receptors, we tested ΦBrmt against a panel of knock-out mutants, revealing that it failed to infect a <i>ΔpilA</i>/<i>ΔfliC</i> double mutant, despite being able to infect each single mutant. Transmission electron microscopy revealed that ΦBrmt adsorbed to the flagella of the Pa12 WT, whereas this adsorption was abolished on the phage-resistant mutants Pa12 mt^ΦBrmt. In contrast, <i>Pbunavirus</i> ΦS12-3 and ΦR26 were unable to infect the Δ<i>galU</i> mutant but formed clear plaques on the Δ<i>pilA</i> and Δ<i>fliC</i> strains. A cocktail combining the pili/flagella-targeting ΦBrmt with an LPS-targeting <i>Pbunavirus</i> phage significantly suppressed the emergence of phage-resistant variants <i>in vitro</i> against representative clinical isolates when compared to single-phage treatments. Our findings demonstrate that combining phages targeting distinct classes of bacterial receptors is a powerful strategy to limit resistance development, indicating that identifying the receptor genes utilized by <i>Pseudomonas</i> phages can be the rational starting point for such design.IMPORTANCEPhage resistance limits the clinical efficacy of phage therapy against <i>P</i>. aeruginosa, a major antimicrobial-resistant pathogen. To address this, we demonstrate that a cocktail combining phages targeting distinct class of receptors effectively suppresses resistance. Through genetic analysis of resistant mutants, we first identified that the phage Brmt (ΦBrmt) uses both Type IV pili and flagella as receptors; a double mutant deficient in both <i>pilA</i> and <i>fliC</i> became completely resistant to infection. We then combined ΦBrmt with an LPS-targeting <i>Pbunavirus</i> phage, whose receptor was confirmed using a <i>ΔgalU</i> mutant. This receptor-diverse cocktail significantly suppressed the emergence of resistant variants across 10 diverse clinical isolates <i>in vitro</i> compared to single-phage treatments. These results underscore the importance of receptor-based molecular characterization as a critical first step in rational phage cocktail design. Our findings provide mechanistic insights into phage-host interactions and highlight a practical strategy for constructing receptor-diverse phage combinations to delay resistance evolution and enhance therapeutic robustness.