Engineering Pseudomonas aeruginosa for optimised isolation and characterisation of functionally diverse phages for phage therapy

Phage therapy is a promising treatment for multidrug resistant bacterial infections, and for patients no longer able to tolerate antibiotic treatments. A major challenge for phage therapy is emergent phage resistance, which bacteria can acquire by structurally modifying or masking phage receptors to prevent adsorption. Functionally diverse phage cocktails that target a broad range of receptors are less prone to resistance as there is a higher fitness cost associated with modifying multiple receptors. Efficiently designing functionally diverse phage cocktails requires comprehensive phage libraries targeting a broad range of receptors. Here, we engineered P. aeruginosa PAO1 to create a panel of unmarked deletion mutants for efficient phage receptor characterisation and optimised isolation of functionally diverse phages. First, we eliminated the defensive restriction endonuclease subunit, HsdR, to create a more promiscuous host for propagating and isolating phages. Phage propagation efficiency increased 2.15-fold and isolation yields from sewage samples increased seven-fold compared to wildtype PAO1. Next, we used ∆hsdR as the parent strain to delete a series of known P. aeruginosa phage receptors. Fifty P. aeruginosa phages from the Citizen Phage Library were screened against the panel of receptor mutants, revealing that all of them required either type IV pili, outer core LPS or O-antigen as their receptor. To isolate phages targeting alternative receptors, we enriched sewage samples on a ∆hsdR ∆pilA ∆galU mutant that lacks these three most common receptors. This led to the isolation of a novel phage, CPL01276, predicted to bind to the LPS inner core. We identified a trade-off between resistance to CPL01276 and another phage, CPL00272, that targets the LPS outer core, mediated by host-derived LPS modifications. Combined treatment with CPL00272 and CPL01276 delayed the emergence of phage resistance for at least three times longer than individual phage treatments. This work highlights the importance of constructing functionally diverse phage libraries and strategically designing cocktails considering phage receptors to minimise the likelihood of emergent phage resistance.<p></p>