Identification and impact on Pseudomonas aeruginosa virulence of mutations conferring resistance to a phage cocktail for phage therapy

Phage therapy represents a promising strategy for curing bacterial infections refractory to antibiotics. However, the success rate of phage therapy may be lowered by the emergence of bacterial resistance to the phages used for therapy. In this work, we studied the resistance to the CK4 cocktail, which is a mixture composed of four phages able to cure Pseudomonas aeruginosa infections in animal models. CK4-resistant mutants were easily isolated from cultures grown in either a standard laboratory medium or an artificial sputum medium mimicking the composition of the airway fluid of cystic fibrosis (CF) patients, who are highly susceptible to P. aeruginosa chronic lung infections. In both cases, CK4-resistant mutants resulted in being defective in lipopolysaccharide (LPS) biosynthesis. Accordingly, all CK4 phages were unable to infect wzy mutants lacking the O-antigen polymerase. A survey of the other 15 P. aeruginosa phages isolated from different environmental sources showed that they all needed either wzy or the Type IV-pilus (T4P) biosynthetic gene pilQ for the infection. Overall, our data suggest that 16 out of the 19 analyzed Pseudomonas phages may use either the LPS or the T4P as a receptor. Interestingly, CK4-resistant mutants devoid of the O-antigen had strongly attenuated virulence in a zebrafish embryo infection model, and the lack of T4P also decreased virulence in zebrafish. With respect to isolates from patients with CF, phages not reproducing in the Δwzy mutant had a wider host range than those requiring pilQ, suggesting that phages dependent on PAO1-type T4P may have limited therapeutic value for treating CF-related infections.