Abstract
Phages show promising application prospects as antibacterial agents for the treatment of drug-resistant bacterial infections. However, the rapid evolution of phage resistance in bacteria has compromised the therapeutic efficacy. Here, we first screened a broad-host-range phage PC13 based on the Salmonella-phage library. Then, phage-resistant Salmonella mutants were obtained by serial co-culture passages of PC13 Salmonella enterica subsp. enterica serovar Enteritidis C2. Whole-genome sequencing analyses were performed on the phages and resistant strains to decipher the potential molecular mechanisms of phage-host interactions and collateral sensitivity. The bacteriostatic and therapeutic effects of the triple phage cocktail (PC13 + PC11+PC16) against S. Enteritidis C2 were evaluated using in vitro bacteriostatic assays and in vivo chicken infection models. The results showed that PC13 lysed 90% (81/90) of the tested chicken-derived Salmonella isolates. PC11 achieved a lysis rate of 95% against all 20 PC13-resistant mutants. Relative to single-phage PC13 and the PC13 + PC11 cocktail, the triple-phage cocktail PC13 + PC11 + PC16 markedly extended bacteriostatic time in vitro (p < 0.01). The phage cocktail significantly prolonged the bacteriostatic time in vitro compared to individual phages (p < 0.01). Comparative genomic analysis revealed that 95% of the PC13-resistant mutants harbored missense, nonsense, or frameshift mutations in genes associated with lipopolysaccharide (LPS) core oligosaccharide synthesis. These PC13-resistant mutants exhibited significantly enhanced susceptibility to PC11 and PC16 relative to the wild-type strain (p < 0.05). We propose that Salmonella evaded PC13 infection by altering LPS structure via these mutations, and these genetic changes simultaneously exposed the putative binding receptors predicted for PC11 and PC16, conferring collateral sensitivity of the resistant mutants to the latter two phages. In the chicken S. Enteritidis infection model, compared with the Salmonella treatment group, the fecal Salmonella load of chicks in the cocktail treatment group decreased by about 4.0 log CFU/g, and there was no significant difference in body weight changes compared with control group. In conclusion, a phage cocktail against S. Enteritidis with significantly enhanced antibacterial activity was rationally formulated constructed by targeting phage-resistant escape mutants generated during phage-bacterial coevolution. The collateral sensitivity effect uncovered in this study explains its superior inhibitory performance, which provides theoretical and practical strategies for developing highly efficient phage therapy.