Temperate phage-antibiotic synergy is widespread, but varies by phage, host, and antibiotic pairing

With a decline in antibiotic effectiveness, there is a renewed interest in bacteriophage (phage) therapy. Phages are bacterial-specific viruses that can be used alone or with antibiotics to reduce bacterial load. Most phages are unsuitable for therapy because they are temperate and can integrate into the host genome, forming a lysogen which is protected from subsequent phage infections. However, integrated phages can be awakened by stressors such as antibiotics. This interaction was previously reported to result in a potent synergy between antibiotic classes and a model E. coli temperate phage, which can readily eradicate the bacterium at sub-lethal concentrations of antibiotics, despite the poor effectiveness of the phage alone. Here we explore the generalizability of this synergy to a clinically relevant pathogen: Pseudomonas aeruginosa. Thirty-six temperate phages isolated from clinical strains were screened for synergy with six antibiotics (ciprofloxacin, levofloxacin, meropenem, piperacillin, tobramycin, polymyxin B), using checkerboard assays. Interestingly, our screen identified phages that can synergize with each antibiotic, despite their widely differing targets - however, these are highly phage-antibiotic and phage-host pairing specific. Screening the strongest pairings across multiple clinical strains reveal that these phages can reduce the antibiotic minimum inhibitory concentration up to 32-fold, even in a resistant isolate, functionally re-sensitizing the bacterium to the antibiotic. When meropenem and tobramycin were effective synergistic agents, they did not reduce the frequency of lysogens, suggesting a mechanism of action independent of the temperate nature of the phages. In contrast, ciprofloxacin and piperacillin were able to reduce the frequency of lysogeny, the former by inducing phages - as previously reported in E. coli. Curiously, synergy with piperacillin reduced the frequency of lysogeny, but not by inducing the phages, and therefore likely acts by biasing the phage away from lysogeny in the initial infection. Overall, our findings indicate that temperate phages can act as adjuvants to antibiotics in clinically relevant pathogens, even in the presence of antibiotic resistance, thereby drastically expanding their therapeutic potential.