Single phage selection pressure results in broad cross-resistance to phage predation in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a bacterial pathogen that causes multiple infections, including pneumonia, ocular and urinary tract infections. Treatment of these infections is complicated by intrinsic and acquired resistance mechanisms to antimicrobial drugs. Bacteriophages, viruses that infect bacteria, offer a potential solution with numerous recent reports of successful phage therapy. However, much as antibiotic resistance emerges rapidly, so does bacteriophage resistance. In order to better understand resistance patterns that emerge from phage selective pressures, we investigated the development of cross-resistance, i.e. that exposure to one phage may cause resistance to both similar and unrelated phages. P. aeruginosa was exposed to selection pressure with highly lytic anti-Pa phages. Interestingly, isolates emerged that were resistant to the original challenge phage, and were also resistant to other different phages, with one isolate proving close to pan-resistant. Resistance was associated with a loss of growth fitness in minimal media, but not rich media, implicating defects in nutrient transporter(s). Whole-genome sequencing of the resistors revealed mutations in genes for LPS and the type IV pilus, known phage receptors, while large-scale deletions were associated with the broadest resistance. Collectively, these data suggest that a single phage selection pressure can result in emergence of resistors that are refractory to lytic killing by many other phages, underscoring the importance of selecting phages in a cocktail or treatment regimen that reduces the probability of developing broad resistance.