The crisis of MDR pathogens has led to rare use of bacteriophages (phages) as a therapeutic. However, bacterial resistance to phage threatens to undermine phage therapy. While well explored in laboratory settings, there is little research regarding the development of phage resistance under therapeutic conditions. In this study, we explore the coevolutionary arms race between bacteria and phages, using clinically-derived isolates of Extraintestinal pathogenic E. coli (ExPEC) and well-studied phages (primarily ϕHP3) which have been used therapeutically for patients with infections recalcitrant to antibiotics.

The development of phage resistance (“resisters”) by the bacteria was studied using two parallel methods: a plate-based method, and a murine model of bacteremia. Resisters isolated from both methods were assessed for viability in simulated host environments. Most murine-derived resisters were attenuated in human urine, and all resisters failed to survive in human blood. Strikingly, tested resisters further failed to cause severe disease in the very model of bacteremia from which they were isolated. Sequencing revealed highly conserved mechanisms of resistance, suggesting ST131 isolates preferentially develop resistance to ϕHP3 through changes to its receptors. Using the Appelman’s method or a continuous bioreactor that we designed, we directed the evolution of ϕHP3. The resulting two phages, ϕHP3.1 and ϕHP3.2, were remarkably similar to parental ϕHP3.

To test the efficacy of phage cocktails to prevent phage resistance, we constructed four phage preparations (three cocktails and a singular phage). Measuring bacterial growth and regrowth revealed that, as is currently believed, phage cocktails are better at preventing the development of phage resistance than single phage preparations. Interestingly, the cocktails containing evolved phages were more efficient at preventing resistance the wild type cocktail. This finding underscores the importance of anticipating resistance mechanisms and adapting phages to common resisters.

In this study, we found that ExPEC resistance to phage is associated with attenuation in host microenvironments and systemic infection. We found that phage-resistant pathogenic E. coli readily develop, but are often attenuated. Further, cocktails of phages can decrease the likelihood of resister development and, likely, impose greater fitness losses in breakthrough resisters. This work lays important groundwork for the use of phage therapeutics while countering bacterial resilience.