Abstract

The evolution of phage resistance poses an inevitable threat to the efficacy of phage therapy. The strategic selection of phage combinations that impose high genetic barriers to resistance and/or high compensatory fitness costs may mitigate this threat. However, for such a strategy to be effective, the evolution of phage resistance must be sufficiently constrained to be consistent. In this study, we isolated lytic phages capable of infecting a modified Klebsiella pneumoniae clinical isolate and characterized a total of 57 phage-resistant mutants that evolved from their prolonged coculture in vitro Single- and double-phage-resistant mutants were isolated from independently evolved replicate cocultures grown in broth or on plates. Among resistant isolates evolved against the same phage under the same conditions, mutations conferring resistance occurred in different genes, yet in each case, the putative functions of these genes clustered around the synthesis or assembly of specific cell surface structures. All resistant mutants demonstrated impaired phage adsorption, providing a strong indication that these cell surface structures functioned as phage receptors. Combinations of phages targeting different host receptors reduced the incidence of resistance, while, conversely, one three-phage cocktail containing two phages targeting the same receptor increased the incidence of resistance (relative to its two-phage, nonredundant receptor-targeting counterpart). Together, these data suggest that laboratory characterization of phage-resistant mutants is a useful tool to help optimize therapeutic phage selection and cocktail design.IMPORTANCE The therapeutic use of bacteriophage (phage) is garnering renewed interest in the setting of difficult-to-treat infections. Phage resistance is one major limitation of phage therapy; therefore, developing effective strategies to avert or lessen its impact is critical. Characterization of in vitro phage resistance may be an important first step in evaluating the relative likelihood with which phage-resistant populations emerge, the most likely phenotypes of resistant mutants, and the effect of certain phage cocktail combinations in increasing or decreasing the genetic barrier to resistance. If this information confers predictive power in vivo, then routine studies of phage-resistant mutants and their in vitro evolution should be a valuable means for improving the safety and efficacy of phage therapy in humans.

Highlights

  • The evolution of phage resistance poses an inevitable threat to the efficacy of phage therapy

  • The utility of such an approach depends on two factors: (i) how consistently phage resistance evolves in vitro and (ii) how closely phage resistance generated in vitro correlates with resistance generated in vivo. We addressed the former by characterizing how consistently phage resistance evolved from a clonal bacterial population exposed to lytic phages, singly or in combination, in independent replicate cocultures

  • Sequence analysis confirmed that P1 and P2 were related to strictly lytic phages and that neither phage harbored an identifiable integrase, toxin, or bacterial virulence factor, satisfying several recommended criteria for therapeutic phage selection [14]

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Summary

Introduction

The evolution of phage resistance poses an inevitable threat to the efficacy of phage therapy. Characterization of in vitro phage resistance may be an important first step in evaluating the relative likelihood with which phage-resistant populations emerge, the most likely phenotypes of resistant mutants, and the effect of certain phage cocktail combinations in increasing or decreasing the genetic barrier to resistance. If this information confers predictive power in vivo, routine studies of phage-resistant mutants and their in vitro evolution should be a valuable means for improving the safety and efficacy of phage therapy in humans. Phage-resistant isolates generated in vitro could conceivably be screened for changes in bacterial fitness, such as growth rates in various tissues and antibiotic susceptibility, which may, in turn, help avert the use of phages that select for more virulent bacterial subpopulations and help identify antibiotics that synergize with phage

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