Phage Resistance in Multidrug-Resistant Klebsiella pneumoniae ST258 Evolves via Diverse Mutations That Culminate in Impaired Adsorption

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.

Keywords: bacteriophage cocktails; bacteriophage resistance; bacteriophage therapy.

FIG 1

Structural characterization and plaque morphology of environmentally isolated phages. (a to f) Transmission electron micrographs (a to c) and plaque appearance (d to f) of Pharr, ϕKpNIH-2, and ϕKpNIH-10. The plated bacterial hosts were MKP103 (d and e) and P1+P2-resistant MKP103 (f).

FIG 2

Phage lysis and resistant bacterial outgrowth. (a and b) Phage lysis of host bacterial cultures in a shaking incubator at a multiplicity of infection (MOI) of 3. Error bars denote standard deviations among triplicate samples. MKP103^P1+P2-R, P1+P2-resistant MKP103. (c and d) Growth of phage-resistant bacterial populations over time in cocultures of MKP103-phage. For each phage or phage combination, data from 10 independent cocultures were plotted. Total MOI remained constant (P1, MOI of 6; P2, MOI of 6; P1+P2, MOI of 3 + 3; P1+P2+P10, MOI of 2 + 2 + 2; P1+P2+P6, MOI of 2 + 2 + 2). P1, Pharr; P2, ϕKpNIH-2; P6, ϕKpNIH-6; P10, ϕKpNIH-10.

FIG 3

P1-, P2-, and P1+P2-resistant mutant generation in planktonic culture and on plates. (a to c) Outgrowth of planktonic bacterial populations with resistance to P1, P2, and P1+P2, respectively. Each panel depicts growth from 10 independent MKP103-phage coculture replicates, from which one colony-purified isolate from each was sequenced. Replicates in panel c were selected from a pool in order to exclude replicates in which phage resistance was not detectable by an increase in optical density. (d to f) Appearance of colonies with resistance to P1, P2, and P1+P2. Photos of phage spots were taken at 40 h (d), 64 h (e), and 80 h (f) after phage plating.

FIG 4

Phage adsorption among resistant isolates. Free phage titers were measured after brief incubation with each phage-resistant bacterial isolate and calculated as a percentage of phage recovered from the medium-only condition. (a and b) Adsorption of phage P1 by P1-resistant and P1+P2-resistant isolates. (c and d) Adsorption of phage P2 by P2-resistant and P1+P2-resistant isolates. Error bars denote standard deviations among triplicate samples. Free phage recovery following incubation with MKP103, denoted by (+), was minimal. *, 1.2% (P1); **, 0.04% (P2).