Interplay among Different Fosfomycin Resistance Mechanisms in Klebsiella pneumoniae

Abstract

The objectives of this study were to characterize the role of the uhpT, glpT, and fosA genes in fosfomycin resistance in Klebsiella pneumoniae and evaluate the use of sodium phosphonoformate (PPF) in combination with fosfomycin. Seven clinical isolates of K. pneumoniae and the reference strain (ATCC 700721) were used, and their genomes were sequenced. ΔuhpT, ΔglpT, and ΔfosA mutants were constructed from two isolates and K. pneumoniae ATCC 700721. Fosfomycin susceptibility testing was done by the gradient strip method. Synergy between fosfomycin and PPF was studied by checkerboard assay and analyzed using SynergyFinder. Spontaneous fosfomycin mutant frequencies at 64 and 512 mg/liter, in vitro activity using growth curves with fosfomycin gradient concentrations (0 to 256mg/liter), and time-kill assays at 64 and 307 mg/liter were evaluated with and without PPF (0.623 mM). The MICs of fosfomycin against the clinical isolates ranged from 16 to ≥1,024 mg/liter. The addition of 0.623 mM PPF reduced fosfomycin MIC between 2- and 8-fold. Deletion of fosA led to a 32-fold decrease. Synergistic activities were observed with the combination of fosfomycin and PPF (most synergistic area at 0.623 mM). The lowest fosfomycin-resistant mutant frequencies were found in ΔfosA mutants, with decreases in frequency from 1.69 × 10^-1 to 1.60 × 10^-5 for 64 mg/liter of fosfomycin. In the final growth monitoring and time-kill assays, fosfomycin showed a bactericidal effect only with the deletion of fosA and not with the addition of PPF. We conclude that fosA gene inactivation leads to a decrease in fosfomycin resistance in K. pneumoniae The pharmacological approach using PPF did not achieve enough activity, and the effect decreased with the presence of fosfomycin-resistant mutations.

Keywords: Klebsiella pneumoniae; antimicrobial resistance; fosfomycin.

FIG 1

(A) Synergistic activity of fosfomycin in combination with PPF against K. pneumoniae ATCC 700721, Kp12, and Kp142 strains. Red and green areas represent synergy (synergy score greater than +10) and antagonism (less than −10), respectively. White rectangles show the maximum synergy area. (B) Fosfomycin and PPF dose-response curves.

FIG 2

Fosfomycin-resistant mutant frequencies of K. pneumoniae ATCC 700721, Kp12, and Kp142 wild-type and isogenic mutant strains. Empty and full boxes show individual results, and black lines represent the median values of the three replicates.

FIG 3

Viability of K. pneumoniae ATCC 700721, Kp12, and Kp142 wild-type and isogenic mutant strains against fosfomycin concentrations from 0 to 256 mg/liter, with and without 0.623 mM PPF after 24 h. Lines stand for mean values of measured viability. Symbols represent single results of the replicates.