Activity of fosfomycin and amikacin against fosfomycin-heteroresistant Escherichia coli strains in a hollow-fiber infection model.

Objectives:To evaluate human-like intravenous doses of fosfomycin (8g/Q8h) and amikacin (15mg/kg/Q24h) efficacy in monotherapy and in combination against six fosfomycin-heteroresistant Escherichia coli isolates using a hollow-fiber infection model (HFIM).Materials and methods:Six fosfomycin-heteroresistant E. coli isolates (4 with strong mutator phenotype) and the control strain E. coli ATCC 25922 were used. Mutant frequencies for rifampin (100mg/L), fosfomycin (50 and 200mg/L) and amikacin (32mg/L) were determined. Fosfomycin and amikacin MICs were assessed by agar dilution (AD), gradient strip (GSA) and broth microdilution (BMD) assays. Fosfomycin and amikacin synergies were studied by checkerboard and time-kill assays at different concentrations. Fosfomycin (8g/Q8h) and amikacin (15mg/kg/Q24h) efficacy alone and in combination were assessed using a HFIM.Results:Five isolates were resistant to fosfomycin by AD and BMD, but all susceptible by GSA. All isolates were considered susceptible to amikacin. Antibiotic combinations were synergistic in two isolates and no antagonism was detected. In time-kill assays, all isolates survived under fosfomycin at 64mg/L, although, at 307mg/L, only the normomutators and two hypermutators survived. Four isolates survived under 16mg/L amikacin and none at 45mg/L. No growth was detected under combination conditions. In HFIM, fosfomycin and amikacin monotherapies failed to sterilise bacterial cultures, however, fosfomycin and amikacin combination showed a rapid eradication.Conclusions.There may be a risk of treatment failure of fosfomycin-heteroresistant E. coli isolates using either amikacin or fosfomycin in monotherapy. These results support that the combination amikacin-fosfomycin can rapidly decrease bacterial burden and prevent the emergence of resistant subpopulations against fosfomycin-heteroresistant strains.

Interplay among Different Fosfomycin Resistance Mechanisms in Klebsiella pneumoniae.

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.

Molecular insights into fosfomycin resistance in Escherichia coli.

Objectives: Fosfomycin activity in Escherichia coli depends on several genes of unknown importance for fosfomycin resistance. The objective was to characterize the role of uhpT , glpT , cyaA and ptsI genes in fosfomycin resistance in E. coli. Methods: WT E. coli BW25113 and null mutants, Δ uhpT , Δ glpT , Δ cyaA , Δ ptsI , Δ glpT-uhpT , Δ glpT-cyaA , Δ glpT-ptsI , Δ uhpT-cyaA , Δ uhpT-ptsI and Δ ptsI-cyaA , were studied. Susceptibility to fosfomycin was tested using CLSI guidelines. Fosfomycin mutant frequencies were determined at concentrations of 64 and 256 mg/L. Fosfomycin in vitro activity was tested using time-kill assays at concentrations of 64 and 307 mg/L (human C max ). Results: Fosfomycin MICs were: WT E. coli BW25113 (2 mg/L), Δ glpT (2 mg/L), Δ uhpT (64 mg/L), Δ cyaA (8 mg/L), Δ ptsI (2 mg/L), Δ glpT-uhpT (256 mg/L), Δ glpT-cyaA (8 mg/L), Δ glpT-ptsI (2 mg/L), Δ uhpT-cyaA (512 mg/L), Δ uhpT-ptsI (64 mg/L) and Δ ptsI-cyaA (32 mg/L). In the mutant frequency assays, no mutants were recovered from BW25113. Mutants appeared in Δ glpT , Δ uhpT , Δ cyaA and Δ ptsI at 64 mg/L and in Δ uhpT and Δ cyaA at 256 mg/L. Δ glpT-ptsI , but not Δ glpT-cyaA , Δ uhpT-cyaA or Δ uhpT-ptsI , increased the mutant frequency compared with the highest frequency found in each single mutant. In time-kill assays, all mutants regrew at 64 mg/L. Initial bacterial reductions of 2-4 log 10 cfu/mL were observed for all strains, except for Δ uhpT-ptsI , Δ glpT-uhpT and Δ uhpT-cyaA . Only Δ glpT and Δ ptsI mutants were cleared using 307 mg/L. Conclusions: Fosfomycin MIC may not be a good efficacy predictor, as highly resistant mutants may appear, depending on other pre-existing mutations with no impact on MIC.

In Vitro Activity of Cefiderocol Compared to Other Antimicrobials against a Collection of Metallo-Beta-Lactamase-Producing Gram-Negative Bacilli from Southern Spain.

In this study, we aimed to comparatively evaluate the in vitro activity of cefiderocol versus other antimicrobials against a well-characterized collection of metallo-beta-lactamase (MBL)-producing Gram-negative bacilli (MBL-GNB) isolates from hospitals in Andalusia, Spain. We recovered 232 MBL-GNB from Andalusian hospitals, including 160 Enterobacterales and 72 nonfermenting Gram-negative bacilli belonging to 44 different clones (2015 to 2020). Cefiderocol and comparator MICs were determined with commercial methods (UMIC [Bruker] and EUMDROXF [Sensititre; Thermo Fisher], respectively). EUCAST breakpoints were used for all antimicrobials tested, and CLSI also was used for cefiderocol. Control strains used were E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853. Cefiderocol showed potent in vitro activity against isolates tested, regardless of breakpoint (susceptibility rates, 85.3% for EUCAST versus 96.6% for CLSI, P < 0.001). MIC ranges for Enterobacterales and nonfermenting Gram-negative bacilli (NF-GNB) were ≤0.03 to 1 mg/L and 0.06 to 2 (IMP), 0.06 to 8 mg/L and 0.06 to 16 (VIM), 0.25 to 16 mg/L and 2 to 16 mg/L (NDM), respectively, and 0.25 to 8 mg/L for double MBL-producing Enterobacterales. By species, all cefiderocol-susceptible rates were over 90%, except Klebsiella oxytoca, Enterobacter cloacae, Escherichia coli, and Acinetobacter spp. Significant differences were observed comparing resistant isolates between Enterobacterales and NF-GNB by EUCAST (19.4% versus 4.2%, P < 0.01), but not by CLSI (4.4% versus 1.4%, P = 0.2). Cefiderocol was the most active antimicrobial tested. Cefiderocol showed excellent in vitro activity against MBL-GNB, especially NF-GNB; almost all isolates resistant to comparators were susceptible. IMPORTANCE This article demonstrates the efficacy of cefiderocol against a large collection of well-characterized metallo-beta-lactamase-producing isolates, some of them even producing double carbapenemases. Furthermore, cefiderocol activity is compared to other novel broad-spectrum antimicrobials with activity against carbapenemases.