Evaluation of temocillin efficacy against KPC-2-producing Klebsiella pneumoniae isolates in a hollow-fibre infection model.

BACKGROUND: Temocillin is an old antimicrobial that is resistant to hydrolysis by ESBLs but has variable activity against carbapenemase-producing Enterobacteriaceae. The current EUCAST susceptibility breakpoints for Enterobacterales are set at ≤16 mg/L (susceptible with increased exposure) based on a dose of 2 g q8h, but there is limited information on the efficacy of this dose against temocillin-susceptible carbapenemase-producing Klebsiella pneumoniae isolates. OBJECTIVES: To evaluate the efficacy of this dose using a hollow-fibre infection model (HFIM) against six KPC-2-producing clinical isolates of K. pneumoniae. METHODS: The isolates were characterized by WGS and temocillin susceptibility was determined using standard and high inoculum temocillin. Mutant frequencies were estimated and temocillin activity was tested in time-kill assays and in the HFIM. At standard conditions, three of the isolates were classified as susceptible (MIC ≤ 16 mg/L) and three as resistant (MIC > 16 mg/L). The HFIM was performed over 3 days to mimic human-like pharmacokinetics of 2 g q8h. Bacterial counts were performed by plating on Mueller-Hinton agar (MHA) and MHA containing 64 mg/L temocillin to detect resistant subpopulations. RESULTS: All isolates showed a reduction in bacterial population of at least 3 log cfu/mL within the first 8 h of simulated treatment in the hollow-fibre assay. Regrowth was observed for the three resistant isolates and one of the susceptible ones. The MIC value for these isolates was higher by at least two dilutions compared with their initial values. CONCLUSIONS: These data suggest that an optimized pharmacokinetic regimen may be of clinical interest for the treatment of KPC-2-producing K. pneumoniae susceptible to temocillin. These data showed activity of temocillin against KPC-2-producing K. pneumoniae susceptible to temocillin; however, a dose of 2g q8h administered over 30 min may be inadequate to prevent the emergence of resistant variants.

Effect of Glycerol on Fosfomycin Activity against Escherichia coli.

Fosfomycin is an antimicrobial that inhibits the biosynthesis of peptidoglycan by entering the bacteria through two channels (UhpT and GlpT). Glycerol is clinically used as a treatment for elevated intracranial pressure and induces the expression of glpT in Escherichia coli. Glycerol might offer synergistic activity by increasing fosfomycin uptake. The present study evaluates the use of glycerol at physiological concentrations in combination with fosfomycin against a collection of isogenic mutants of fosfomycin-related genes in E. coli strains. Induction of fosfomycin transporters, susceptibility tests, interaction assays, and time-kill assays were performed. Our results support the notion that glycerol allows activation of the GlpT transporter, but this induction is delayed over time and is not homogeneous across the bacterial population, leading to contradictory results regarding the enhancement of fosfomycin activity. The susceptibility assays showed an increase in fosfomycin activity with glycerol in the disk diffusion assay but not in the agar dilution or broth microdilution assays. Similarly, in the time-kill assays, the effect of glycerol was absent by the emergence of fosfomycin-resistant subpopulations. In conclusion, glycerol may not be a good candidate for use as an adjuvant with fosfomycin.

Role of inorganic phosphate concentrations in in vitro activity of fosfomycin.

OBJECTIVES: The objective of this study was to evaluate the in vitro activity of fosfomycin under different physiological concentrations of inorganic phosphate (Pi). METHODS: The wild-type BW25113 strain, four isogenic mutants (ΔglpT, ΔuhpT, ΔglpT-uhpT, and ΔphoB) and six clinical isolates of Escherichia coli with different fosfomycin susceptibilities were used. EUCAST breakpoints were used. Susceptibility was evaluated by agar dilution using standard Mueller-Hinton agar (Pi concentration of 1 mM similar to human plasma concentration) and supplemented with Pi (13 and 42 mM, minimum and maximum urinary Pi concentrations) and/or glucose-6-phosphate (25 mg/L). Fosfomycin transporter promoter activity was assayed using PglpT::gfpmut2 or PuhpT::gfpmut2 promoter fusions in standard Mueller-Hinton Broth (MHB), supplemented with Pi (13 or 42 mM) ± glucose-6-phosphate. Fosfomycin activity was quantified, estimating fosfomycin EC50 under different Pi concentrations (1, 13 and 42 mM + glucose-6-phosphate) and in time-kill assays using fosfomycin concentrations of 307 (maximum plasma concentration (Cmax)), 1053 and 4415 mg/L (urine Cmax range), using MHB with 28 mM Pi (mean urine Pi concentration) + 25 mg/L glucose-6-phosphate. RESULTS: All the strains showed decreased susceptibility to fosfomycin linked to increased Pi concentrations: 1-4 log2 dilution differences from 1 to 13 mM, and 1-8 log2 dilution differences at 42 mM Pi. Changes in phosphate concentration did not affect the expression of fosfomycin transporters. By increasing Pi concentrations higher fosfomycin EC50 bacterial viability was observed, except against ΔglpT-uhpT. The increase in Pi reduced the bactericidal effect of fosfomycin. DISCUSSION: Pi variations in physiological fluids may reduce fosfomycin activity against E. coli. Elevated Pi concentrations in urine may explain oral fosfomycin failure in non-wild-type but fosfomycin-susceptible E. coli strains.

Contribution of hypermutation to fosfomycin heteroresistance in Escherichia coli.

OBJECTIVES: To explore the effect of combining defects in DNA repair systems with the presence of fosfomycin-resistant mechanisms to explain the mechanisms underlying fosfomycin heteroresistance phenotypes in Enterobacteriaceae. MATERIALS AND METHODS: We used 11 clinical Escherichia coli isolates together with isogenic single-gene deletion mutants in the E. coli DNA repair system or associated with fosfomycin resistance, combined with double-gene deletion mutants. Fosfomycin MICs were determined by gradient strip assay (GSA) and broth microdilution (BMD). Mutant frequencies for rifampicin (100 mg/L) and fosfomycin (50 and 200 mg/L) were determined. Using two starting inocula, in vitro fosfomycin activity was assessed over 24 h in growth (0.5-512 mg/L) and time-kill assays (64 and 307 mg/L). RESULTS: Strong and weak mutator clinical isolates and single-gene deletion mutants, except for ΔuhpT and ΔdnaQ, were susceptible by GSA. By BMD, the percentage of resistant clinical isolates reached 36%. Single-gene deletion mutants showed BMD MICs similar to those for subpopulations by GSA. Strong mutators showed a higher probability of selecting fosfomycin mutants at higher concentrations. By combining the two mechanisms of mutation, MICs and ranges of resistant subpopulations increased, enabling strains to survive at higher fosfomycin concentrations in growth monitoring assays. In time-kill assays, high inocula increased survival by 37.5% at 64 mg/L fosfomycin, compared with low starting inocula. CONCLUSIONS: The origin and variability of the fosfomycin heteroresistance phenotype can be partially explained by high mutation frequencies together with mechanisms of fosfomycin resistance. Subpopulations should be considered until clinical meaning is established.