Synergistic Effect of SOS Response and GATC Methylome Suppression on Antibiotic Stress Survival in Escherichia coli.

The suppression of the SOS response has been shown to enhance the in vitro activity of quinolones. Furthermore, Dam-dependent base methylation has an impact on susceptibility to other antimicrobials affecting DNA synthesis. Here, we investigated the interplay between these two processes, alone and in combination, in terms of antimicrobial activity. A genetic strategy was used employing single- and double-gene mutants for the SOS response (recA gene) and the Dam methylation system (dam gene) in isogenic models of Escherichia coli both susceptible and resistant to quinolones. Regarding the bacteriostatic activity of quinolones, a synergistic sensitization effect was observed when the Dam methylation system and the recA gene were suppressed. In terms of growth, after 24 h in the presence of quinolones, the Δdam ΔrecA double mutant showed no growth or delayed growth compared to the control strain. In bactericidal terms, spot tests showed that the Δdam ΔrecA double mutant was more sensitive than the ΔrecA single mutant (about 10- to 102-fold) and the wild type (about 103- to 104-fold) in both susceptible and resistant genetic backgrounds. Differences between the wild type and the Δdam ΔrecA double mutant were confirmed by time-kill assays. The suppression of both systems, in a strain with chromosomal mechanisms of quinolone resistance, prevents the evolution of resistance. This genetic and microbiological approach demonstrated the enhanced sensitization of E. coli to quinolones by dual targeting of the recA (SOS response) and Dam methylation system genes, even in a resistant strain model.

RecA inactivation as a strategy to reverse the heteroresistance phenomenon in clinical isolates of Escherichia coli.

RecA inhibition could be an important strategy to combat antimicrobial resistance because of its key role in the SOS response, DNA repair and homologous recombination contributing to bacterial survival. This study evaluated the impact of RecA inactivation on heteroresistance in clinical isolates of Escherichia coli and their corresponding recA-deficient isogenic strains to multiple classes of antimicrobial agents. A high frequency (>30%) of heteroresistance was observed in this collection of clinical isolates. Deletion of the recA gene led to a marked reduction in heteroresistant subpopulations, especially against quinolones or ß-lactams. The molecular basis of heteroresistance was associated with an increase in copy number of plasmid-borne resistance genes (blaTEM-1B) or tandem gene amplifications (qnrA1). Of note, in the absence of the recA gene, the increase in copy number of resistance genes was suppressed. This makes the recA gene a promising target for combating heteroresistance.

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.

Effect of RecA inactivation and detoxification systems on the evolution of ciprofloxacin resistance in Escherichia coli.

BACKGROUND: Suppression of SOS response and overproduction of reactive oxygen species (ROS) through detoxification system suppression enhance the activity of fluoroquinolones. OBJECTIVES: To evaluate the role of both systems in the evolution of resistance to ciprofloxacin in an isogenic model of Escherichia coli. METHODS: Single-gene deletion mutants of E. coli BW25113 (wild-type) (ΔrecA, ΔkatG, ΔkatE, ΔsodA, ΔsodB), double-gene (ΔrecA-ΔkatG, ΔrecA-ΔkatE, ΔrecA-ΔsodA, ΔrecA-ΔsodB, ΔkatG-ΔkatE, ΔsodB-ΔsodA) and triple-gene (ΔrecA-ΔkatG-ΔkatE) mutants were included. The response to sudden high ciprofloxacin pressure was evaluated by mutant prevention concentration (MPC). The gradual antimicrobial pressure response was evaluated through experimental evolution and antibiotic resistance assays. RESULTS: For E. coli BW25113 strain, ΔkatE, ΔsodB and ΔsodB/ΔsodA mutants, MPC values were 0.25 mg/L. The ΔkatG, ΔsodA, ΔkatG/katE and ΔrecA mutants showed 2-fold reductions (0.125 mg/L). The ΔkatG/ΔrecA, ΔkatE/ΔrecA, ΔsodA/ΔrecA, ΔsodB/ΔrecA and ΔkatG/ΔkatE/ΔrecA strains showed 4-8-fold reductions (0.03-0.06 mg/L) relative to the wild-type. Gradual antimicrobial pressure increased growth capacity for ΔsodA and ΔsodB and ΔsodB/ΔsodA mutants (no growth in 4 mg/L) compared with the wild-type (no growth in the range of 0.5-2 mg/L). Accordingly, increased growth was observed with the mutants ΔrecA/ΔkatG (no growth in 2 mg/L), ΔrecA/ΔkatE (no growth in 2 mg/L), ΔrecA/ΔsodA (no growth in 0.06 mg/L), ΔrecA/ΔsodB (no growth in 0.25 mg/L) and ΔrecA/ΔkatG/ΔkatE (no growth in 0.5 mg/L) compared with ΔrecA (no growth in the range of 0.002-0.015 mg/L). CONCLUSIONS: After RecA inactivation, gradual exposure to ciprofloxacin reduces the evolution of resistance. After suppression of RecA and detoxification systems, sudden high exposure to ciprofloxacin reduces the evolution of resistance in E. coli.

Synergistic Quinolone Sensitization by Targeting the recA SOS Response Gene and Oxidative Stress.

Suppression of the recA SOS response gene and reactive oxygen species (ROS) overproduction have been shown, separately, to enhance fluoroquinolone activity and lethality. Their putative synergistic impact as a strategy to potentiate the efficacy of bactericidal antimicrobial agents such as fluoroquinolones is unknown. We generated Escherichia coli mutants that exhibited a suppressed ΔrecA gene in combination with inactivated ROS detoxification system genes (ΔsodA, ΔsodB, ΔkatG, ΔkatE, and ΔahpC) or inactivated oxidative stress regulator genes (ΔoxyR and ΔrpoS) to evaluate the interplay of both DNA repair and detoxification systems in drug response. Synergistic sensitization effects, ranging from 7.5- to 30-fold relative to the wild type, were observed with ciprofloxacin in double knockouts of recA and inactivated detoxification system genes. Compared to recA knockout, inactivation of an additional detoxification system gene reduced MIC values up to 8-fold. In growth curves, no growth was evident in mutants doubly deficient for recA gene and oxidative detoxification systems at subinhibitory concentrations of ciprofloxacin, in contrast to the recA-deficient strain. There was a marked reduction of viable bacteria in a short period of time when the recA gene and other detoxification system genes (katG, sodA, or ahpC) were inactivated (using absolute ciprofloxacin concentrations). At 4 h, a bactericidal effect of ciprofloxacin was observed for ΔkatG ΔrecA and ΔahpC ΔrecA double mutants compared to the single ΔrecA mutant (Δ3.4 log10 CFU/ml). Synergistic quinolone sensitization, by targeting the recA gene and oxidative detoxification stress systems, reinforces the role of both DNA repair systems and ROS in antibiotic-induced bacterial cell death, opening up a new pathway for antimicrobial sensitization.

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.

Effect of RecA inactivation on quinolone susceptibility and the evolution of resistance in clinical isolates of Escherichia coli.

BACKGROUND: SOS response suppression (by RecA inactivation) has been postulated as a therapeutic strategy for potentiating antimicrobials against Enterobacterales. OBJECTIVES: To evaluate the impact of RecA inactivation on the reversion and evolution of quinolone resistance using a collection of Escherichia coli clinical isolates. METHODS: Twenty-three E. coli clinical isolates, including isolates belonging to the high-risk clone ST131, were included. SOS response was suppressed by recA inactivation. Susceptibility to fluoroquinolones was determined by broth microdilution, growth curves and killing curves. Evolution of quinolone resistance was evaluated by mutant frequency and mutant prevention concentration (MPC). RESULTS: RecA inactivation resulted in 2-16-fold reductions in fluoroquinolone MICs and modified EUCAST clinical category for several isolates, including ST131 clone isolates. Growth curves and time-kill curves showed a clear disadvantage (up to 10 log10 cfu/mL after 24 h) for survival in strains with an inactivated SOS system. For recA-deficient mutants, MPC values decreased 4-8-fold, with values below the maximum serum concentration of ciprofloxacin. RecA inactivation led to a decrease in mutant frequency (≥103-fold) compared with isolates with unmodified SOS responses at ciprofloxacin concentrations of 4×MIC and 1 mg/L. These effects were also observed in ST131 clone isolates. CONCLUSIONS: While RecA inactivation does not reverse existing resistance, it is a promising strategy for increasing the effectiveness of fluoroquinolones against susceptible clinical isolates, including high-risk clone isolates.

Role of low-level quinolone resistance in generating tolerance in Escherichia coli under therapeutic concentrations of ciprofloxacin.

BACKGROUND: Tolerance (including persistence) and resistance result in increased survival under antibiotic pressure. OBJECTIVES: We evaluated the interplay between resistance and tolerance to ciprofloxacin under therapeutic and killing conditions to determine the contribution of low-level quinolone resistance (LLQR) mechanisms to tolerance. We also determined how the interaction between resistance (LLQR phenotypes) and tolerance was modified under SOS response suppression. METHODS: Twelve isogenic Escherichia coli strains harbouring quinolone resistance mechanisms combined with SOS response deficiency and six clinical E. coli isolates (LLQR or non-LLQR) were evaluated. Survival (tolerance or persistence) assays were used to measure surviving bacteria after a short period (up to 4 h) of bactericidal antibiotic treatment under therapeutic and killing concentrations of ciprofloxacin [1 mg/L, EUCAST/CLSI breakpoint for resistance; and 2.5 mg/L, peak serum concentration (Cmax) of this drug]. RESULTS: QRDR substitutions (S83L in GyrA alone or combined with S80R in ParC) significantly increased the fraction of tolerant bacteria (2-4 log10 cfu/mL) after exposure to ciprofloxacin at clinically relevant concentrations. The impact on tolerant bacteria due to SOS response suppression (including persistence mediated by the tisB gene) was reversed by LLQR mechanisms at therapeutic concentrations. Furthermore, no reduction in the fraction of tolerant bacteria due to SOS response suppression was observed when S83L in GyrA plus S80R in ParC were combined. CONCLUSIONS: Tolerance and quinolone resistance mutations interact synergistically, giving LLQR mechanisms an additional role in allowing bacterial survival and evasion of therapeutic antimicrobial conditions by a combination of the two strategies. At clinically relevant concentrations, LLQR mechanisms reverse further impact of SOS response suppression in reducing bacterial tolerance.

Suppression of the SOS response modifies spatiotemporal evolution, post-antibiotic effect, bacterial fitness and biofilm formation in quinolone-resistant Escherichia coli.

Background: Suppression of the SOS response has been proposed as a therapeutic strategy for potentiating quinolones against susceptible, low-level quinolone-resistant (LLQR) and resistant Enterobacteriaceae. Objectives: To monitor the functionality of the SOS response in the evolution towards clinical quinolone resistance and study its impact on the evolution of spatiotemporal resistance. Methods: An isogenic collection of Escherichia coli (derived from the strain ATCC 25922) carrying combinations of chromosomally and plasmid-mediated quinolone resistance mechanisms (including susceptible, LLQR and resistant phenotypes) and exhibiting a spectrum of SOS activity was used. Relevant clinical parameters such as mutation rate, mutant prevention concentration (MPC), bacterial fitness, biofilm formation and post-antibiotic effect (PAE) were evaluated. Results: Inactivating the SOS response (recA deletion) led to a decrease in mutation rate (∼103 fold) in LLQR compared with WT strains at ciprofloxacin concentrations of 1 mg/L (the EUCAST breakpoint for resistance) and 2.5 mg/L (Cmax), as well as a remarkable delay in the spatiotemporal evolution of quinolone resistance. For all strains, there was an 8-fold decrease in MPC in RecA-deficient strains, with values for LLQR strains decreasing below the Cmax of ciprofloxacin. Inactivation of the SOS response reduced competitive fitness by 33%-50%, biofilm production by 22%-80% and increased the PAE by ∼3-4 h at sub-MIC concentrations of ciprofloxacin. Conclusions: Our data indicate that suppression of the SOS response affects key bacterial traits and is a promising strategy for reversing and tackling the evolution of antibiotic resistance in E. coli, including low-level and resistant phenotypes at therapeutic quinolone concentrations.

Population pharmacokinetics and pharmacodynamics of fosfomycin in non-critically ill patients with bacteremic urinary infection caused by multidrug-resistant Escherichia coli.

OBJECTIVES: To describe the population pharmacokinetics of fosfomycin for patients with bacteraemic urinary tract infection (BUTI). The analysis identified optimal regimens on the basis of pharmacodynamic targets and assessed the adequacy of Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) susceptibility breakpoints for Escherichia coli. METHODS: Data of 16 patients with BUTI caused by multidrug-resistant E. coli (FOREST clinical trial) received intravenous fosfomycin (4 g every 6 hours) were analysed. A population pharmacokinetic analysis was performed, and Monte Carlo simulations were undertaken using 4 g every 6 hours and 8 g every 8 hours. The probability of pharmacodynamic target attainment was assessed using pharmacodynamic targets for E. coli for static effect, 1-log drop in bacterial burden and resistance suppression. RESULTS: Sixty-four plasma samples were collected over a single dosing interval (day 2 or 3 after starting fosfomycin treatment). Fosfomycin concentrations were highly variable. Pharmacodynamic target attainment analysis showed mild improvement by increasing fosfomycin dosing (4 g every 6 hours vs. every 8 hours). These dosages showed success for decreasing 1-log bacterial burden in 89% to 96% (EUCAST breakpoints) and 33% to 54% (CLSI breakpoints) of patients, but they were unable to reach bacterial resistance suppression targets. CONCLUSIONS: Fosfomycin concentrations are highly variable-a fact partially explained by renal impairment. The present work supports the use of 4 g every 6 hours as an effective regimen for the treatment of non-critically ill patients with BUTI caused by multidrug-resistant E. coli, as higher dosages might increase toxicity but may not significantly increase efficacy. The current information may suggest that fosfomycin susceptibility breakpoints need to be reappraised.

Urinary Tract Conditions Affect Fosfomycin Activity against Escherichia coli Strains Harboring Chromosomal Mutations Involved in Fosfomycin Uptake.

The steps by which Escherichia coli strains harboring mutations related to fosfomycin (FOS) resistance arise and spread during urinary tract infections (UTIs) are far from being understood. The aim of this study was to evaluate the effects of urine, pH, and anaerobiosis on FOS activity against a set of isogenic strains carrying the most prevalent chromosomal mutations conferring FOS resistance (ΔuhpT, ΔglpT, ΔcyaA, and ΔptsI), either singly or in combination. We also studied fosfomycin-resistant E. coli clinical isolates from patients with UTI. Our results demonstrate that urinary tract physiological conditions might have a profound impact on FOS activity against strains with chromosomal FOS resistance mutations. Specifically, acidic pH values and anaerobiosis convert most of the strains categorized as resistant to fosfomycin according to the international guidelines to a susceptible status. Therefore, urinary pH values may have practical interest in the management of UTIs. Finally, our results, together with the high fitness cost associated with FOS resistance mutations, might explain the low prevalence of fosfomycin-resistant E. coli variants in UTIs.

Quinolone Resistance Reversion by Targeting the SOS Response.

Suppression of the SOS response has been postulated as a therapeutic strategy for potentiating antimicrobial agents. We aimed to evaluate the impact of its suppression on reversing resistance using a model of isogenic strains of Escherichia coli representing multiple levels of quinolone resistance. E. coli mutants exhibiting a spectrum of SOS activity were constructed from isogenic strains carrying quinolone resistance mechanisms with susceptible and resistant phenotypes. Changes in susceptibility were evaluated by static (MICs) and dynamic (killing curves or flow cytometry) methodologies. A peritoneal sepsis murine model was used to evaluate in vivo impact. Suppression of the SOS response was capable of resensitizing mutant strains with genes encoding three or four different resistance mechanisms (up to 15-fold reductions in MICs). Killing curve assays showed a clear disadvantage for survival (Δlog10 CFU per milliliter [CFU/ml] of 8 log units after 24 h), and the in vivo efficacy of ciprofloxacin was significantly enhanced (Δlog10 CFU/g of 1.76 log units) in resistant strains with a suppressed SOS response. This effect was evident even after short periods (60 min) of exposure. Suppression of the SOS response reverses antimicrobial resistance across a range of E. coli phenotypes from reduced susceptibility to highly resistant, playing a significant role in increasing the in vivo efficacy.IMPORTANCE The rapid rise of antibiotic resistance in bacterial pathogens is now considered a major global health crisis. New strategies are needed to block the development of resistance and to extend the life of antibiotics. The SOS response is a promising target for developing therapeutics to reduce the acquisition of antibiotic resistance and enhance the bactericidal activity of antimicrobial agents such as quinolones. Significant questions remain regarding its impact as a strategy for the reversion or resensitization of antibiotic-resistant bacteria. To address this question, we have generated E. coli mutants that exhibited a spectrum of SOS activity, ranging from a natural SOS response to a hypoinducible or constitutively suppressed response. We tested the effects of these mutations on quinolone resistance reversion under therapeutic concentrations in a set of isogenic strains carrying different combinations of chromosome- and plasmid-mediated quinolone resistance mechanisms with susceptible, low-level quinolone resistant, resistant, and highly resistant phenotypes. Our comprehensive analysis opens up a new strategy for reversing drug resistance by targeting the SOS response.

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.

Role of inoculum and mutant frequency on fosfomycin MIC discrepancies by agar dilution and broth microdilution methods in Enterobacteriaceae.

OBJECTIVES: Fosfomycin is re-evaluated as a treatment of multidrug-resistant Enterobacteriaceae infections. However, MIC differences have been described among the different susceptibility testing. The aim was to study the role of the different inoculum size used in agar dilution with respect to broth microdilution, according to CLSI, in the fosfomycin MIC discrepancies. METHODS: Fosfomycin MICs were determined using agar dilution (reference) and broth microdilution in 220 Escherichia coli (n=81) and Klebsiella pneumoniae (n=139) clinical isolates. Fosfomycin mutant frequencies were determined in 21 E. coli (MIC=1mg/L) and 21 K. pneumoniae (MIC=16mg/L). The emergence of resistant subpopulations of five E. coli strains (MIC=1mg/L) was monitored over the time by microdilution assay using 0, 4 and 8 mg/L of fosfomycin, and eight different inocula (5×105-3.91×103 CFU/well, 1 : 2 dilutions). RESULTS: For E. coli, 86.4% of categorical agreement (CA), 9.1% very major errors (VME), 3.3% major errors (ME) and 9.9% minor errors (mE) were found. For K. pneumoniae, CA was 51.1%, VME 15.7%, ME 28.4% and mE 25.2%. Essential agreement (±1-log2) was observed in 55.45%. By microdilution, 35.9% of the MICs showed discrepancies of ≥2 dilutions. Initial inoculum used was 5.63 times higher in the microdilution method, in range with CLSI methodology for both techniques. Fosfomycin mutant frequencies were 6.05×10-5 (4×MIC) to 5.59×10-7 (256×MIC) for E. coli, and 1.49×10-4 (4×MIC) to 1.58×10-5 (16×MIC) for K. pneumoniae. Resistant subpopulations arose mainly after 8 h of incubation with inocula >3.13×104 CFU/well. CONCLUSIONS: The higher inoculum used in the microdilution method enriched the initial inoculum with resistant subpopulations and could partially explain the fosfomycin MIC discrepancies with respect to the agar dilution method.

Pharmacodynamics of vancomycin for CoNS infection: experimental basis for optimal use of vancomycin in neonates.

OBJECTIVES: CoNS are the most common cause of neonatal late-onset sepsis. Information on the vancomycin pharmacokinetics/pharmacodynamics against CoNS is limited. The aim of this study was to characterize vancomycin pharmacokinetic/pharmacodynamic relationships for CoNS and investigate neonatal optimal dosage regimens. METHODS: A hollow fibre and a novel rabbit model of neonatal central line-associated bloodstream CoNS infections were developed. The results were then bridged to neonates by use of population pharmacokinetic techniques and Monte Carlo simulations. RESULTS: There was a dose-dependent reduction in the total bacterial population and C-reactive protein levels. The AUC/MIC and Cmax/MIC ratios were strongly linked with total and mutant resistant cell kill. Maximal amplification of resistance was observed in vitro at an fAUC/MIC of 200 mg ·â€Šh/L. Simulations predicted that neonates <29 weeks post-menstrual age are underdosed with standard regimens with respect to older age groups. CONCLUSIONS: The AUC/MIC and Cmax/MIC ratios are the pharmacodynamic indices that best explain total and resistant cell kill in CoNS infection. This suggests that less-fractionated regimens are appropriate for clinical use and continuous infusions may be associated with increased risk of emergence of antimicrobial resistance. This study has provided the pharmacodynamic evidence to inform an optimized neonatal dosage regimen to take into a randomized controlled trial.

Pharmacodynamics of fosfomycin: insights into clinical use for antimicrobial resistance.

The aim of this study was to improve the understanding of the pharmacokinetic-pharmacodynamic relationships of fosfomycin against extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli strains that have different fosfomycin MICs. Our methods included the use of a hollow fiber infection model with three clinical ESBL-producing E. coli strains. Human fosfomycin pharmacokinetic profiles were simulated over 4 days. Preliminary studies conducted to determine the dose ranges, including the dose ranges that suppressed the development of drug-resistant mutants, were conducted with regimens from 12 g/day to 36 g/day. The combination of fosfomycin at 4 g every 8 h (q8h) and meropenem at 1 g/q8h was selected for further assessment. The total bacterial population and the resistant subpopulations were determined. No efficacy was observed against the Ec42444 strain (fosfomycin MIC, 64 mg/liter) at doses of 12, 24, or 36 g/day. All dosages induced at least initial bacterial killing against Ec46 (fosfomycin MIC, 1 mg/liter). High-level drug-resistant mutants appeared in this strain in response to 12, 15, and 18 g/day. In the study arms that included 24 g/day, once or in a divided dose, a complete extinction of the bacterial inoculum was observed. The combination of meropenem with fosfomycin was synergistic for bacterial killing and also suppressed all fosfomycin-resistant clones of Ec2974 (fosfomycin MIC, 1 mg/liter). We conclude that fosfomycin susceptibility breakpoints (≤64 mg/liter according to CLSI [for E. coli urinary tract infections only]) should be revised for the treatment of serious systemic infections. Fosfomycin can be used to treat infections caused by organisms that demonstrate lower MICs and lower bacterial densities, although relatively high daily dosages (i.e., 24 g/day) are required to prevent the emergence of bacterial resistance. The ratio of the area under the concentration-time curve for the free, unbound fraction of fosfomycin versus the MIC (fAUC/MIC) appears to be the dynamically linked index of suppression of bacterial resistance. Fosfomycin with meropenem can act synergistically against E. coli strains in preventing the emergence of fosfomycin resistance.

Population pharmacokinetics of teicoplanin in children.

Teicoplanin is frequently administered to treat Gram-positive infections in pediatric patients. However, not enough is known about the pharmacokinetics (PK) of teicoplanin in children to justify the optimal dosing regimen. The aim of this study was to determine the population PK of teicoplanin in children and evaluate the current dosage regimens. A PK hospital-based study was conducted. Current dosage recommendations were used for children up to 16 years of age. Thirty-nine children were recruited. Serum samples were collected at the first dose interval (1, 3, 6, and 24 h) and at steady state. A standard 2-compartment PK model was developed, followed by structural models that incorporated weight. Weight was allowed to affect clearance (CL) using linear and allometric scaling terms. The linear model best accounted for the observed data and was subsequently chosen for Monte Carlo simulations. The PK parameter medians/means (standard deviation [SD]) were as follows: CL, [0.019/0.023 (0.01)] × weight liters/h/kg of body weight; volume, 2.282/4.138 liters (4.14 liters); first-order rate constant from the central to peripheral compartment (Kcp), 0.474/3.876 h(-1) (8.16 h(-1)); and first-order rate constant from peripheral to central compartment (Kpc), 0.292/3.994 h(-1) (8.93 h(-1)). The percentage of patients with a minimum concentration of drug in serum (Cmin) of <10 mg/liter was 53.85%. The median/mean (SD) total population area under the concentration-time curve (AUC) was 619/527.05 mg · h/liter (166.03 mg · h/liter). Based on Monte Carlo simulations, only 30.04% (median AUC, 507.04 mg · h/liter), 44.88% (494.1 mg · h/liter), and 60.54% (452.03 mg · h/liter) of patients weighing 50, 25, and 10 kg, respectively, attained trough concentrations of >10 mg/liter by day 4 of treatment. The teicoplanin population PK is highly variable in children, with a wider AUC distribution spread than for adults. Therapeutic drug monitoring should be a routine requirement to minimize suboptimal concentrations. (This trial has been registered in the European Clinical Trials Database Registry [EudraCT] under registration number 2012-005738-12.).

Impact of qnrA1, qnrB1 and qnrS1 on the efficacy of ciprofloxacin and levofloxacin in an experimental pneumonia model caused by Escherichia coli with or without the GyrA mutation Ser83Leu.

OBJECTIVES: The aim of this study was to evaluate the impact of qnrA1, qnrB1 and qnrS1 on the in vivo efficacies of ciprofloxacin and levofloxacin in an experimental model of pneumonia caused by Escherichia coli. METHODS: Two isogenic groups of E. coli transformants, based on two ATCC 25922 strains, with or without the GyrA mutation Ser83Leu, and carrying qnrA1, qnrB1 or qnrS1, were used in an experimental pneumonia model. The efficacies of ciprofloxacin (40 mg/kg/day) and levofloxacin (50 and 150 mg/kg/day) were evaluated. RESULTS: For the pneumonia caused by the parental strains lacking qnr genes, both fluoroquinolones significantly (P<0.05) reduced the bacterial lung concentration by >7 log10 cfu/g against E. coli ATCC/pBK and between 5.09 and 6.34 log10 cfu/g against E. coli ATCC-S83L/pBK. The presence of any qnr genes in the strains of both isogenic groups diminished the reduction of bacterial lung concentration with any therapy (P<0.05). Furthermore, all therapeutic schemes reduced the percentage of positive blood cultures in both isogenic groups (P<0.05). Finally, the survival results suggest a higher mortality with the strains expressing qnr genes. CONCLUSIONS: The presence of qnrA1, qnrB1 and qnrS1 in E. coli reduced the efficacy of ciprofloxacin and levofloxacin in a murine pneumonia model.

Inoculum effect on the efficacies of amoxicillin-clavulanate, piperacillin-tazobactam, and imipenem against extended-spectrum ß-lactamase (ESBL)-producing and non-ESBL-producing Escherichia coli in an experimental murine sepsis model.

Escherichia coli is commonly involved in infections with a heavy bacterial burden. Piperacillin-tazobactam and carbapenems are among the recommended empirical treatments for health care-associated complicated intra-abdominal infections. In contrast to amoxicillin-clavulanate, both have reduced in vitro activity in the presence of high concentrations of extended-spectrum ß-lactamase (ESBL)-producing and non-ESBL-producing E. coli bacteria. Our goal was to compare the efficacy of these antimicrobials against different concentrations of two clinical E. coli strains, one an ESBL-producer and the other a non-ESBL-producer, in a murine sepsis model. An experimental sepsis model {~5.5 log10 CFU/g [low inoculum concentration (LI)] or ~7.5 log(10) CFU/g [high inoculum concentration (HI)]} using E. coli strains ATCC 25922 (non-ESBL producer) and Ec1062 (CTX-M-14 producer), which are susceptible to the three antimicrobials, was used. Amoxicillin-clavulanate (50/12.5 mg/kg given intramuscularly [i.m.]), piperacillin-tazobactam (25/3.125 mg/kg given intraperitoneally [i.p.]), and imipenem (30 mg/kg i.m.) were used. Piperacillin-tazobactam and imipenem reduced spleen ATCC 25922 strain concentrations (-2.53 and -2.14 log10 CFU/g [P < 0.05, respectively]) in the HI versus LI groups, while amoxicillin-clavulanate maintained its efficacy (-1.01 log10 CFU/g [no statistically significant difference]). Regarding the Ec1062 strain, the antimicrobials showed lower efficacy in the HI than in the LI groups: -0.73, -1.89, and -1.62 log10 CFU/g (P < 0.05, for piperacillin-tazobactam, imipenem, and amoxicillin-clavulanate, respectively, although imipenem and amoxicillin-clavulanate were more efficacious than piperacillin-tazobactam). An adapted imipenem treatment (based on the time for which the serum drug concentration remained above the MIC obtained with a HI of the ATCC 25922 strain) improved its efficacy to -1.67 log10 CFU/g (P < 0.05). These results suggest that amoxicillin-clavulanate could be an alternative to imipenem treatment of infections caused by ESBL- and non-ESBL-producing E. coli strains in patients with therapeutic failure with piperacillin-tazobactam.

In vitro and in vivo reduced fitness and virulence in ciprofloxacin-resistant Acinetobacter baumannii.

Limited data on relative fitness and virulence of antimicrobial-resistant Acinetobacter baumannii are known. We aimed to study the virulence and fitness cost of ciprofloxacin-resistance in A. baumannii (CipR) compared with the susceptible parental wild-type strain (CipS). Human lung epithelial cells were infected with CipS and CipR for 24 h. Competition fitness was monitored in vitro and in vivo in a murine peritoneal sepsis model. We showed that CipR induced less cell death than CipS and CipR growth was slow when in competition with CipS. Altogether, acquisition of ciprofloxacin resistance confers a biological fitness cost and reduces virulence in A. baumannii.