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.

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.

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.

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.