67 research outputs found
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) (ÎrecA, ÎkatG, ÎkatE, ÎsodA, ÎsodB), double-gene (ÎrecA-ÎkatG, ÎrecA-ÎkatE, ÎrecA-ÎsodA, ÎrecA-ÎsodB, ÎkatG-ÎkatE, ÎsodB-ÎsodA) and triple-gene (ÎrecA-ÎkatG-ÎkatE) 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, ÎkatE, ÎsodB and ÎsodB/ÎsodA mutants, MPC values were 0.25 mg/L. The ÎkatG, ÎsodA, ÎkatG/katE and ÎrecA mutants showed 2-fold reductions (0.125 mg/L). The ÎkatG/ÎrecA, ÎkatE/ÎrecA, ÎsodA/ÎrecA, ÎsodB/ÎrecA and ÎkatG/ÎkatE/ÎrecA strains showed 4â8-fold reductions (0.03â0.06 mg/L) relative to the wild-type. Gradual antimicrobial pressure increased growth capacity for ÎsodA and ÎsodB and ÎsodB/ÎsodA mutants (no growth in 4 mg/L) compared with the wild-type (no growth in the range of 0.5â2 mg/L). Accordingly, increased growth was observed with the mutants ÎrecA/ÎkatG (no growth in 2 mg/L), ÎrecA/ÎkatE (no growth in 2 mg/L), ÎrecA/ÎsodA (no growth in 0.06 mg/L), ÎrecA/ÎsodB (no growth in 0.25 mg/L) and ÎrecA/ÎkatG/ÎkatE (no growth in 0.5 mg/L) compared with ÎrecA (no growth in the range of 0.002â0.015 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.Plan Nacional de I+D+i 2013-2016 and the Instituto de Salud Carlos III (projects and PI17/01501 and PI20-00239)SubdirecciĂłn General de Redes y Centros de InvestigaciĂłn Cooperativa, Ministerio de EconomĂa, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI; RD16/0016/0001 and REIPI RD16/ 0016/0009
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 (Îlog10 CFU per milliliter [CFU/ml] of 8 log units after 24 h), and the in vivo efficacy of ciprofloxacin was significantly enhanced (Îlog10 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
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 fosfomycinresistant clones of Ec2974 (fosfomycin MIC, 1 mg/liter). We conclude that fosfomycin susceptibility breakpoints (â€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.ConsejerĂa de Igualdad, Salud y PolĂticas Sociales Junta de AndalucĂa PI-0044-2013FEDER REIPI RD12/001
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