96 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
Impaired Virulence and In Vivo Fitness of Colistin-Resistant Acinetobacter baumannii
4 pĂĄginas, 2 figuras. Presentado en parte: 20 Âș Congreso Europeo de MicrobiologĂa ClĂnica y Enfermedades Infecciosas, Resumen 1389, Viena, Austria, 10-13 de abril de 2010.Acinetobacter baumannii (American Type Culture Collection strain 19606) acquires mutations in the pmrB gene during the in vitro development of resistance to colistin. The colistin-resistant strain has lower affinity for colistin, reduced in vivo fitness (competition index, .016), and decreased virulence, both in terms of mortality (0% lethal dose, 6.9 vs 4.9 log colony-forming units) and survival in a mouse model of peritoneal sepsis. These results may explain the low incidence and dissemination of colistin resistance in A. baumannii in clinical settings.This work was supported by the European Development Regional Fund âA way to achieve Europeâ (ERDF); the Spanish Network for the Research in Infectious Diseases (REIPI RD06/0008), Instituto de Salud Carlos III, Ministerio de Ciencia e InnovaciĂłn, Spain; and the Autonomous government of Madrid (COMBACT S-BIO-0260/2006, L.R.).Peer reviewe
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. DuhpT, DglpT, and DfosA 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 512mg/liter, in vitro activity using growth curves with fosfomycin gradient concentrations (0 to 256mg/liter), and time-kill assays at 64 and 307mg/liter were evaluated with and without PPF (0.623mM). The MICs of fosfomycin against the clinical isolates ranged from 16 to â„1,024mg/liter. The addition of 0.623mM 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 0.623mM). The lowest fosfomycin-resistant mutant frequencies were found in ÎfosA mutants, with decreases in frequency from 1.69Ă10-1 to 1.60Ă10-5 for 64mg/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.Ministerio de EconomĂa y Competitividad PI16/01824, REIPI RD12/0015/0010, EIPI RD16/0016/0001Junta de AndalucĂa PI-0044Innovative Medicines Initiative 115523, 115620, 11573
Impact of suppression of the SOS response on protein expression in clinical isolates of Escherichia coli under antimicrobial pressure of ciprofloxacin
Introduction/objective: Suppression of the SOS response in combination with drugs damaging DNA has been proposed as a potential target to tackle antimicrobial resistance. The SOS response is the pathway used to repair bacterial DNA damage induced by antimicrobials such as quinolones. The extent of lexA-regulated protein expression and other associated systems under pressure of agents that damage bacterial DNA in clinical isolates remains unclear. The aim of this study was to assess the impact of this strategy consisting on suppression of the SOS response in combination with quinolones on the proteome profile of Escherichia coli clinical strains.
Materials and methods: Five clinical isolates of E. coli carrying different chromosomally- and/or plasmid-mediated quinolone resistance mechanisms with different phenotypes were selected, with E. coli ATCC 25922 as control strain. In addition, from each clinical isolate and control, a second strain was created, in which the SOS response was suppressed by deletion of the recA gene. Bacterial inocula from all 12 strains were then exposed to 1xMIC ciprofloxacin treatment (relative to the wild-type phenotype for each isogenic pair) for 1âh. Cell pellets were collected, and proteins were digested into peptides using trypsin. Protein identification and label-free quantification were done by liquid chromatography-mass spectrometry (LCâMS) in order to identify proteins that were differentially expressed upon deletion of recA in each strain. Data analysis and statistical analysis were performed using the MaxQuant and Perseus software.
Results: The proteins with the lowest expression levels were: RecA (as control), AphA, CysP, DinG, DinI, GarL, PriS, PsuG, PsuK, RpsQ, UgpB and YebG; those with the highest expression levels were: Hpf, IbpB, TufB and RpmH. Most of these expression alterations were strain-dependent and involved DNA repair processes and nucleotide, protein and carbohydrate metabolism, and transport. In isolates with suppressed SOS response, the number of underexpressed proteins was higher than overexpressed proteins.
Conclusion: High genomic and proteomic variability was observed among clinical isolates and was not associated with a specific resistant phenotype. This study provides an interesting approach to identify new potential targets to combat antimicrobial resistance
Effect of RecA inactivation on quinolone susceptibility and the evolution of resistance in clinical isolates of Escherichia coli
This study was presented in part at the Twenty-Ninth European Congress of Clinical Microbiology and Infectious Diseases, Amsterdam, The Netherlands, 2019 (Poster Presentation P1339).[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 2â16-fold reductions in fluoroquinolone MICs and modified EUCAST clinical category for several isolates, including ST131 clone isolates. Growth curves and timeâkill curves showed a clear disadvantage (up to 10 log10 cfu/mL after 24âh) for survival in strains with an inactivated SOS system. For recA-deficient mutants, MPC values decreased 4â8-fold, with values below the maximum serum concentration of ciprofloxacin. RecA inactivation led to a decrease in mutant frequency (â„103-fold) compared with isolates with unmodified SOS responses at ciprofloxacin concentrations of 4ĂMIC and 1âmg/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.This study was funded by the Instituto de Salud Carlos III, Ministerio de EconomĂa y Competitividadâco-financed by European Development Regional Fund âA way to achieve Europeâ ERDF, Spanish Network for Research in Infectious Diseases (REIPI RD12/0015 and RD16/0016).
Supported by Plan Nacional de I+D+i 2013â2016 and Instituto de Salud Carlos III, SubdirecciĂłn General de Redes y Centros de InvestigaciĂłnCooperativa, Ministerio de EconomĂa, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (PI14/00940, PI17/01501, AC16/00072, RD16/0016/0001 and REIPI RD16/0016/0009) â co-financed by European Development Regional Fund âA way to achieve Europeâ, Operative Programme Intelligent Growth 2014â2020.Peer reviewe
Impact of suppression of the SOS response on protein expression in clinical isolates of Escherichia coli under antimicrobial pressure of ciprofloxacin
Introduction/objective: Suppression of the SOS response in combination with drugs damaging DNA has been proposed as a potential target to tackle antimicrobial resistance. The SOS response is the pathway used to repair bacterial DNA damage induced by antimicrobials such as quinolones. The extent of lexA-regulated protein expression and other associated systems under pressure of agents that damage bacterial DNA in clinical isolates remains unclear. The aim of this study was to assess the impact of this strategy consisting on suppression of the SOS response in combination with quinolones on the proteome profile of Escherichia coli clinical strains. Materials and methods: Five clinical isolates of E. coli carrying different chromosomally- and/or plasmid-mediated quinolone resistance mechanisms with different phenotypes were selected, with E. coli ATCC 25922 as control strain. In addition, from each clinical isolate and control, a second strain was created, in which the SOS response was suppressed by deletion of the recA gene. Bacterial inocula from all 12 strains were then exposed to 1xMIC ciprofloxacin treatment (relative to the wild-type phenotype for each isogenic pair) for 1 h. Cell pellets were collected, and proteins were digested into peptides using trypsin. Protein identification and label-free quantification were done by liquid chromatography-mass spectrometry (LCâMS) in order to identify proteins that were differentially expressed upon deletion of recA in each strain. Data analysis and statistical analysis were performed using the MaxQuant and Perseus software. Results: The proteins with the lowest expression levels were: RecA (as control), AphA, CysP, DinG, DinI, GarL, PriS, PsuG, PsuK, RpsQ, UgpB and YebG; those with the highest expression levels were: Hpf, IbpB, TufB and RpmH. Most of these expression alterations were strain-dependent and involved DNA repair processes and nucleotide, protein and carbohydrate metabolism, and transport. In isolates with suppressed SOS response, the number of underexpressed proteins was higher than overexpressed proteins. Conclusion: High genomic and proteomic variability was observed among clinical isolates and was not associated with a specific resistant phenotype. This study provides an interesting approach to identify new potential targets to combat antimicrobial resistance
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