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.Plan Nacional de I+D+i 2013-2016 and the Instituto de Salud Carlos III (projects and PI17/01501 and PI20-00239)Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI; RD16/0016/0001 and REIPI RD16/ 0016/0009

Combined inactivation of the SOS response with TCA fumarases and the adaptive response enhances antibiotic susceptibility against Escherichia coli

IntroductionTargeting bacterial DNA damage responses such as the SOS response represents a promising strategy for enhancing the efficacy of existing antimicrobials. This study focuses on a recently discovered DNA damage response mechanism involving tricarboxylic acid cycle (TCA) fumarases and the adaptive response, crucial for Escherichia coli survival in the presence of genotoxic methyl methanesulfonate (MMS). We investigated whether this pathway contributes to protection against antibiotics, either separately or in combination with the SOS response.MethodsAn isogenic collection of E. coli BW25113 mutants was used, including strains deficient in fumarases (ΔfumA, ΔfumB, ΔfumC) and the adaptive response (ΔalkA, ΔalkB, ΔaidB). Additional SOS response inactivation (ΔrecA) was conducted by P1 phage transduction. All mutants were subjected to antimicrobial susceptibility testing, growth curve analysis, survival and evolution assays. To validate the relevance of these findings, experiments were also performed in a quinolone-resistant E. coli ST131 clinical isolate.Results and discussionOverall, no significant differences or only moderate increases in susceptibility were observed in the single mutants, with ΔfumC and ΔaidB mutants showing the highest susceptibility. To enhance this effect, these genes were then inactivated in combination with the SOS response by constructing ΔfumC/ΔrecA and ΔaidB/ΔrecA mutants. These combinations exhibited significant differences in susceptibility to various antimicrobials, particularly cephalosporins and quinolones, and especially in the ΔfumC/ΔrecA strain. To further assess these results, we constructed an E. coli ST131 ΔfumC/ΔrecA mutant, in which a similar trend was observed. Together, these findings suggest that co-targeting the SOS response together with fumarases or the adaptive response could enhance the effectiveness of antibiotics against E. coli, potentially leading to new therapeutic strategies

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 (LC–MS) 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

Outbreaks by Klebsiella oxytoca in neonatal intensive care units: Analysis of an outbreak in a tertiary hospital and systematic review

Objective: Klebsiella oxytoca can cause nosocomial infections, affecting vulnerable newborns. There are few studies describing nosocomial outbreaks in the neonatal intensive care units (NICU). In this study, a systematic review of the literature was carried out to know the main characteristics of these outbreaks and the evolution of one is described. Methods: We conducted a systematic review in the Medline database up to July 2022, and present a descriptive study of an outbreak with 21 episodes in the NICU of a tertiary hospital, between September 2021 and January 2022. Results: 9 articles met the inclusion criteria. The duration of outbreaks was found to be variable, of which 4 (44.4%) lasted for a year or more. Colonization (69%) was more frequent than infections (31%) and the mortality rate was 22.4%. In studies describing sources, the most frequent was the environmental origin (57.1%). In our outbreak there were 15 colonizations and 6 infections. The infections were mild conjunctivitis without sequelae. Molecular typing analysis made it possible to detect 4 different clusters. Conclusions: There is an important variability in the evolution and results of the published outbreaks, highlighting a greater number of colonized, use of PFGE (pulsed-field gel electrophoresis) techniques for molecular typing and implementation of control measures. Finally, we describe an outbreak in which 21 neonates were affected with mild infections, resolved without sequelae and whose control measures were effective.Objetivos: Klebsiella oxytoca puede causar infecciones nosocomiales y afectar a recién nacidos vulnera bles. Existen escasos trabajos que describan brotes nosocomiales en las unidades de cuidados intensivos neonatales (UCIN). En este estudio se realiza una revisión sistemática de la literatura para conocer las características principales de estos brotes y se describe la evolución de uno. Métodos: Se realizó una revisión sistemática en la base de datos Medline hasta julio de 2022, y un estudio descriptivo de un brote con 21 episodios en la UCIN de un hospital de tercer nivel, desde septiembre de 2021 a enero de 2022. Resultados: Nueve artículos cumplían los criterios de inclusión. Se encontró que la duración de los brotes fue variable, y que 4 (44,4%) se prolongaron durante un ano˜ o más. Las colonizaciones (69%) fueron más frecuentes que las infecciones (31%) y la tasa de mortalidad fue del 22,4%. En los estudios que describen las fuentes lo más frecuente fue el origen ambiental (57,1%). En nuestro brote hubo 15 colonizaciones y 6 infecciones. Las infecciones fueron conjuntivitis leves sin secuelas. El análisis mediante tipado molecular permitió detectar 4 clústeres diferentes. Conclusiones: Existe una variabilidad importante en la evolución y resultados de los brotes publicados, destacando un mayor número de colonizados, uso de técnicas de electroforesis en campo pulsado para tipado molecular e implantación de medidas de control. Finalmente, describimos un brote en el que se afectaron 21 neonatos con infecciones leves, resueltas sin secuelas y cuyas medidas para su control resultaron eficaces

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 (LC–MS) 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

Identification and antimicrobial susceptibility testing of positive blood culture isolates from briefly incubated solid medium cultures

[ES] Introducción: La espectrometría de masas Matrix-Assisted Laser Desorption-Ionization Time-of-Flight (MALDI-TOF) permite la identificación rápida de los microorganismos causantes de bacteriemia. Se requieren métodos fiables y rápidos que permitan acortar el tiempo necesario hasta disponer de los resultados de sensibilidad a antibióticos de los aislados de hemocultivos. Métodos: Se evalúa la fiabilidad de un método que combina la identificación con MALDI-TOF y el estudio de sensibilidad en paneles de microdilución inoculados a partir de un subcultivo incubado durante solo 4 h. Resultados: La concordancia de los resultados de sensibilidad a antibióticos de la técnica evaluada frente a la técnica de referencia fue del 99,3%, sin que se observaran errores máximos. Conclusión: La inoculación de paneles de microdilución a partir de un subcultivo de solo 4 h de incubación es un método fiable y fácil de realizar que permite acortar el tiempo de informe de hemocultivos positivos.[EN] Introduction: Mass spectrometry Matrix-Assisted Laser Desorption-Ionization Time-of-Flight (MALDI-TOF) helps in the rapid identification of microorganisms causing blood stream infection. Rapid and reliable methods are required to decrease the turnaround time for reporting antimicrobial susceptibility results from blood culture isolates. Methods: An evaluation was performed on the reliability of a method for antimicrobial susceptibility testing of positive blood culture isolates from briefly incubated solid medium cultures. Results: The agreement between the evaluated and standard methods was 99.3%. The major and minor error rates were 0.4% and 0.3%, respectively, and no very major errors were observed. Conclusion: The inoculation of briefly incubated solid medium cultures into antimicrobial susceptibility testing panels is an easy and reliable technique, and helps to decrease the turnaround time for reporting antimicrobial susceptibility results of positive blood cultures.Peer reviewe

Disbalancing Envelope Stress Responses as a Strategy for Sensitization of Escherichia coli to Antimicrobial Agents

Disbalancing envelope stress responses was investigated as a strategy for sensitization of Escherichia coli to antimicrobial agents. Seventeen isogenic strains were selected from the KEIO collection with deletions in genes corresponding to the σE, Cpx, Rcs, Bae, and Psp responses. Antimicrobial activity against 20 drugs with different targets was evaluated by disk diffusion and gradient strip tests. Growth curves and time-kill curves were also determined for selected mutant-antimicrobial combinations. An increase in susceptibility to ampicillin, ceftazidime, cefepime, aztreonam, ertapenem, and fosfomycin was detected. Growth curves for Psp response mutants showed a decrease in optical density (OD) using sub-MIC concentrations of ceftazidime and aztreonam (ΔpspA and ΔpspB mutants), cefepime (ΔpspB and ΔpspC mutants) and ertapenem (ΔpspB mutant). Time-kill curves were also performed using 1xMIC concentrations of these antimicrobials. For ceftazidime, 2.9 log10 (ΔpspA mutant) and 0.9 log10 (ΔpspB mutant) decreases were observed at 24 and 8 h, respectively. For aztreonam, a decrease of 3.1 log10 (ΔpspA mutant) and 4 log1010 (ΔpspB mutant) was shown after 4–6 h. For cefepime, 4.2 log10 (ΔpspB mutant) and 2.6 log10 (ΔpspC mutant) decreases were observed at 8 and 4 h, respectively. For ertapenem, a decrease of up to 6 log10 (ΔpspB mutant) was observed at 24 h. A deficient Psp envelope stress response increased E. coli susceptibility to beta-lactam agents such as cefepime, ceftazidime, aztreonam and ertapenem. Its role in repairing extensive inner membrane disruptions makes this pathway essential to bacterial survival, so that disbalancing the Psp response could be an appropriate target for sensitization strategies.This study was supported by the Instituto de Salud Carlos III, Ministerio de Economía y Competitividad—co-financed by European Development Regional Fund “A way to achieve Europe” ERDF, Spanish Network for Research in Infectious Diseases [REIPI RD12/0015 and RD16/0016]. Supported by the Plan Nacional de I+D+i 2013–2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (PI14/00940, PI17/01501, PI20/00239, RD16/0016/0001, and REIPI RD16/0016/0009)—co-financed by European Development Regional Fund “A way to achieve Europe,” Operative Programme Intelligent Growth 2014–2020