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

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 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.This study was funded 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 Plan Nacional de I+D+i 2013‐2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de InvestigaciónCooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (PI14/00940, PI17/01501, AC16/00072, 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.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 (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

Cellular Response to Ciprofloxacin in Low-Level Quinolone-Resistant Escherichia coli

Bactericidal activity of quinolones has been related to a combination of DNA fragmentation, reactive oxygen species (ROS) production and programmed cell death (PCD) systems. The underlying molecular systems responsible for reducing bactericidal effect during antimicrobial therapy in low-level quinolone resistance (LLQR) phenotypes need to be clarified. To do this and also define possible new antimicrobial targets, the transcriptome profile of isogenic Escherichia coli harboring quinolone resistance mechanisms in the presence of a clinical relevant concentration of ciprofloxacin was evaluated. A marked differential response to ciprofloxacin of either up- or downregulation was observed in LLQR strains. Multiple genes implicated in ROS modulation (related to the TCA cycle, aerobic respiration and detoxification systems) were upregulated (sdhC up to 63.5-fold) in mutants with LLQR. SOS system components were downregulated (recA up to 30.7-fold). yihE, a protective kinase coding for PCD, was also upregulated (up to 5.2-fold). SdhC inhibition sensitized LLQR phenotypes (up to ΔLog = 2.3 after 24 h). At clinically relevant concentrations of ciprofloxacin, gene expression patterns in critical systems to bacterial survival and mutant development were significantly modified in LLQR phenotypes. Chemical inhibition of SdhC (succinate dehydrogenase) validated modulation of ROS as an interesting target for bacterial sensitization.This work was supported by the Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III (projects PI11-00934 and PI14/00940) and the Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía (P11-CTS-7730), Spain, by the Plan Nacional de I+D+i 2008–2011 and the Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, the Spanish Network for Research in Infectious Diseases (REIPI RD12/0015)—co-financed by European Development Regional Fund ‘A way to achieve Europe’ ERDF.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

Impact of AAC(6′)-Ib-cr in combination with chromosomal-mediated mechanisms on clinical quinolone resistance in Escherichia coli

[Objectives] aac(6′)-Ib-cr is the most prevalent plasmid-mediated fluoroquinolone (FQ) resistance mechanism in Enterobacteriaceae. We aimed to analyse the interplay between this plasmid-mediated gene and chromosomal-mediated quinolone resistance mechanisms on both FQ resistance and bacterial fitness in Escherichia coli.[Methods] E. coli ATCC 25922 and derived isogenic strains carrying chromosomal-mediated quinolone resistance modifications (Ser83Leu–Asp87Asn in GyrA, Ser80Arg in ParC and/or a marR gene deletion) were electroporated with a pBK-CMV vector encoding AAC(6′)-Ib-cr. The MICs of FQs were determined by microdilution and bactericidal activity was determined using time–kill curves. A peritoneal sepsis murine model was used to evaluate the in vivo impact. Bacterial fitness was analysed using growth curves and competition assays.[Results] The presence of the aac(6′)-Ib-cr gene increased the MICs of ciprofloxacin and norfloxacin 4–8-fold for all E. coli genotypes, independently of the initial resistance level. Combination of the aac(6′)-Ib-cr gene with three or four chromosomal mechanisms was necessary to reach MIC values above the susceptible category. Killing curve assays showed a clear selective advantage for survival in strains harbouring the aac(6′)-Ib-cr gene (up to 7 log10 cfu/mL after 24 h). AAC(6′)-Ib-cr significantly reduced the ciprofloxacin efficacy in vivo. In terms of bacterial fitness cost, maximal OD was significantly lower for all strains harbouring the aac(6′)-Ib-cr gene, independently of chromosomal mutations associated.[Conclusions] The aac(6′)-Ib-cr gene, in spite of producing low-level resistance by itself, plays a relevant role in acquisition of a clinical level of ciprofloxacin and norfloxacin resistance, when combined with three or four chromosomal mutations, both in vitro and in vivo.This work was supported by the Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III (projects PI11-00934 and PI14/00940) and the Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía (P11-CTS-7730), Spain, by the Plan Nacional de I + D + i 2008–2011 and the Instituto de Salud Carlos III, Subdirección General de Redes y Centros de InvestigaciónCooperativa, Ministerio de Economía y Competitividad, the Spanish Network for Research in Infectious Diseases (REIPI RD12/0015)—co-financed by European Development Regional Fund ‘A way to achieve Europe’ ERDF. F. D.-P. is supported by a VPPI-US fellowship from the University of Seville.Peer reviewe

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.This work was supported by the 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)‐co‐financed by European Development Regional Fund ‘A way to achieve Europe’, Operative program Intelligent Growth 2014‐2020. Sara Diaz-Diaz is supported by a PFIS Grant from the Instituto de Salud Carlos III (FI18/00086).Peer reviewe

Nosocomial outbreak linked to a flexible gastrointestinal endoscope contaminated with an amikacin-resistant ST17 clone of Pseudomonas aeruginosa

Endoscope contamination is infrequent but can be the source of nosocomial infections and outbreaks. In August 2016, an unexpected increase in the incidence of amikacin-resistant P. aeruginosa isolates (AK-Pae) was observed at a tertiary care center in the south of Spain. An epidemiological and microbiological investigation (August-October 2016) was performed to explain this finding. Isolates from clinical and environmental samples (2 endoscopes used for retrograde cholangiopancreatography; ERCP) were identified by MALDI-TOF. Antimicrobial susceptibility testing was performed using the MicroScan system. Whole-Genome-Sequencing (Miseq, Illumina) was performed to determine the resistome and virulome. Clonal relatedness among isolates was assessed by SpeI-PFGE and MLST. A Caenorhabditis elegans killing assay was performed for virulence testing. Biofilm formation was performed using a colorimetric assay. Four of the 5 patients infected and/or colonized with AK-Pae in August 2016 had undergone ERCP ≤5 days before sample collection. Two endoscopes were contaminated with AK-Pae. Isolates from one endoscope showed an identical PFGE pattern to 9 isolates (cluster I) and differed (1–2 bands) to 5 isolates (cluster II). Isolates from these clusters belonged to the ST17 clone. This S17 clone was characterized by its low virulence in the C. elegans killing assay, and its biofilm-forming ability, slightly superior to that of high-risk clones of P. aeruginosa ST175 and ST235. This outbreak was caused by an endoscope used for ERCP contaminated with an invasive, moderately virulent, biofilm-forming AK-Pae ST17 clone, suggesting the possible emergence of a new high-risk lineage of this clone