Heterogeneity of SOS response expression in clinical isolates of Escherichia coli influences adaptation to antimicrobial stress.

In recent years, new evidence has shown that the SOS response plays an important role in the response to antimicrobials, with involvement in the generation of clinical resistance. Here we evaluate the impact of heterogeneous expression of the SOS response in clinical isolates of Escherichia coli on response to the fluoroquinolone, ciprofloxacin. In silico analysis of whole genome sequencing data showed remarkable sequence conservation of the SOS response regulators, RecA and LexA. Despite the genetic homogeneity, our results revealed a marked differential heterogeneity in SOS response activation, both at population and single-cell level, among clinical isolates of E. coli in the presence of subinhibitory concentrations of ciprofloxacin. Four main stages of SOS response activation were identified and correlated with cell filamentation. Interestingly, there was a correlation between clinical isolates with higher expression of the SOS response and further progression to resistance. This heterogeneity in response to DNA damage repair (mediated by the SOS response) and induced by antimicrobial agents could be a new factor with implications for bacterial evolution and survival contributing to the generation of antimicrobial resistance.

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.

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.

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.

Identificación y determinación de sensibilidad a antibióticos de aislados de hemocultivos a partir de subcultivos de corta incubación / Identification and antimicrobial susceptibility testing of positive blood culture isolates from briefly incubated solid medium cultures

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 4h. 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 4h de incubación es un método fiable y fácil de realizar que permite acortar el tiempo de informe de hemocultivos positivos (AU)

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 (AU)

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.

Molecular topology: A new strategy for antimicrobial resistance control.

The control of antimicrobial resistance (AMR) seems to have come to an impasse. The use and abuse of antibacterial drugs has had major consequences on the genetic mutability of both pathogenic and nonpathogenic microorganisms, leading to the development of new highly resistant strains. Because of the complexity of this situation, an in silico strategy based on QSAR molecular topology was devised to identify synthetic molecules as antimicrobial agents not susceptible to one or several mechanisms of resistance such as: biofilms formation (BF), ionophore (IA) activity, epimerase (EI) activity or SOS system (RecA inhibition). After selecting a group of 19 compounds, five of them showed significant antimicrobial activity against several strains of Staphylococcus (2 S. aureus, including 1 methicillin resistant, and 1 S. epidermidis), with MIC values between 16 and 32 mg/L. Among the compounds active on RecA, one showed a marked activity in decreasing RecA gene expression in Escherichia coli.

Cytotoxic Effect Associated with Overexpression of QNR Proteins in Escherichia coli.

OBJECTIVE: The objective was to evaluate the cytotoxic effect associated with overexpression of multiple Qnr-like plasmid-mediated quinolone resistance (PMQR) mechanisms in Escherichia coli. METHODS: Coding regions of different PMQR genes (qnrA1, qnrB1, qnrC, qnrD1, qnrS1, and qepA2) and efsqnr were cloned into pET29a(+) vector and overexpressed in E. coli BL21. E. coli BL21 with and without an empty pET29a(+) vector were used as controls. The cytotoxic effect associated with PMQR mechanism overexpression was determined by transmission electron microscopy and viability assays. RESULTS: Overexpressed qnr genes produced loss of bacterial viability in the range of 77-97% compared with the controls, comparable with loss of viability associated with EfsQnr overexpression (97%). No loss of viability was observed in E. coli overexpressing QepA2. In transmission electron microscopy assays, signs of cytotoxicity were observed in E. coli cells overexpressing EfsQnr and Qnr proteins (30-45% of the bacterial population showed morphological changes). Morphological changes were observed in less than 5% of bacterial populations from the control strains and E. coli overexpressing QepA2. CONCLUSIONS: Overexpression of qnr genes produces a cytotoxic cellular and structural effect in E. coli, the magnitude of which varies depending on the family of Qnr proteins.

Identification and antimicrobial susceptibility testing of positive blood culture isolates from briefly incubated solid medium cultures. / Identificación y determinación de sensibilidad a antibióticos de aislados de hemocultivos a partir de subcultivos de corta incubación.

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.

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.