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.

Implications of two-component systems EnvZ/OmpR and BaeS/BaeR in in vitro temocillin resistance in Escherichia coli.

BACKGROUND: BaeS/BaeR is a two-component system of Escherichia coli that controls the expression of porins and efflux pumps. Its role in beta-lactam resistance is limited. OBJECTIVES: To study the role of baeS/baeR two-component system in temocillin resistance in E. coli. METHODS: E. coli strain BW25113 and single-gene deletion mutants related to two-component systems were collected from the KEIO collection. Double-gen deletion mutants were generated. Temocillin-resistant mutant frequencies were determined at 32 mg/L. E. coli BW25113 mutants were selected by selective pressure from serial passages. Biological costs were analysed by growth curves. Genomes of the generated mutants were sequenced. The expression level of the mdtA, mdtB, mdtC, acrD and tolC in the ΔbaeS mutant was determined by RT-PCR (with/without temocillin exposure). RESULTS: The frequency of temocillin mutants ranged from 2.12 × 10-8 to 4.51 × 10-8 in single-porin mutants. No mutants were recovered from E. coli BW25113 (>10-9). Selection of temocillin-resistant variants by serial passage yielded mutants up to 128 mg/L. Mutations were found in the baeS gene. Temocillin MICs ranged from 4 to 32 mg/L (highest MICs for ΔbaeS and ΔompR). The efflux pumps mdtA, mdtB, mdtC and acrD pumps were overexpressed 3-10-fold in the presence of temocillin in ΔbaeS compared to control. CONCLUSIONS: Mutations in the sensor histidine kinase, baeS, may be involved in temocillin resistance through the expression of the efflux pumps mdtABC and acrD. In addition, the low mutation rate may be a good predictor of temocillin activity.

Evaluation of temocillin efficacy against KPC-2-producing Klebsiella pneumoniae isolates in a hollow-fibre infection model.

BACKGROUND: Temocillin is an old antimicrobial that is resistant to hydrolysis by ESBLs but has variable activity against carbapenemase-producing Enterobacteriaceae. The current EUCAST susceptibility breakpoints for Enterobacterales are set at ≤16 mg/L (susceptible with increased exposure) based on a dose of 2 g q8h, but there is limited information on the efficacy of this dose against temocillin-susceptible carbapenemase-producing Klebsiella pneumoniae isolates. OBJECTIVES: To evaluate the efficacy of this dose using a hollow-fibre infection model (HFIM) against six KPC-2-producing clinical isolates of K. pneumoniae. METHODS: The isolates were characterized by WGS and temocillin susceptibility was determined using standard and high inoculum temocillin. Mutant frequencies were estimated and temocillin activity was tested in time-kill assays and in the HFIM. At standard conditions, three of the isolates were classified as susceptible (MIC ≤ 16 mg/L) and three as resistant (MIC > 16 mg/L). The HFIM was performed over 3 days to mimic human-like pharmacokinetics of 2 g q8h. Bacterial counts were performed by plating on Mueller-Hinton agar (MHA) and MHA containing 64 mg/L temocillin to detect resistant subpopulations. RESULTS: All isolates showed a reduction in bacterial population of at least 3 log cfu/mL within the first 8 h of simulated treatment in the hollow-fibre assay. Regrowth was observed for the three resistant isolates and one of the susceptible ones. The MIC value for these isolates was higher by at least two dilutions compared with their initial values. CONCLUSIONS: These data suggest that an optimized pharmacokinetic regimen may be of clinical interest for the treatment of KPC-2-producing K. pneumoniae susceptible to temocillin. These data showed activity of temocillin against KPC-2-producing K. pneumoniae susceptible to temocillin; however, a dose of 2g q8h administered over 30 min may be inadequate to prevent the emergence of resistant variants.

The mef(A)/msr(D)-carrying streptococcal prophage Φ1207.3 encodes an SOS-like system, induced by UV-C light, responsible for increased survival and increased mutation rate.

Bacterial SOS response is an inducible system of DNA repair and mutagenesis. Streptococci lack a canonical SOS response, but an SOS-like response was reported in some species. The mef(A)-msr(D)-carrying prophage Ф1207.3 of Streptococcus pyogenes contains a region, spanning orf6 to orf11, showing homology to characterized streptococcal SOS-like cassettes. Genome-wide homology search showed the presence of the whole Φ1207.3 SOS-like cassette in three S. pyogenes prophages, while parts of it were found in other bacterial species. To investigate whether this cassette confers an SOS-mutagenesis phenotype, we constructed Streptococcus pneumoniae R6 isogenic derivative strains: (i) FR172, streptomycin resistant, (ii) FR173, carrying Φ1207.3, and (iii) FR174, carrying a recombinant Φ1207.3, where the SOS-like cassette was deleted. These strains were used in survival and mutation rate assays using a UV-C LED instrument, for which we designed and 3D-printed a customized equipment, constituted of an instrument support and swappable-autoclavable mini-plates and lids. Upon exposure to UV fluences ranging from 0 to 6,400 J/m2 at four different wavelengths, 255, 265, 275, and 285 nm, we found that the presence of Φ1207.3 SOS-like cassette increases bacterial survival up to 34-fold. Mutation rate was determined by measuring rifampicin resistance acquisition upon exposure to UV fluence of 50 J/m2 at the four wavelengths by fluctuation test. The presence of Φ1207.3 SOS-like cassette resulted in a significant increase in the mutation rate (up to 18-fold) at every wavelength. In conclusion, we demonstrated that Φ1207.3 carries a functional SOS-like cassette responsible for an increased survival and increased mutation rate in S. pneumoniae. IMPORTANCE Bacterial mutation rate is generally low, but stress conditions and DNA damage can induce stress response systems, which allow for improved survival and continuous replication. The SOS response is a DNA repair mechanism activated by some bacteria in response to stressful conditions, which leads to a temporary hypermutable phenotype and is usually absent in streptococcal genomes. Here, using a reproducible and controlled UV irradiation system, we demonstrated that the SOS-like gene cassette of prophage Φ1207.3 is functional, responsible for a temporary hypermutable phenotype, and enhances bacterial survival to UV irradiation. Prophage Φ1207.3 also carries erythromycin resistance genes and can lysogenize different pathogenic bacteria, constituting an example of a mobile genetic element which can confer multiple phenotypes to its host.

Effect of Glycerol on Fosfomycin Activity against Escherichia coli.

Fosfomycin is an antimicrobial that inhibits the biosynthesis of peptidoglycan by entering the bacteria through two channels (UhpT and GlpT). Glycerol is clinically used as a treatment for elevated intracranial pressure and induces the expression of glpT in Escherichia coli. Glycerol might offer synergistic activity by increasing fosfomycin uptake. The present study evaluates the use of glycerol at physiological concentrations in combination with fosfomycin against a collection of isogenic mutants of fosfomycin-related genes in E. coli strains. Induction of fosfomycin transporters, susceptibility tests, interaction assays, and time-kill assays were performed. Our results support the notion that glycerol allows activation of the GlpT transporter, but this induction is delayed over time and is not homogeneous across the bacterial population, leading to contradictory results regarding the enhancement of fosfomycin activity. The susceptibility assays showed an increase in fosfomycin activity with glycerol in the disk diffusion assay but not in the agar dilution or broth microdilution assays. Similarly, in the time-kill assays, the effect of glycerol was absent by the emergence of fosfomycin-resistant subpopulations. In conclusion, glycerol may not be a good candidate for use as an adjuvant with fosfomycin.

Prevalence, Incidence, and Risk Factors for Intestinal Colonization Due to Fluoroquinolone-Resistant ST131 Escherichia coli: a Longitudinal Study in Highly Dependent, Long-Term Care Facility Residents.

Escherichia coli ST131 clade C is an important driver for fluoroquinolone resistance (FQ-R). We conducted a prospective observational study in residents from two long-term care facilities (LTCFs) in Seville, Spain, in 2018. Fecal swabs and environmental samples were obtained. E. coli isolates were screened for clade C, FQ-R ST131 by PCR, and molecular typing by PFGE; representatives from pulsotypes were studied by whole-genome-sequencing (WGS) and assigned to lineages (cgSTs). Prevalence of colonization at each time point, incidence density, and risk factors for acquisition were studied. Seventy-six FQ-R ST131 E. coli isolates belonging to 34 cgSTs were obtained; 24 belonging to subclade C1 (116 isolates, 65.9%) and 10 to C2 (60, 34.1%). C1 lineages showed lower virulence scores than C2 (median [IQR], 19 [18 to 20] versus 21 [20 to 21.5], P = 0.001) and higher number of plasmids (4 [3 to 5] versus 2 [2 to 3], P = 0.01). aac(6')-Ib-cr and blaOXA-1 were less frequent in C1 than C2 (2 [8.3%] versus 6 [60%], P = 0.003 for both); ESBL genes were detected in eight (33.3%) C1 (5 blaCTX-M-27) and three (30%) C2 (all blaCTX-M-15). Of the 82 residents studied, 49 were colonized at some point (59.7%), with a pooled prevalence of 38.6%. Incidence density of new lineage acquisition was 2.22 per 100 resident weeks (1.28 and 0.93 C1 and C2 subclades, respectively). Independent risk factors for acquisitions were having a colonized roommate (HR = 4.21; 95% CI = 1.71 to 10.36; P = 0.002) and urinary or fecal incontinence (HR = 2.82; 95% CI = 1.21 to 6.56; P = 0.01). LTCFs are important reservoirs of clade C ST131 E. coli. The risk factors found suggest that cross-transmission is the most relevant transmission mechanisms. IMPORTANCE We aimed at investigating the microbiological and epidemiological features of clade C fluoroquinolone-resistant ST131 E. coli isolates colonizing highly dependent residents in long-term care facilities (LTCFs) during 40 weeks and the risk factors of acquisition. Isolates from C1 and C2 subclades were characterized in this environment. The clonality of the isolates was characterized and they were assigned to lineages (cgSTs), Resistance genes, virulence factors, and plasmids were also described. This study suggests that cross-transmission is the most relevant transmission mechanisms; however, environmental colonization might also play a role. We believe the data provide useful information to depict the epidemiology of these bacteria by merging detailed microbiological and epidemiological information.

Higher prevalence of CTX-M-27-producing Escherichia coli belonging to ST131 clade C1 among residents of two long-term care facilities in Southern Spain.

Recently, the emergence of an international lineage of the CTX-M-27-producing clade C1 of Escherichia coli ST131 is being observed. The aim is to see if this strain has also been introduced in our area. Twenty-eight (33%) out of 86 individuals from two LTCFs in Seville were found to be colonized with fluoroquinolone-resistant E. coli ST131 and 46% isolates were ESBL/pAmpC producers. C1 isolates were more common than C2 and more frequently produced blaESBL/pAmpC genes (53% vs 33%). Strain sharing was observed in 6 groups of 2-5 cases (61%). A differentiated cluster of 5 C1-CTX-M-27 isolates was found which lacked the M27PP1 region.

Interplay between IncF plasmids and topoisomerase mutations conferring quinolone resistance in the Escherichia coli ST131 clone: stability and resistance evolution.

The Escherichia coli ST131 H30-Rx subclone vehicles CTX-M-15 plasmids and mutations in gyrA and parC conferring multidrug resistance successfully in the clinical setting. The aim of this study was (1) to investigate the relationship of specific topoisomerase mutations on the stability of IncF (CTX-M producing) plasmids using isogenic E. coli mutants and (2) to investigate the impact of the IncF-type plasmids present in the E. coli clone ST131 on the evolution of quinolone resistance. E. coli ATCC 25922 (background strain) and derived mutants encoding specific QRDR substitutions were used. Also, NGS-characterized IncFIA and IncFIB plasmids (encoding CTX-M genes) were included. Plasmid stability was evaluated by sequential dilutions into Luria broth medium without antibiotics for 7 days. Mutant frequency to ciprofloxacin was also evaluated. Moderate differences in the IncF plasmids stability were observed among E. coli ATCC 25922 and isogenic mutants. Under our experimental conditions, the fluctuation of bacteria harboring plasmids was less than 0.5-log(10) in all cases. In the mutant frequency tests, it was observed that the presence of these IncF plasmids increased this value significantly (10-1000-fold). Quinolone resistance substitutions in gyrA or parC genes, frequently found associated with E. coli clone ST131, do not modify the stability of ST131-associated IncFIA and IncFIB plasmids under in vitro conditions. IncF-type plasmids present in E. coli clone ST131 facilitate the selection of resistance to quinolones. These results are consistent with the clinical scenario in which the combination of resistance to quinolones and beta-lactams is highly frequent in the E. coli clone ST131.

Clinical characteristics and outcome of bacteraemia caused by Enterobacter cloacae and Klebsiella aerogenes: more similarities than differences.

OBJECTIVES: The genus Enterobacter is a common cause of nosocomial infections. Historically, the most frequent Enterobacter species were those of Enterobacter cloacae complex and Enterobacter aerogenes. In 2019, E. aerogenes was re-classified as Klebsiella aerogenes owing to its higher genotypic similarity with the genus Klebsiella. Our objective was to characterise and compare the clinical profiles of bacteraemia caused by E. cloacae and K. aerogenes. METHODS: This 3-year multicentre, prospective cohort study enrolled consecutive patients with bacteraemia by E. cloacae or K. aerogenes. Baseline characteristics, bacteraemia features (source, severity, treatment), antibiotic susceptibility, resistance mechanisms and mortality were analysed. RESULTS: The study included 285 patients with bacteraemia [196 (68.8%) E. cloacae and 89 (31.2%) K. aerogenes]. The groups showed no differences in age, sex, previous use of invasive devices, place of acquisition, sources or severity at onset. The Charlson score was higher among patients with E. cloacae bacteraemia [2 (1-4) vs. 1 (0.5-3); P = 0.018], and previous antibiotic therapy was more common in patients with K. aerogenes bacteraemia (57.3% vs. 41.3%; P = 0.01). Mortality was 19.4% for E. cloacae and 20.2% for K. aerogenes (P = 0.869). Antibiotic susceptibility was similar for both species, and the incidence of multidrug resistance or ESBL production was low (6% and 5.3%, respectively), with no differences between species. CONCLUSION: Bacteraemias caused by E. cloacae and K. aerogenes share similar patient profiles, presentation and prognosis. Patients with E. cloacae bacteraemia had more co-morbidities and those with K. aerogenes bacteraemia had received more antibiotics.

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.

Detection of Low-Level Fosfomycin-Resistant Variants by Decreasing Glucose-6-Phosphate Concentration in Fosfomycin Susceptibility Determination.

Mutations that confer low-level fosfomycin resistance (LLFR) but not clinical resistance in Escherichia coli are increasingly reported. LLFR strains can become clinically resistant under urinary tract physiological conditions or may act as gateways for highly resistant subpopulations by the selection of additional LLFR mutations. Nevertheless, most LLFR strains are impossible to detect under routine fosfomycin susceptibility determinations. Here, we have explored the possibility of detecting LLFR variants by reducing glucose-6-phosphate (G6P) concentration in fosfomycin susceptibility testing for E. coli strains. As a proof of concept, fosfomycin minimal inhibitory concentrations (MICs) and disk diffusion susceptibility tests were performed for E. coli strain BW25113 and 10 isogenic derivatives carrying the most prevalent LLFR chromosomal mutations (∆uhpT, ∆glpT, ∆cyaA, and ∆ptsI) and their double combinations. Whereas standard G6P concentrations detected only ∆uhpT single and double variants, assays with reduced G6P detected all LLFR variants. In addition, G6P levels were determined to be ≤5 µg/mL in urine samples from 30 patients with urinary tract infection (UTI) caused by E. coli and 10 healthy volunteers, suggesting that most bacterial cells in uncomplicated UTIs are facing fosfomycin under low G6P concentration. Reducing G6P allows for the detection of LLFR variants, which may suppose a risk for future resistance development, especially in UTIs.

Contribution of hypermutation to fosfomycin heteroresistance in Escherichia coli.

OBJECTIVES: To explore the effect of combining defects in DNA repair systems with the presence of fosfomycin-resistant mechanisms to explain the mechanisms underlying fosfomycin heteroresistance phenotypes in Enterobacteriaceae. MATERIALS AND METHODS: We used 11 clinical Escherichia coli isolates together with isogenic single-gene deletion mutants in the E. coli DNA repair system or associated with fosfomycin resistance, combined with double-gene deletion mutants. Fosfomycin MICs were determined by gradient strip assay (GSA) and broth microdilution (BMD). Mutant frequencies for rifampicin (100 mg/L) and fosfomycin (50 and 200 mg/L) were determined. Using two starting inocula, in vitro fosfomycin activity was assessed over 24 h in growth (0.5-512 mg/L) and time-kill assays (64 and 307 mg/L). RESULTS: Strong and weak mutator clinical isolates and single-gene deletion mutants, except for ΔuhpT and ΔdnaQ, were susceptible by GSA. By BMD, the percentage of resistant clinical isolates reached 36%. Single-gene deletion mutants showed BMD MICs similar to those for subpopulations by GSA. Strong mutators showed a higher probability of selecting fosfomycin mutants at higher concentrations. By combining the two mechanisms of mutation, MICs and ranges of resistant subpopulations increased, enabling strains to survive at higher fosfomycin concentrations in growth monitoring assays. In time-kill assays, high inocula increased survival by 37.5% at 64 mg/L fosfomycin, compared with low starting inocula. CONCLUSIONS: The origin and variability of the fosfomycin heteroresistance phenotype can be partially explained by high mutation frequencies together with mechanisms of fosfomycin resistance. Subpopulations should be considered until clinical meaning is established.

A prospective, multicenter case control study of risk factors for acquisition and mortality in Enterobacter species bacteremia.

BACKGROUND: Enterobacter is among the main etiologies of hospital-acquired infections. This study aims to identify the risk factors of acquisition and attributable mortality of Enterobacter bacteremia. METHODS: Observational, case-control study for risk factors and prospective cohort for outcomes of consecutive cases with Enterobacter bacteremia. This study was conducted in five hospitals in Spain over a three-year period. Matched controls were patients with negative blood cultures and same sex, age, and hospitalization area. RESULTS: The study included 285 cases and 570 controls. E. cloacae was isolated in 198(68.8%) cases and E. aerogenes in 89(31.2%). Invasive procedures (hemodialysis, nasogastric tube, mechanical ventilation, surgical drainage tube) and previous antibiotics or corticosteroids were independently associated with Enterobacter bacteremia. Its attributable mortality was 7.8%(CI95%2.7-13.4%), being dissimilar according to a McCabe index: non-fatal=3.2%, ultimately fatal=12.9% and rapidly fatal=0.12%. Enterobacter bacteremia remained an independent risk factor for mortality among cases with severe sepsis or septic shock (OR 5.75 [CI95%2.57-12.87], p<0.001), with an attributable mortality of 40.3%(CI95%25.7-53.3). Empiric therapy or antibiotic resistances were not related to the outcome among patients with bacteremia. CONCLUSIONS: Invasive procedures, previous antibiotics and corticosteroids predispose to acquire Enterobacter bacteremia. This entity increases mortality among fragile patients and those with severe infections. Antibiotic resistances did not affect the outcome.

Prevalence of ST131 Clone Producing Both ESBL CTX-M-15 and AAC(6')Ib-cr Among Ciprofloxacin-Resistant Escherichia coli Isolates from Yemen.

Objective: The aim of this study was to characterize the O25b/ST131 clone in ciprofloxacin-resistant Escherichia coli isolates from Yemen. Materials and Methods: A total of 41 ciprofloxacin-resistant E. coli strains were collected from clinical samples of inpatients and outpatients from Sana'a (Yemen) from January to December 2013. Antimicrobial susceptibility testing, polymerase chain reaction amplification, and sequencing were used for detection of plasmid-mediated quinolone resistance determinants, extended-spectrum beta-lactamases genes and mutations in the quinolone resistance-determining regions of the target genes gyrA and parC. Genetic relatedness of E. coli isolates was determined by pulsed-field gel electrophoresis (PFGE). O25b/ST131 clone detection was performed using polymerase chain reaction of O25b rfb and allele 3 of the pabB gene and by a multilocus sequence typing. Results: All E. coli isolates contained the aac(6')Ib-cr gene associated with blaCTX-M-15 and qnrS genes in 63.4% and 12.2%, respectively. A rate of 36.6% (15/41) of O25b/ST131 E. coli isolates were identified belonging to the H30-Rx subclone producing both CTX-M-15 and Aac(6')Ib-cr enzymes and carrying two substitutions in GyrA (Ser83Leu/Asp87Asn) and two substitutions in ParC (Ser80Ile/Glu84Val). Most of them were uropathogenic unrelated E. coli isolates recovered from outpatients. Conclusion: This is the first report of a high prevalence of E. coli O25b/ST131 from Yemen.

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.

Molecular Characterization of Fluoroquinolone-Resistant Haemophilus parainfluenzae Clinical Isolates in Spain.

OBJECTIVE: The objective was to characterize a group of clinical isolates of fluoroquinolone-resistant Haemophilus parainfluenzae collected in Northern Spain (March-December 2014). METHODS: Twelve clinical isolates of H. parainfluenzae were studied by performing antimicrobial susceptibility testing and PCR amplification and nucleotide sequencing of the QRDR (quinolone resistance-determining region) of gyrA, parC, gyrB, and parE genes. Screening for plasmid-mediated quinolone resistance (PMQR) was also studied. Pulsed-field gel electrophoresis (PFGE) was used for molecular typing. RESULTS: Antimicrobial susceptibility testing showed that all the isolates were resistant to the fluoroquinolones tested (ciprofloxacin, levofloxacin, norfloxacin, and moxifloxacin). Analysis of the QRDR demonstrated that all the isolates presented mutations in gyrA and parC. A Glu88Lys substitution in ParC is reported for the first time in H. parainfluenzae. No PMQR gene was detected. PFGE results showed that isolates were not clonally related. CONCLUSION: Multiple H. parainfluenzae fluoroquinolone-resistant isolates grouped in the same area in a short period of time showed diverse substitutions in QRDR of gyrA/parC and were not clonally related, indicating individual emergence. In addition, we described the first report of Glu88Lys substitution in ParC.