Gonococcal Strains Research Articles

Arginine- and Polyamine-Induced Lactic Acid Resistance in Neisseria gonorrhoeae.

Microbe-derived lactic acid protects women from pathogens in their genital tract. The purpose of this study was to determine lactic acid susceptibility of Neisseria gonorrhoeae, and identify potential acid resistance mechanisms present in this pathogen. Tested in vitro, lactic acid killed all 10 gonococcal strains analyzed in a low pH-dependent manner. Full inactivation occurred at pH 4.5. At low pH, lactic acid treatment resulted in the entry of the DNA-binding fluorochrome propidium iodide into the microbial cells, suggesting that hydrogen ions from lactic acid compromise the integrity of the bacterial cell wall/membrane. Most likely, hydrogen ions also inactivate intracellular proteins since arginine rendered significant protection against lactic acid presumably through action of the gonococcal arginine decarboxylase, an enzyme located in the bacterial cytoplasm. Surprisingly, arginine also lessened lactic acid-mediated cell wall/membrane disruption. This effect is probably mediated by agmatine, a triamine product of arginine decarboxylase, since agmatine demonstrated a stronger protective effect on GC than arginine at equal molar concentration. In addition to agmatine, diamines cadaverine and putrescine, which are generated by bacterial vaginosis-associated microbes, also induced significant resistance to lactic acid-mediated GC killing and cell wall/membrane disruption. These findings suggest that the arginine-rich semen protects gonococci through both neutralization-dependent and independent mechanisms, whereas polyamine-induced acid resistance contributes to the increased risk of gonorrhea in women with bacterial vaginosis.

Novel Genes Related to Ceftriaxone Resistance Found among Ceftriaxone-Resistant Neisseria gonorrhoeae Strains Selected In Vitro

The emergence of ceftriaxone-resistantNeisseria gonorrhoeaeis currently a global public health concern. However, the mechanism of ceftriaxone resistance is not yet fully understood. To investigate the potential genes related to ceftriaxone resistance inNeisseria gonorrhoeae, we subcultured six gonococcal strains with increasing concentrations of ceftriaxone and isolated the strains that became resistant. After analyzing several frequently reported genes involved in ceftriaxone resistance, we found only a single mutation inpenA(A501V). However, differential analysis of the genomes and transcriptomes between pre- and postselection strains revealed many other mutated genes as well as up- and downregulated genes. Transformation of the mutatedpenAgene into nonresistant strains increased the MIC between 2.0- and 5.3-fold, and transformation of mutatedftsXincreased the MIC between 3.3- and 13.3-fold. Genes encoding the ABC transporters FarB, Tfq, Hfq, and ExbB were overexpressed, whilepilM,pilN, andpilQwere downregulated. Furthermore, the resistant strain developed cross-resistance to penicillin and cefuroxime, had an increased biochemical metabolic rate, and presented fitness defects such as prolonged growth time and downregulated PilMNQ. In conclusion, antimicrobial pressure could result in the emergence of ceftriaxone resistance, and the evolution of resistance ofNeisseria gonorrhoeaeto ceftriaxone is a complicated process at both the pretranscriptional and posttranscriptional levels, involving several resistance mechanisms of increased efflux and decreased entry.

An outbreak of high-level azithromycin resistant Neisseria gonorrhoeae in England

ObjectivesTo investigate a potential outbreak of high-level azithromycin resistant (HL-AziR) gonococcal infections diagnosed in eight patients attending a sexual health clinic in Leeds, North England, between November 2014 and March...

Gonococcal infections in Austria: a long-term observation of prevalence and resistance profiles from 1999 to 2014.

The increase in minimum inhibitory concentrations (MICs) of cephalosporins for Neisseria gonorrhoeae has given rise to concerns regarding potentially untreatable gonococcal infections. The goal was to ascertain the prevalence of gonorrhea in a Viennese patient group and determine resistance patterns. Another objective was to evaluate resistance profiles and MIC values of gonococcal isolates in an Austria-wide surveillance project. From 1999 to 2014, 350,000 individuals were tested for gonococci at the Viennese Outpatient Clinic. In addition, from 2010 to 2014, the MICs of recommended antibiotics was determined in 3,584 gonococcal isolates, initially by agar dilution and breakpoint determination, and, from 2012 onwards, by Etest®. During the observation period, the prevalence of gonorrhea increased eightfold, with a significantly greater number of quinolone, penicillin, and tetracycline- resistant strains. In gonococcal strains isolated from across Austria, there was an increase in cefixime and ceftriaxone MICs toward breakpoints. Twenty-one isolates showed cefixime resistance, and while there was an increase in azithromycin resistance from 0.9 % (2013) to 3.2 % (2014), no resistance to ceftriaxone was observed. Currently, there is no imminent risk of untreatable gonorrhea in Austria. However, continuing the use of gonococcal cultures as a diagnostic tool for establishing resistance profiles is essential in order to monitor trends in the development of Neisseria (N.) gonorrhoeae resistance.

006.2 High in vitro susceptibility to the novel spiropyrimidinetrione azd0914 among 873 contemporary clinical neisseria gonorrhoeae isolates in 21 european countries during 2012–2014

Introduction Resistance in Neisseria gonorrhoeae has emerged to all antimicrobials available for treatment of gonorrhoea. The first gonococcal strains with high-level resistance to ceftriaxone, the last option for first-line empirical antimicrobial monotherapy, were recently described. Consequently, new treatment options are essential. In this study, the in vitro activity of the novel spiropyrimidinetrione AZD0914, a DNA topoisomerase II inhibitor, among contemporary consecutive clinical N. gonorrhoeae isolates obtained in 21 European countries was investigated and compared to the activities of antimicrobials currently or previously recommended for treatment. Methods Consecutive clinical N. gonorrhoeae isolates (n = 873) cultured in 21 European countries during 2012–2014 were examined for their susceptibility to AZD0914. The MICs of AZD0914 were determined using agar dilution method. For comparison, the MICs of ceftriaxone, cefixime, azithromycin and ciprofloxacin were determined using Etest or agar dilution method. Results For AZD0914, the MIC range, modal MIC, MIC50 and MIC90 was ≤0.002–0.25 mg/L, 0.125 mg/L, 0.064 mg/L and 0.125 mg/L, respectively. The MIC values were substantially lower than those of the fluoroquinolone ciprofloxacin and most other antimicrobials examined. No cross-resistance with any other examined antimicrobial was observed. Conclusion The in vitro susceptibility to the novel spiropyrimidinetrione AZD0914 among 873 contemporary clinical isolates from 21 European countries was high and no cross-resistance to antimicrobials currently or previously used for gonorrhoea treatment was indicated. Additional studies investigating the in vitro and in vivo induction and mechanisms of AZD0914 resistance in gonococci, pharmacokinetics/pharmacodynamics in modelling/simulations and in humans, and performance in randomised controlled gonorrhoea treatment trials, are essential. Disclosure of interest statement This work was funded by the Orebro County Council Research Committee and the Foundation for Medical Research at Orebro University Hospital, Sweden. We are grateful to Michael Huband and John Mueller, AstraZeneca for providing the AZD0914 compound and to ECDC, particularly Gianfranco Spiteri and Andrew Amato-Gauci, for funding and coordinating the European Gonococcal Antimicrobial Surveillance Programme (Euro-GASP).

S11.2 Efflux pumps inneisseria gonorrhoeae– cause of resistance and targets for therapeutics and vaccine?

Neisseria gonorrhoeae is the cause of the sexually transmitted infection termed gonorrhoea, which afflicts over 100 million people worldwide each year. Since the mid-1940s, beginning with the availability of penicillin (Pen), antibiotic therapy has been the mainstay for curing infection and halting the spread of the gonococcus in the community. Unfortunately, gonococci developed resistance to Pen and many other antibiotics that were brought into clinical practice to counter act the growing problem of Pen (and other antibiotics) resistance. With the recent emergence of strains expressing resistance to the third-generation cephalosporins (cefixime and ceftriaxone) or other important antibiotics (macrolides and fluoroquinolones) there is considerable fear that without new antibiotics, gonorrhoea will become more difficult to treat; indeed, some have warned of the possibility of untreatable infections. In order to address this public health crisis of antibiotic resistant gonococci, it is essential that new bacterial targets are identified so as to facilitate the development of novel therapeutic drugs. I will discuss the role of the MtrC-MtrD-MtrE efflux pump in the development of gonococcal resistance to antibiotics and host-derived antimicrobials (e.g., cationic antimicrobial peptides). These phenotypes are augmented in gonococcal strains that have mutations that result in enhanced transcription of the mtrCDE efflux pump gene complex. I will provide evidence that this efflux pump is critical for the ability of gonococci to resist certain antibiotics as well as surviving during infection. Against this background, I will propose that this pump offers new targets for drug development (efflux pump inhibitors) and vaccine development to combat antibiotic resistant gonococcal strains and to prevent infections.

001.3 High-level azithromycin resistance inneisseria gonorrhoeaeclinical isolates in nanjing, china, 2013–2014

Introduction Dual antimicrobial therapy with ceftriaxone plus azithrothmycin has recently been recommended for uncomplicated gonorrhoea in the United States, the United Kingdom and Canada because of increases in MICs of N. gonorrhoeae for extended-spectrum cephalosporins. However, emergence of high-level azithromycin resistance in N. gonorrhoeae have been reported in many countries. No high-level azithromycin-resistant isolates of N.gonorrhoeae have been reported in China. Azithromycin has been added into antibiotic susceptibility panel since 2013. In this study, we present the results of amtimicrobial susceptibility testing of 384 gonococcal strains isolated between 2013 and 2014 and evidence of high-level azithromycin resistance in Nanjing, China. Methods 384 N. gonorrhoeae isolates were isolated sequentially from male adults with symptoms/signs of urethritis attending a single STD clinic in Nanjing, China between 2013 and 2014. Minimum inhibitory concentrations (MICs) of N. gonorrhoeae to penicillin, tetracycline, ciprofloxacin, spectinomycin, azithromycin, cefixime and ceftriaxone were determined by the agar dilution technique. b-lactamase production was determined by paper acidometric testing. Results Resistance to penicillin and tetracycline was 72.1% (277/384) and 85.9% (330/384), respectively; 46.9% (180/384) of strains were PPNG and 34.6% (133/384) were TRNG. All isolates (100%) were resistant to ciprofloxacin (MIC ≥1 μg/ml). A total of 32.3% (124/384) of isolates were resistant to azithromycin (MIC ≥1μg/ml), among them 25% (31/124) isolates displayed high-level azithromycin resistance (MIC ≥256 μg/ml). All isolates were susceptible to spectinomycin, cefixime and ceftriaxone. However, 16 isolates (4.3%) had elevated MICs (≥0.125 μg/ml) for cefixime and 38 isolates (10.1%) had an MIC of 0.125 μg/ml for ceftriaxone. Conclusion The present study shows a high prevalence of N. gonorrhoeae isolates displaying resistance to penicillin, tetracycline, azithromycin and ciprofloxacin and reduced susceptibility to extended-spectrum cephalosporins. High-level azithromycin resistance in N. gonorrhoeae has emerged in Nanjing, China. Disclosure of interest statement Nothing to declare

005.2 Diagnostic and clinical implications of genotypic fluoroquinolone susceptibility detection forneisseria gonorrhoeae

Introduction Antibiotic resistance poses major challenges to empirical treatment of Neisseria gonorrhoeae (NG), potentially addressable if antimicrobial susceptibility point of care (POC) tests were available. The performance of a POC compatible real-time PCR assay (GCSNP), enabling detection of fluoroquinolone susceptible NG directly on clinical samples from multiple anatomical sites and on diverse circulating strains, was evaluated. Methods Residual routine nucleic acid amplification test samples, derived from patients who were also culture positive for NG at the same clinical attendance were GCSNP tested. Assay performance was further verified using a phenotypically characterised fluoroquinolone resistant and susceptible strain panel which was sequence-typed using NG-MAST (NG Multi Antigen Sequence Typing). Results 290 residual samples derived from 222 clinical episodes (56 female; 166 male) were tested by GCSNP, yielding result in 90% (n = 262/290), with assay failure more likely in non-genital compared to genital samples (16.4% vs. 5.2%, p = 0.002). 29.7% (n = 66/222) of NG cases were attributable to fluoroquinolone resistant strains in at least one anatomical site. GCSNP predictive values for fluoroquinolone susceptibility were 100% (95% CI: 95.9–100%) and 100% (82.8–100%), respectively for urogenital (n = 173) and rectal samples (n = 37). In four episodes of multi-anatomical-site infection (3 male, 1 female) different antimicrobial susceptibility profiles were observed across sample sites but all were correctly genotyped using GCSNP. GCSNP panel testing correctly identified all of 92 phenotypically susceptible (n = 52) and resistant (n = 40) strains. A total of 66 diverse NG-MAST sequence types were observed in the panel. Conclusion GCSNP testing enables accurate genotypic detection of fluoroquinolone susceptible NG from clinical samples, from multiple anatomical sites and across diverse circulating gonococcal strains, enabling use of tailored anti-gonococcal therapy following NAAT positivity. If multi-site infection is suspected, genotypic testing on all anatomical sites is necessary in order to account for the presence of infections with mixed susceptibility. Disclosure of interest statement This study was funded under the UKCRC Translational Infection Research (TIR) Initiative supported by the Medical Research Council (Grant Number G0901608) with contributions to the Grant from the Biotechnology and Biological Sciences Research Council, the National Institute for Health Research on behalf of the Department of Health, the Chief Scientist Office of the Scottish Government Health Directorates and the Wellcome Trust.

Evaluation of the new AmpliSens multiplex real-time PCR assay for simultaneous detection of Neisseriagonorrhoeae, Chlamydiatrachomatis, Mycoplasmagenitalium, and Trichomonasvaginalis.

In this study, we performed an evaluation of the new CE-marked multiplex real-time AmpliSens N.gonorrhoeae/C.trachomatis/M.genitalium/T.vaginalis-MULTIPRIME-FRT PCR assay compared to APTIMA tests, i.e., APTIMA COMBO 2assay, APTIMA Trichomonasvaginalis assay (FDA-approved), and two different APTIMA Mycoplasmagenitalium assays (research use only; one of them only used for discrepancy analysis). Vaginal swabs (n=209) and first-void urine (FVU) specimens from females (n=498) and males (n=554), consecutive attendees (n=1261) at a dermatovenerological clinic in Sweden, were examined. The sensitivity of the AmpliSens PCR assay for detection of C.trachomatis (6.3% prevalence), M.genitalium (5.7% prevalence), N.gonorrhoeae (0.3% prevalence), and T.vaginalis (0.08% prevalence) was 97.5% (95% confidence interval (CI): 91.2-99.6%), 81.9% (95% CI: 70.7-89.7%), 100% (95% CI: 40.2-100%) and 100% (95% CI: 16.5-100%), respectively. The specificity of the AmpliSens PCR assay was 100% (95% CI: 99.6-100%) for all agents. The analytical sensitivity and specificity for N.gonorrhoeae detection was excellent, i.e., 55 international gonococcal strains detected and 135 isolates of 13 non-gonococcal Neisseria species were negative. In conclusion, the multiplex real-time AmpliSens N.gonorrhoeae/C.trachomatis/M.genitalium/T.vaginalis-MULTIPRIME-FRT PCR assay demonstrated high sensitivity and excellent specificity for the detection of C.trachomatis, N.gonorrhoeae, and T.vaginalis, and excellent specificity but suboptimal sensitivity for M.genitalium detection.

Characterization of a spermine/spermidine transport system reveals a novel DNA sequence duplication in Neisseria gonorrhoeae.

During infection, Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea, comes into contact with numerous host compounds including polyamines (e.g. spermine and spermidine). Here, we show that spermine and spermidine concentrations in the growth medium decrease to undetectable levels in the presence of gonococci over time, but not when proteins of the putative polyamine transport system are lost due to mutation. We propose that gonococci have a functional and sole polyamine transport system (PotFGHI) that specifically imports spermine and spermidine. Bioinformatics and molecular analyses showed that the transporter's potGHI genes are organized as an operon while the gene encoding the necessary cognate periplasmic polyamine-binding protein (PotF) is located elsewhere on the chromosome. Interestingly, within the potGHI locus, we identified a novel duplicated sequence, which we term the Pot-Gene-Associated-Duplication-Element, present in variable copy numbers in different gonococcal strains that was likely formed from the 5(') and 3(') ends of the coding sequences of the tandemly linked potH and potG genes, respectively.

Emergence of decreased susceptibility and resistance to extended-spectrum cephalosporins in Neisseria gonorrhoeae in Korea.

Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a major concern globally; however, no comprehensive AMR data for gonococcal isolates cultured after 2006 in Korea have been published internationally. We determined the susceptibility of N. gonorrhoeae isolates cultured in 2011-13, the mechanism of extended-spectrum cephalosporin (ESC) resistance and the molecular epidemiology of gonococcal strains in Korea. In 2011-13, 210 gonococcal isolates were collected in Korea and their AMR profiles were examined by the agar dilution method. The penA, mtrR, penB, ponA and pilQ genes were sequenced in 25 isolates that were resistant to ESCs and 70 randomly selected isolates stratified by year. For molecular epidemiology, N. gonorrhoeae multiantigen sequence typing and MLST were performed. None of the N. gonorrhoeae isolates was susceptible to penicillin G and most were resistant to tetracycline (50%) and ciprofloxacin (97%). The rates of resistance to ceftriaxone, azithromycin, cefpodoxime and cefixime were 3%, 5%, 8% and 9%, respectively. However, all isolates were susceptible to spectinomycin. Twenty-one (84%) of the 25 ESC-resistant isolates contained the non-mosaic PBP2 XIII allele; however, the remaining 4 (16%) possessed the mosaic PBP2 X allele, which has been previously associated with ESC resistance including treatment failures. In Korea, susceptibility to spectinomycin remains high. However, the recent emergence of ESC-resistant N. gonorrhoeae strains, including strains possessing the PBP2 mosaic X and non-mosaic XIII alleles, is a major concern and enhanced AMR surveillance is necessary to prevent transmission of these strains.

High in vitro susceptibility to the novel spiropyrimidinetrione ETX0914 (AZD0914) among 873 contemporary clinical Neisseria gonorrhoeae isolates from 21 European countries from 2012 to 2014.

Resistance in Neisseria gonorrhoeae against all antimicrobials available for the treatment of gonorrhea has emerged. The first gonococcal strains with high-level resistance to ceftriaxone, the last option for first-line empirical antimicrobial monotherapy, were recently described. Consequently, new treatment options are essential. In this study, the in vitro activity of the novel spiropyrimidinetrione ETX0914 (AZD0914), a DNA topoisomerase II inhibitor, was investigated among contemporary consecutive clinical N. gonorrhoeae isolates obtained in 21 European countries and compared to the activities of antimicrobials currently or previously recommended for treatment. Consecutive clinical N. gonorrhoeae isolates (n = 873) cultured in 21 European countries from 2012 to 2014 were examined for their susceptibility to ETX0914. The MICs of ETX0914 were determined using the agar dilution method. For comparison, the MICs of ceftriaxone, cefixime, azithromycin, and ciprofloxacin were determined using Etest or the agar dilution method. For ETX0914, the MIC range, modal MIC, MIC50, and MIC90 were ≤0.002 to 0.25 mg/liter, 0.125 mg/liter, 0.064 mg/liter, and 0.125 mg/liter, respectively. The MIC values were substantially lower than those of the fluoroquinolone ciprofloxacin and most other antimicrobials examined. No cross-resistance with any other examined antimicrobial was observed. In conclusion, the in vitro susceptibility to the novel spiropyrimidinetrione ETX0914 (AZD0914) among 873 contemporary clinical isolates from 21 European countries was high, and no cross-resistance to antimicrobials currently or previously used for gonorrhea treatment was indicated. Additional studies investigating the in vitro and in vivo induction and mechanisms of ETX0914 resistance in gonococci, pharmacokinetics/pharmacodynamics in modeling/simulations and in humans, and performance in randomized controlled gonorrhea treatment trials are essential.

P7 The epidemiology of unique ng-masts differs from that of commonly circulating ng-masts in lothian, scotland

Background/introduction Surveillance of gonococcal infection in Scotland has demonstrated variation in the prevalence of Neisseria gonorrhoeae multi-antigen sequence types. Some appear to circulate extensively, other sequence types (STs) may be recorded only once. Aim(s)/objectives We aimed to review the epidemiology of gonococcal infection resulting from unique STs and examine for any association between unique STs and antibiotic resistance patterns. Methods All gonococcal isolates from Lothian with a ST unique to Scotland, identified July 2006–October 2013, were included in the study. A control group of 76 patients infected with commonly circulating STs was also identified from the same study period. Results 92 cases of N. gonorrhoae with unique STs were identified. Of these, 55 were truly distinct and categorised as ‘unique and different’. The remaining 33 cases had STs which differed only slightly from locally circulating types and were likely to have evolved due to mutation of common strains. These were categorised as ‘unique and similar’. Patients infected with ‘unique and different’ STs were significantly more likely than controls (p Discussion/conclusion Gonococcal strains from non-European countries may be associated with antibiotic resistance. Identification of a ‘unique and different’ sequence type raises the possibility of an imported strain of gonococcal infection and demands particular vigilance in looking for antibiotic resistance.

Will use of combination cephalosporin/azithromycin therapy forestall resistance to cephalosporins in Neisseria gonorrhoeae?

The history of antimicrobial therapy of Neisseria gonorrhoeae infections has been one of recurrent development of resistance towards the antibiotics recommended as first-line therapies.1 ,2 The initial discovery of penicillin was lifesaving for those who suffered from serious staphylococcal and streptococcal infections, but also provided the first truly effective therapy against disseminated gonococccal infections. The emergence of gonococcal penicillin resistance associated with increasing clinical use of penicillin to treat gonococcal infections led to more use of tetracycline for treatment, which was associated with the emergence and spread of tetracycline resistance. Similarly for the use of spectinomycin. Once prevalent, resistance determinants persist even when use of the associated antibiotic declines, and re-emerge rapidly on reintroduction of the antibiotic. Early use of ceftriaxone was not associated with emergence of resistance, but its widespread use diminished with the introduction of oral cephalosporins such as cefixime and the clinical availability of the fluoroquinolones. Unfortunately, substantial resistance emerged to both fluoroquinolones and oral cephalosporins, resulting in a return of ceftriaxone as recommended first-line therapy for gonococcal infections. With the recent widespread use of ceftriaxone for first-line therapy, we are now observing the emergence of gonococcal strains resistant to this agent.3 As there are no other drugs predictably effective as single doses for resistant strains, we face the prospect of worldwide spread of untreatable gonorrhoea. The exodus of many large pharmaceutical firms from the antibacterial therapeutic area in recent decades has heightened the urgency of the gonococcal resistance problem.4 Put simply, there are a few drugs in the pipeline likely to be effective in single doses against multiresistant gonococcal strains. As such, there is increasing interest in strategies to forestall the spread of gonococcal ceftriaxone resistance. Common strategies to forestall resistance, such as withholding antibiotics pending culture confirmation of infection, are impractical …

Bacteriocins and other bioactive substances of probiotic lactobacilli as biological weapons against Neisseria gonorrhoeae.

In the search of new antimicrobial agents against Neisseria gonorrhoeae, the bacteriocins-producing probiotic lactobacilli deserve special attention. The inhibitory effects of biosubstances such as organic acids, hydrogen peroxide and each bacteriocin-like inhibitory substance (BLIS) L23 and L60 on the growth of different gonococcal strains were investigated. Different non-treated and treated cell-free supernatants of two probiotic lactobacilli containing these metabolites were used. The aims of this work were (i) to evaluate the antimicrobial activity of the biosubstances produced by two probiotic lactobacilli, estimating the proportion in which each of them is responsible for the inhibitory effect, (ii) to define their minimum inhibitory concentrations (MICs) and, (iii) to determine the potential interactions between these biosubstances against N. gonorrhoeae. The main antimicrobial metabolites were the BLIS-es L23 and L60 in comparison with other biosubstances. Proportionally, their contributions to the inhibition on the gonococcal growth were 87.28% and 80.66%, respectively. The MIC values of bacteriocins were promising since these substances, when diluted, showed considerable inhibitory activity for all gonococci. In the interaction between bacteriocins, 100% of synergism was found on the gonococcal growth. In summary, this study indicates that both L23 and L60 could potentially serve to design new bioproducts against N. gonorrhoeae.

Fluoroquinolone resistance in Neisseria gonorrhoeae after cessation of ciprofloxacin usage in San Francisco: using molecular typing to investigate strain turnover.

Ciprofloxacin resistance (CipR) among gonococcal strains in San Francisco (SF) increased between 2001 and 2006 and decreased between 2007 and 2009. Molecular typing of isolates obtained from 2005 to 2009 was performed to elucidate changes in CipR prevalence. A total of 2526 samples were collected at the SF City Clinic between 2001 and 2009. Minimum inhibitory concentrations to ciprofloxacin were obtained by agar dilution. Prevalences of CipR strains were determined, with corresponding confidence intervals (CIs). Between 2005 and 2009, 460 isolates were selected for molecular typing using Neisseria gonorrhoeae multiantigen sequence typing. Between 2001 and 2006, the prevalence of CipR increased from 3.4% (95% CI, 1.3%-5.4%) to 44% (95% CI, 39%-50%). However, in 2007 prevalence began to decrease, reaching 9.6% (95% CI, 6.0%-13%) by 2009. Of the 203 strain types identified between 2005 and 2009, 126 genogroups of closely related strain types were formed (varying by ≤1% at both target loci). Levels of CipR within the data set correlate with the prevalence of 3 major genogroups (G): G437, G1407, and G3112. Molecular typing reveals that CipR within the tested population is maintained by strain turnover between resistant genogroups. Despite early recommendation in 2002 to stop ciprofloxacin use in California, CipR in SF increased through 2006. The subsequent decrease in CipR corresponds with the 2007 national recommendation to cease ciprofloxacin treatment of gonorrhea, which suggests that national recommendations are potentially more effective at reducing CipR than regional recommendations in areas with high strain turnover.

Antimicrobial susceptibility of Neisseria gonorrhoeae isolates from symptomatic men attending the Nanjing sexually transmitted diseases clinic (2011-2012): genetic characteristics of isolates with reduced sensitivity to ceftriaxone.

BackgroundEvolving gonococcal antimicrobial resistance (AMR) poses a serious threat to public health. The aim of this study was to: update antimicrobial susceptibility data of Neisseria gonorrhoeae recently isolated in Nanjing, China and identify specific deteminants of antimicrobial resistance and gentoypes of isolates with decreased sensitivity to ceftriaxone.Methods334 N. gonorrhoeae isolates were collected consecutively from symptomatic men attending the Nanjing STD Clinic between April 2011 and December 2012. The minimum inhibitory concentrations (MICs) for penicillin, tetracycline, ciprofloxacin, spectinomycin and ceftriaxone were determined by agar plate dilution for each isolate. Penicillinase-producing N. gonorrhoeae (PPNG) and tetracycline-resistant N. gonorrhoeae (TRNG) were examined and typed for β-lactamase and tetM encoding plasmids respectively. Isolates that displayed elevated MICs to ceftriaxone (MIC ≥0.125 mg/L) were also tested for mutations in penA, mtrR, porB1b, ponA and pilQ genes and characterized by Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST).Results98.8% (330/334) of N. gonorrhoeae isolates were resistant to ciprofloxacin; 97.9% (327/334) to tetracycline and 67.7% (226/334) to penicillin. All isolates were susceptible to ceftriaxone (MIC ≤0.25 mg/L) and spectinomycin (MIC ≤32 mg/L). Plasmid mediated resistance was exhibited by 175/334 (52%) of isolates: 120/334 (36%) of isolates were PPNG and 104/334 (31%) were TRNG. 90.0% (108/120) of PPNG isolates carried the Asia type β-lactamase encoding plasmid and 96% (100/104) of TRNG isolates carried the Dutch type tetM containing plasmid. Elevated MICs for ceftriaxone were present in 15 (4.5%) isolates; multiple mutations were found in penA, mtrR, porB1b and ponA genes. The 15 isolates were distributed into diverse NG-MAST sequence types; four different non-mosaic penA alleles were identified, including one new type.ConclusionsN. gonorrhoeae isolates in Nanjing generally retained similar antimicrobial resistance patterns to isolates obtained five years ago. Fluctuations in resistance plasmid profiles imply that genetic exchange among gonococcal strains is ongoing and is frequent. Ceftriaxone and spectinomycin remain treatments of choice of gonorrhea in Nanjing, however, decreased susceptibility to ceftriaxone and rising MICs for spectinomycin of N. gonorrhoeae isolates underscore the importance of maintaining surveillance for AMR (both phenotypic and genotypic).Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-014-0622-0) contains supplementary material, which is available to authorized users.

Characterisation of blaTEM genes and types of β-lactamase plasmids in Neisseria gonorrhoeae - the prevalent and conserved blaTEM-135 has not recently evolved and existed in the Toronto plasmid from the origin.

BackgroundAntimicrobial resistance (AMR) in Neisseria gonorrhoeae is a major concern worldwide. It has been recently feared that the blaTEM-1 gene is, via blaTEM-135, evolving into an extended-spectrum β-lactamase (ESBL), which could degrade all cephalosporins including ceftriaxone. The aims of the present study were to characterize the blaTEM genes, types of β-lactamase plasmids, the degradation of ampicillin by TEM-135 compared to TEM-1, and to perform molecular epidemiological typing of β-lactamase-producing N. gonorrhoeae strains internationally.Methodsβ-lactamase producing N. gonorrhoeae isolates (n = 139) cultured from 2000 to 2011 in 15 countries were examined using antibiograms, blaTEM gene sequencing, β-lactamase plasmid typing, and N. gonorrhoeae multiantigen sequence typing (NG-MAST). Furthermore, the blaTEM gene was sequenced in the first described Toronto plasmid (pJD7), one of the first Asian plasmids (pJD4) and African plasmids (pJD5) isolated in Canada. The degradation of ampicillin by TEM-135 compared to TEM-1 was examined using a MALDI-TOF MS hydrolysis assay.ResultsSix different blaTEM sequences were identified (among isolates with 125 different NG-MAST STs), i.e. blaTEM-1 (in 104 isolates), blaTEM-135 (in 30 isolates), and four novel blaTEM sequences (in 5 isolates). The blaTEM-1 allele was only found in the African and Asian plasmids, while all Rio/Toronto plasmids possessed the blaTEM-135 allele. Most interesting, the first described gonococcal Toronto plasmid (pJD7), identified in 1984, also possessed the highly conserved blaTEM-135 allele. The degradation of ampicillin by TEM-135 compared to TEM-1 was indistinguishable in the MALDI-TOF MS hydrolysis assay.ConclusionsblaTEM-135, encoding TEM-135, is predominantly and originally associated with the Rio/Toronto plasmid and prevalent among the β-lactamase producing gonococcal strains circulating globally. blaTEM-135 does not appear, as previously hypothesized, to have recently evolved due to some evolutionary selective pressure, for example, by the extensive use of extended-spectrum cephalosporins worldwide. On the contrary, the present study shows that blaTEM-135 existed in the Toronto plasmid from its discovery and that blaTEM-135 is highly conserved (not further evolved in the past >30 years). Nevertheless, international studies for monitoring the presence of different blaTEM alleles, the possible evolution of the blaTEM-135 allele, and the types of β-lactamase producing plasmids, remain imperative.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2334-14-454) contains supplementary material, which is available to authorized users.

Antimicrobial resistance in Neisseria gonorrhoeae in the 21st century: past, evolution, and future.

Neisseria gonorrhoeae is evolving into a superbug with resistance to previously and currently recommended antimicrobials for treatment of gonorrhea, which is a major public health concern globally. Given the global nature of gonorrhea, the high rate of usage of antimicrobials, suboptimal control and monitoring of antimicrobial resistance (AMR) and treatment failures, slow update of treatment guidelines in most geographical settings, and the extraordinary capacity of the gonococci to develop and retain AMR, it is likely that the global problem of gonococcal AMR will worsen in the foreseeable future and that the severe complications of gonorrhea will emerge as a silent epidemic. By understanding the evolution, emergence, and spread of AMR in N. gonorrhoeae, including its molecular and phenotypic mechanisms, resistance to antimicrobials used clinically can be anticipated, future methods for genetic testing for AMR might permit region-specific and tailor-made antimicrobial therapy, and the design of novel antimicrobials to circumvent the resistance problems can be undertaken more rationally. This review focuses on the history and evolution of gonorrhea treatment regimens and emerging resistance to them, on genetic and phenotypic determinants of gonococcal resistance to previously and currently recommended antimicrobials, including biological costs or benefits; and on crucial actions and future advances necessary to detect and treat resistant gonococcal strains and, ultimately, retain gonorrhea as a treatable infection.

A high-throughput method to examine protein-nucleotide interactions identifies targets of the bacterial transcriptional regulatory protein fur.

The Ferric uptake regulatory protein (Fur) is a transcriptional regulatory protein that functions to control gene transcription in response to iron in a number of pathogenic bacteria. In this study, we applied a label-free, quantitative and high-throughput analysis method, Interferometric Reflectance Imaging Sensor (IRIS), to rapidly characterize Fur-DNA interactions in vitro with predicted Fur binding sequences in the genome of Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea. IRIS can easily be applied to examine multiple protein-protein, protein-nucleotide and nucleotide-nucleotide complexes simultaneously and demonstrated here that seventy percent of the predicted Fur boxes in promoter regions of iron-induced genes bound to Fur in vitro with a range of affinities as observed using this microarray screening technology. Combining binding data with mRNA expression levels in a gonococcal fur mutant strain allowed us to identify five new gonococcal genes under Fur-mediated direct regulation.