Associations between antimicrobial susceptibility/resistance of Neisseria gonorrhoeae isolates in European Union/European Economic Area and patients' gender, sexual orientation and anatomical site of infection, 2009-2016.

BACKGROUND: The emergence and spread of antimicrobial resistance (AMR) in Neisseria gonorrhoeae, nationally and internationally, is a serious threat to the management and control of gonorrhoea. Limited and conflicting data regarding the epidemiological drivers of gonococcal AMR internationally have been published. We examined the antimicrobial susceptibility/resistance of gonococcal isolates (n = 15,803) collected across 27 European Union/European Economic Area (EU/EEA) countries in 2009-2016, in conjunction to epidemiological and clinical data of the corresponding patients, to elucidate associations between antimicrobial susceptibility/resistance and patients' gender, sexual orientation and anatomical site of infection. METHODS: In total, 15,803 N. gonorrhoeae isolates from the European Gonococcal Antimicrobial Surveillance Programme (Euro-GASP), 2009-2016, were examined. Associations between gonococcal susceptibility/resistance and patients' gender, sexual orientation and anatomical site of infection were investigated using univariate and multivariate logistic regression analysis. Statistical significance was determined by Pearson χ2-test or Fisher's exact test with two-tailed p-values of < 0.05 indicating significance. RESULTS: The overall gonococcal resistance from 2009 to 2016 was 51.7% (range during the years: 46.5-63.5%), 7.1% (4.5-13.2%), 4.3% (1.8-8.7%), and 0.2% (0.0-0.5%) to ciprofloxacin, azithromycin, cefixime, and ceftriaxone, respectively. The level of resistance combined with decreased susceptibility to ceftriaxone was 10.2% (5.7-15.5%). Resistance to cefixime and ciprofloxacin, and resistance combined with decreased susceptibility to ceftriaxone were positively associated with urogenital infections and heterosexual males, males with sexual orientation not reported and females (except for ciprofloxacin), i.e. when compared to men-who-have-sex-with-men (MSM). Azithromycin resistance was positively associated with heterosexual males, but no association was significant regarding anatomical site of infection. CONCLUSIONS: Overall, sexual orientation was the main variable associated with gonococcal AMR. Strongest positive associations were identified with heterosexual patients, particularly males, and not MSM. To provide evidence-based understanding and mitigate gonococcal AMR emergence and spread, associations between antimicrobial susceptibility/resistance and patients' gender, sexual orientation and anatomical site of infection need to be further investigated in different geographic settings. In general, these insights will support identification of groups at increased risk and targeted public health actions such as intensified screening, 3-site testing using molecular diagnostics, sexual contact tracing, and surveillance of treatment failures.

What's left in the cupboard? Older antimicrobials for treating gonorrhoea.

BACKGROUND: Neisseria gonorrhoeae has developed resistance to all antimicrobials used to treat gonorrhoea, with even ceftriaxone being undermined. It is therefore important to examine any potential to redeploy older antimicrobials routinely used for other infections to treat ceftriaxone-resistant gonococcal infections. OBJECTIVES: We examined the susceptibility of N. gonorrhoeae to aztreonam, chloramphenicol, co-trimoxazole, fosfomycin, piperacillin/tazobactam and rifampicin. METHODS: N. gonorrhoeae isolates (n = 94) were selected to include a range of antimicrobial susceptibilities: 58 were collected in the Gonococcal Resistance to Antimicrobials Surveillance Programme; 17 were clinical isolates referred to the PHE reference laboratory; and 19 were control strains. MICs were determined by agar dilution for the six study antimicrobials and for ceftriaxone and azithromycin as comparators. RESULTS: There was correlation between piperacillin/tazobactam and ceftriaxone MICs, but all five isolates with high ceftriaxone MICs (>0.5 mg/L) were inhibited by piperacillin/tazobactam at 0.06-0.5 mg/L. Aztreonam MICs for ceftriaxone-resistant isolates exceeded those of ceftriaxone. Among non-ß-lactams, fosfomycin and co-trimoxazole had low, tightly clustered MICs, suggesting widespread susceptibility, rifampicin split the collection into highly susceptible and highly resistant groups and chloramphenicol had a wide MIC distribution. CONCLUSIONS: Although unsuitable for empirical use, piperacillin/tazobactam, fosfomycin, co-trimoxazole, rifampicin and, possibly, chloramphenicol could be considered for individual patients with ceftriaxone-resistant gonococcal infection once MICs are known. Wider surveillance of the susceptibility of N. gonorrhoeae to these agents is needed, along with clinical trials and the establishment of clinical breakpoints for N gonorrhoeae.

Genomic and Phenotypic Variability in Neisseria gonorrhoeae Antimicrobial Susceptibility, England.

Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a global concern. Phylogenetic analyses resolve uncertainties regarding genetic relatedness of isolates with identical phenotypes and inform whether AMR is due to new mutations and clonal expansion or separate introductions by importation. We sequenced 1,277 isolates with associated epidemiologic and antimicrobial susceptibility data collected during 2013-2016 to investigate N. gonorrhoeae genomic variability in England. Comparing genetic markers and phenotypes for AMR, we identified 2 N. gonorrhoeae lineages with different antimicrobial susceptibility profiles and 3 clusters with elevated MICs for ceftriaxone, varying mutations in the penA allele, and different epidemiologic characteristics. Our results indicate N. gonorrhoeae with reduced antimicrobial susceptibility emerged independently and multiple times in different sexual networks in England, through new mutation or recombination events and by importation. Monitoring and control for AMR in N. gonorrhoeae should cover the entire population affected, rather than focusing on specific risk groups or locations.

Gentamicin, azithromycin and ceftriaxone in the treatment of gonorrhoea: the relationship between antibiotic MIC and clinical outcome.

OBJECTIVES: To investigate the relationship between MIC and clinical outcome in a randomized controlled trial that compared gentamicin 240 mg plus azithromycin 1 g with ceftriaxone 500 mg plus azithromycin 1 g. MIC analysis was performed on Neisseria gonorrhoeae isolates from all participants who were culture positive before they received treatment. METHODS: Viable gonococcal cultures were available from 279 participants, of whom 145 received ceftriaxone/azithromycin and 134 received gentamicin/azithromycin. Four participants (6 isolates) and 14 participants (17 isolates) did not clear infection in the ceftriaxone/azithromycin and gentamicin/azithromycin arms, respectively. MICs were determined by Etest on GC agar base with 1% Vitox. The geometric mean MICs of azithromycin, ceftriaxone and gentamicin were compared using logistic and linear regression according to treatment received and N. gonorrhoeae clearance. RESULTS: As the azithromycin MIC increased, gentamicin/azithromycin treatment was less effective than ceftriaxone/azithromycin at clearing N. gonorrhoeae. There was a higher geometric mean MIC of azithromycin for isolates from participants who had received gentamicin/azithromycin and did not clear infection compared with those who did clear infection [ratio 1.95 (95% CI 1.28-2.97)], but the use of categorical MIC breakpoints did not accurately predict the treatment response. The geometric mean MIC of azithromycin was higher in isolates from the pharynx compared with genital isolates. CONCLUSIONS: We found that categorical resistance to azithromycin or ceftriaxone in vitro, and higher gentamicin MICs in the absence of breakpoints, were poorly predictive of treatment failure.

Detection in the United Kingdom of the Neisseria gonorrhoeae FC428 clone, with ceftriaxone resistance and intermediate resistance to azithromycin, October to December 2018.

We describe detection in the United Kingdom (UK) of the drug-resistant Neisseria gonorrhoeae FC428 clone, with ceftriaxone resistance and intermediate azithromycin resistance. Two female patients developed infection following contact with UK-resident men from the same sexual network linked to travel to Ibiza, Spain. One case failed treatment with ceftriaxone, and azithromycin and gentamicin, before successful treatment with ertapenem. Both isolates had indistinguishable whole-genome sequences. Urgent action is essential to contain this drug-resistant strain.

Genetic relatedness of ceftriaxone-resistant and high-level azithromycin resistant Neisseria gonorrhoeae cases, United Kingdom and Australia, February to April 2018.

Between February and April 2018, three ceftriaxone-resistant and high-level azithromycin-resistant Neisseria gonorrhoeae cases were identified; one in the United Kingdom and two in Australia. Whole genome sequencing was used to show that the isolates from these cases belong to a single gonococcal clone, which we name the A2543 clone.

Stably high azithromycin resistance and decreasing ceftriaxone susceptibility in Neisseria gonorrhoeae in 25 European countries, 2016.

BACKGROUND: The European Gonococcal Antimicrobial Surveillance Programme (Euro-GASP) performs annual sentinel surveillance of Neisseria gonorrhoeae susceptibility to therapeutically relevant antimicrobials across the European Union/European Economic Area (EU/EEA). We present the Euro-GASP results from 2016 (25 countries), linked to patient epidemiological data, and compared with data from previous years. METHODS: Agar dilution and minimum inhibitory concentration (MIC) gradient strip methodologies were used to determine the antimicrobial susceptibility (using EUCAST breakpoints) of 2660 N. gonorrhoeae isolates from 25 countries across the EU/EEA. Significance of differences compared with Euro-GASP results in previous years was analysed using Z-tests. RESULTS: No isolates with resistance to ceftriaxone (MIC > 0.125 mg/L) were detected in 2016 (one in 2015). However, the proportion of isolates with decreased susceptibility to ceftriaxone (MICs from 0.03 mg/L to 0.125 mg/L) increased significantly (p = 0.01) from 2015 to 2016. There were 14 (0.5%) isolates with ceftriaxone MICs 0.125 mg/L (on the resistance breakpoint), of which one isolate was resistant to azithromycin and four showed intermediate susceptibility to azithromycin. Cefixime resistance was detected in 2.1% of isolates in 2016 compared with 1.7% in 2015 (p = 0.26) and azithromycin resistance in 7.5% in 2016 compared with 7.1% in 2015 (p = 0.74). Seven (0.3%) isolates from five countries displayed high-level azithromycin resistance (MIC≥256 mg/L) in 2016 compared with five (0.2%) isolates in 2015. Resistance rate to ciprofloxacin was 46.5% compared with 49.4% in 2015 (p = 0.06). No isolates were resistant to spectinomycin and the MICs of gentamicin remained stable compared with previous years. CONCLUSIONS: Overall AMR rates in gonococci in EU/EEA remained stable from 2015 to 2016. However, the ceftriaxone MIC distribution shifted away from the most susceptible (≤0.016 mg/L) and the proportion of isolates with decreased susceptibility to ceftriaxone increased significantly. This development is of concern as current European gonorrhoea management guideline recommends ceftriaxone 500 mg plus azithromycin 2 g as first-line therapy. With azithromycin resistance at 7.5%, the increasing ceftriaxone MICs might soon threaten the effectiveness of this therapeutic regimen and requires close monitoring.

Gonorrhoea treatment failure caused by a Neisseria gonorrhoeae strain with combined ceftriaxone and high-level azithromycin resistance, England, February 2018.

We describe a gonorrhoea case with combined high-level azithromycin resistance and ceftriaxone resistance. In February 2018, a heterosexual male was diagnosed with gonorrhoea in the United Kingdom following sexual intercourse with a locally resident female in Thailand and failed treatment with ceftriaxone plus doxycycline and subsequently spectinomycin. Resistance arose from two mechanisms combining for the first time in a genetic background similar to a commonly circulating strain. Urgent action is essential to prevent further spread.

Public health surveillance of multidrug-resistant clones of Neisseria gonorrhoeae in Europe: a genomic survey.

BACKGROUND: Traditional methods for molecular epidemiology of Neisseria gonorrhoeae are suboptimal. Whole-genome sequencing (WGS) offers ideal resolution to describe population dynamics and to predict and infer transmission of antimicrobial resistance, and can enhance infection control through linkage with epidemiological data. We used WGS, in conjunction with linked epidemiological and phenotypic data, to describe the gonococcal population in 20 European countries. We aimed to detail changes in phenotypic antimicrobial resistance levels (and the reasons for these changes) and strain distribution (with a focus on antimicrobial resistance strains in risk groups), and to predict antimicrobial resistance from WGS data. METHODS: We carried out an observational study, in which we sequenced isolates taken from patients with gonorrhoea from the European Gonococcal Antimicrobial Surveillance Programme in 20 countries from September to November, 2013. We also developed a web platform that we used for automated antimicrobial resistance prediction, molecular typing (N gonorrhoeae multi-antigen sequence typing [NG-MAST] and multilocus sequence typing), and phylogenetic clustering in conjunction with epidemiological and phenotypic data. FINDINGS: The multidrug-resistant NG-MAST genogroup G1407 was predominant and accounted for the most cephalosporin resistance, but the prevalence of this genogroup decreased from 248 (23%) of 1066 isolates in a previous study from 2009-10 to 174 (17%) of 1054 isolates in this survey in 2013. This genogroup previously showed an association with men who have sex with men, but changed to an association with heterosexual people (odds ratio=4·29). WGS provided substantially improved resolution and accuracy over NG-MAST and multilocus sequence typing, predicted antimicrobial resistance relatively well, and identified discrepant isolates, mixed infections or contaminants, and multidrug-resistant clades linked to risk groups. INTERPRETATION: To our knowledge, we provide the first use of joint analysis of WGS and epidemiological data in an international programme for regional surveillance of sexually transmitted infections. WGS provided enhanced understanding of the distribution of antimicrobial resistance clones, including replacement with clones that were more susceptible to antimicrobials, in several risk groups nationally and regionally. We provide a framework for genomic surveillance of gonococci through standardised sampling, use of WGS, and a shared information architecture for interpretation and dissemination by use of open access software. FUNDING: The European Centre for Disease Prevention and Control, The Centre for Genomic Pathogen Surveillance, Örebro University Hospital, and Wellcome.

Phenotypic antimicrobial susceptibility testing of Chlamydia trachomatis isolates from patients with persistent or successfully treated infections.

Objectives: Antimicrobial susceptibility data for Chlamydia trachomatis are lacking. Methodologies for susceptibility testing in C. trachomatis are not well-defined, standardized or performed routinely owing to its intracellular growth requirements. We sought to develop an assay for the in vitro susceptibility testing of C. trachomatis isolates from two patient cohorts with different clinical outcomes. Methods: Twenty-four clinical isolates (11 from persistently infected and 13 from successfully treated patients) were overlaid with media containing two-fold serial dilutions of azithromycin or doxycycline. After incubation, aliquots were removed from the stock inoculum (SI) and each antimicrobial concentration for total RNA extraction, complementary DNA generation and real-time PCR. The MIC was defined as the lowest antimicrobial concentration where a 95% reduction in transcription was evident in comparison with the SI for each isolate. Results: MICs of azithromycin were comparable for isolates from the two patient groups (82% ≤ 0.25 mg/L for persistently infected and 100% ≤ 0.25 mg/L for successfully treated patients). Doxycycline MICs were at least two-fold lower for isolates from the successfully treated patients (53.9% ≤ 0.064 mg/L) than for the persistently infected patients (100% ≥ 0.125 mg/L) (P = 0.006, Fisher's exact test). Overall, 96% of isolates gave reproducible MICs when re-tested. Conclusions: A reproducible assay was developed for antimicrobial susceptibility testing of C. trachomatis. MICs of azithromycin were generally comparable for the two different patient groups. MICs of doxycycline were significantly higher in the persistently infected patients. However, interpretation of elevated MICs in C. trachomatis is extremely challenging in the absence of breakpoints, or wild-type and treatment failure MIC distribution data.

An evaluation of Neisseria gonorrhoeae antimicrobial susceptibility testing in the UK.

The only method currently available to perform Neisseria gonorrhoeae antimicrobial susceptibility testing (Ng-AST) requires a viable organism obtained by culture. Reports of in vitro resistance to extended-spectrum cephalosporins, the treatment of choice for gonorrhoea, coupled with increasing gonorrhoea diagnoses is worrying. The aim of this study was to identify various methodologies employed by the UK microbiology laboratories to perform Ng-AST. Of the 118 laboratories that responded, 114 offered Ng-AST; the majority (82.5%, 94/114) of the laboratories used British Society for Antimicrobial Chemotherapy methodology for Ng-AST. The other main findings were infrequent use of quality control procedures and inconsistent susceptibility testing of the antibiotics used routinely for treatment.

Combination therapy for gonorrhoea: in vitro synergy testing.

OBJECTIVES: Antimicrobial resistance in Neisseria gonorrhoeae is an increasing problem worldwide and combinations of antimicrobial agents have been recommended to delay the onset of treatment failures. The objective of this study was to obtain in vitro data on the activity of current (ceftriaxone or cefixime plus azithromycin) and alternative (gentamicin plus azithromycin) regimens. METHODS: A panel of 64 gonococcal isolates displaying various cefixime MICs was selected for inclusion in the study. Determination of the activities of the antimicrobial combinations of ceftriaxone, cefixime or gentamicin with azithromycin was performed using the agar dilution method and subsequent calculation of the fractional inhibitory concentration index (FICI) values. RESULTS: No antagonism for any of the antimicrobial combinations was detected among the 64 gonococcal isolates. When cefixime or ceftriaxone was combined with azithromycin all isolates showed additivity/indifference with a mean FICI of 2.0. All gonococcal isolates also showed additivity/indifference with the antimicrobial combination of gentamicin with azithromycin, but with a lower mean FICI of 1.7. No significant difference in the mean FICI between isolates fully susceptible to cefixime and isolates with decreased susceptibility to cefixime was observed. CONCLUSIONS: The results obtained support the gonorrhoea treatment currently recommended in the UK national guidelines and suggest that gentamicin with azithromycin could be a future treatment option. The in vivo activity and efficacy of these combinations remain unknown and prospective clinical studies should be addressed.

European surveillance of antimicrobial resistance in Neisseria gonorrhoeae.

OBJECTIVE: To perform a European sentinel surveillance study for antimicrobial resistance (AMR) in Neisseria gonorrhoeae as part of the European Surveillance of Sexually Transmitted Infections Programme. METHODS: From 2006 to 2008 17 countries participated in the AMR surveillance programme. The susceptibility of a total of 3528 consecutive isolates was tested using the agar dilution breakpoint technique or Etests for ciprofloxacin, penicillin, tetracycline, azithromycin, spectinomycin and ceftriaxone. Nitrocefin was used to detect ß-lactamase activity. RESULTS: Rates of resistance to ciprofloxacin, the previously recommended treatment, were high across Europe (42-52%), indicating that usage is no longer appropriate. Although resistance to the currently recommended treatment, ceftriaxone, was not demonstrated, a concerning upward drift in the minimal inhibitory concentration (MIC) distribution was identified since an earlier European study in 2004. No resistance to spectinomycin was seen, whereas azithromycin resistance varied from 2% to 7% and isolates from Scotland (n=4) and Ireland (n=1) showed high-level resistance (MIC >256 mg/l). High-level resistance to tetracycline and penicillin remained relatively constant at 16% and 12%, respectively. CONCLUSIONS: AMR is an ongoing problem in Europe, with high rates of resistance to many previously recommended therapeutic agents observed in many European countries. Continual European and global surveillance of AMR in N gonorrhoeae is essential to monitor for increasing, emerging and high-level resistance to therapeutically relevant agents and to inform treatment guidelines so optimum treatments are administered.