SummaryBackgroundTraditional 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.MethodsWe 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.FindingsThe 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.InterpretationTo 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.FundingThe European Centre for Disease Prevention and Control, The Centre for Genomic Pathogen Surveillance, Örebro University Hospital, and Wellcome.

Gonococci with ceftriaxone and cefixime MICs of 0.125-0.25 mg/L are accumulating in the UK. These MICs lie on the edge of likely responsiveness to current regimens, which need review. Possible responses include: (i) higher cephalosporin doses; (ii) multidose cephalosporin regimens; (iii) multidrug regimens; (iv) microbiologically directed treatment; or, in the future, (v) drug cycling. The practicalities of these approaches are discussed.

High-level azithromycin resistance (AZM-HR), defined as a MIC of >256 mg/liter, emerged in Neisseria gonorrhoeae in the United Kingdom in 2004. To determine the mechanism of this novel phenotype, isolates from the United Kingdom that were AZM-HR (n, 19), moderately AZM resistant (MICs, 2 to 8 mg/liter) (n, 26), or sensitive (MICs, 0.12 to 0.25 mg/liter) (n, 4) were screened for methylase (erm) genes and for mutations in the mtrR promoter region, associated with efflux pump upregulation. All AZM-resistant isolates and 12 sensitive isolates were screened for mutations in domain V of each 23S rRNA allele. All AZM-HR isolates contained the A2059G mutation (Escherichia coli numbering) in three (3 isolates) or four (16 isolates) 23S rRNA alleles. Most (22/26) moderately AZM resistant isolates contained the C2611T mutation in at least 3/4 alleles. The remainder contained four wild-type alleles, as did 8/12 sensitive isolates, while one allele was mutated in the remaining four sensitive isolates. Serial passage of AZM-sensitive colonies on an erythromycin-containing medium selected AZM-HR if the parent strain already contained mutation A2059G in one 23S rRNA allele. The resultant AZM-HR strains contained four mutated alleles. Eight isolates (five moderately AZM resistant and three AZM-HR) contained mutations in the mtrR promoter. No methylase genes were detected. This is the first evidence that AZM-HR in gonococci may result from a single point mutation (A2059G) in the peptidyltransferase loop in domain V of the 23S rRNA gene. Mutation of a single allele is insufficient to confer AZM-HR, but AZM-HR can develop under selection pressure. The description of a novel resistance mechanism will aid in screening for the AZM-HR phenotype.

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A novel PCR detection assay that amplifies the Helicobacter pylori-specific vacuolating cytotoxin gene (vacA) and thus enables rapid diagnosis of infection is described. Additionally, a real-time probe hybridization melting point analysis assay to detect all three mutations in the 23S rRNA gene associated with clarithromycin resistance was applied directly to antral gastric biopsy samples. Comparison with culture and an alternative PCR assay targeting the 16S rrn gene showed that the vacA assay was sensitive and specific when tested on biopsy samples from 121 patients. Clarithromycin susceptibilities could be determined in the majority (92.3%) of culture-positive gastric biopsy samples analyzed, four of which generated melting peaks indicative of clarithromycin resistance by either an A3G or A3C mutation. The presence of the mutations correlated with the clarithromycin disk diffusion sensitivities of matched cultures. This PCR-based system was simple to perform and could be completed in 3 to 4 h, thereby overcoming the delays associated with conventional culture methods for H. pylori identification and susceptibility testing.Helicobacter pylori, a major cause of chronic gastritis, is strongly associated with the development of gastric and duodenal ulcers and has been linked with gastric adenocarcinoma and B-cell mucosa-associated lymphoid tissue lymphoma (15,17,18). Infection can be eradicated in up to 90% of patients using current combination triple therapies, of which the macrolide antibiotic clarithromycin is a key component (5). Rates of resistance to clarithromycin of 1 to 9% have been reported in several European countries and the United States, with even higher rates in some countries, such as France and Belgium (24). The development of clarithromycin resistance in H. pylori is recognized as a significant contributing factor in treatment failure (8,14), and the mechanism is attributed to single point mutations in the peptidyltransferase region of the 23S rRNA gene (25). Adenine residues at either position 2143 or 2144 can mutate. Transition to guanine (A2143G and A2144G) is the most common mutation type, with the transversion mutation to cytosine (A2143C) less common (16,21,22,25). Although culture of gastric biopsy samples allows further H. pylori strain analysis, including determination of antibiotic susceptibility, tests can take up to 2 weeks to complete. The aim of the present study was to develop a PCR-based system using conventional and real-time techniques enabling same-day diagnosis of H. pylori infection and determination of clarithromycin resistance. MATERIALS AND METHODSGastric biopsy samples and strain isolation. Two sets of gastric biopsy samples were used in this study. First, we examined a series of preserved (Ϫ20°C) gastric biopsy samples from 39 dyspeptic patients attending an open-access endoscopy clinic in Chelmsford during 1995 and 1996. These biopsy samples were confirmed positive for H. pylori by both culture and histology, and the patients were also confirmed seropositive for H. py...

Epidemiological and microbiological investigations confirm that an outbreak of a gonococcal strain showing HL-AziR is ongoing in the North of England. Every effort should be made to identify and curtail dissemination of this strain as it presents a significant threat to the current recommended front-line dual therapy.

This is the first reported evidence that the European gonococcal population susceptibility to gentamicin is similar to that reported in other world regions. Clinical trials to evaluate the therapeutic efficacy of gentamicin may be warranted.

High-level azithromycin resistance has emerged in England and Wales. Contact tracing and typing data suggest this may have originated from Scotland. Surveillance of azithromycin resistance will be key in controlling its further dissemination.