| 1. |
- Sánchez-Busó, Leonor, et al.
(författare)
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pyngoST : fast, simultaneous and accurate multiple sequence typing of Neisseria gonorrhoeae genome collections
- 2024
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Ingår i: Microbial Genomics. - : Microbiology Society. - 2057-5858. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- Extensive gonococcal surveillance has been performed using molecular typing at global, regional, national and local levels. The three main genotyping schemes for this pathogen, multi-locus sequence typing (MLST), Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) and N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR), allow inter-laboratory and inter-study comparability and reproducibility and provide an approximation to the gonococcal population structure. With whole-genome sequencing (WGS), we obtain a substantially higher and more accurate discrimination between strains compared to previous molecular typing schemes. However, WGS remains unavailable or not affordable in many laboratories, and thus bioinformatic tools that allow the integration of data among laboratories with and without access to WGS are imperative for a joint effort to increase our understanding of global pathogen threats. Here, we present pyngoST, a command-line Python tool for fast, simultaneous and accurate sequence typing of N. gonorrhoeae from WGS assemblies. pyngoST integrates MLST, NG-MAST and NG-STAR, and can also designate NG-STAR clonal complexes, NG-MAST genogroups and penA mosaicism, facilitating multiple sequence typing from large WGS assembly collections. Exact and closest matches for existing alleles and sequence types are reported. The implementation of a fast multi-pattern searching algorithm allows pyngoST to be rapid and report results on 500 WGS assemblies in under 1 min. The mapping of typing results on a core genome tree of 2375 gonococcal genomes revealed that NG-STAR is the scheme that best represents the population structure of this pathogen, emphasizing the role of antimicrobial use and antimicrobial resistance as a driver of gonococcal evolution. This article contains data hosted by Microreact.
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| 2. |
- Golparian, Daniel, 1984-, et al.
(författare)
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Antimicrobial resistance prediction and phylogenetic analysis of Neisseria gonorrhoeae isolates using the Oxford Nanopore MinION sequencer
- 2018
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 8:1
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Tidskriftsartikel (refereegranskat)abstract
- Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is common, compromising gonorrhoea treatment internationally. Rapid characterisation of AMR strains could ensure appropriate and personalised treatment, and support identification and investigation of gonorrhoea outbreaks in nearly real-time. Whole-genome sequencing is ideal for investigation of emergence and dissemination of AMR determinants, predicting AMR, in the gonococcal population and spread of AMR strains in the human population. The novel, rapid and revolutionary long-read sequencer MinION is a small hand-held device that generates bacterial genomes within one day. However, accuracy of MinION reads has been suboptimal for many objectives and the MinION has not been evaluated for gonococci. In this first MinION study for gonococci, we show that MinION-derived sequences analysed with existing open-access, web-based sequence analysis tools are not sufficiently accurate to identify key gonococcal AMR determinants. Nevertheless, using an in house-developed CLC Genomics Workbench including de novo assembly and optimised BLAST algorithms, we show that 2D ONT-derived sequences can be used for accurate prediction of decreased susceptibility or resistance to recommended antimicrobials in gonococcal isolates. We also show that the 2D ONT-derived sequences are useful for rapid phylogenomic-based molecular epidemiological investigations, and, in hybrid assemblies with Illumina sequences, for producing contiguous assemblies and finished reference genomes.
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| 3. |
- Golparian, degn, 1984-, et al.
(författare)
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Antimicrobial-resistant Neisseria gonorrhoeae in Europe in 2020 compared with in 2013 and 2018 : a retrospective genomic surveillance study
- 2024
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Ingår i: The Lancet. Microbe. - : Elsevier. - 2666-5247. ; 5:5, s. e478-e488
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Regular quality-assured whole-genome sequencing linked to antimicrobial resistance (AMR) and patient metadata is imperative to elucidate the shifting gonorrhoea epidemiology, both nationally and internationally. We aimed to examine the gonococcal population in the European Economic Area (EEA) in 2020, elucidate emerging and disappearing gonococcal lineages associated with AMR and patient metadata, compare with 2013 and 2018 whole-genome sequencing data, and explain changes in gonococcal AMR and gonorrhoea epidemiology.METHODS: In this retrospective genomic surveillance study, we analysed consecutive gonococcal isolates that were collected in EEA countries through the European Gonococcal Antimicrobial Surveillance Programme (Euro-GASP) in 2020, and made comparisons with Euro-GASP data from 2013 and 2018. All isolates had linked AMR data (based on minimum inhibitory concentration determination) and patient metadata. We performed whole-genome sequencing and molecular typing and AMR determinants were derived from quality-checked whole-genome sequencing data. Links between genomic lineages, AMR, and patient metadata were examined.FINDINGS: 1932 gonococcal isolates collected in 2020 in 21 EEA countries were included. The majority (81·2%, 147 of 181 isolates) of azithromycin resistance (present in 9·4%, 181 of 1932) was explained by the continued expansion of the Neisseria gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) clonal complexes (CCs) 63, 168, and 213 (with mtrD/mtrR promoter mosaic 2) and the novel NG-STAR CC1031 (semi-mosaic mtrD variant 13), associated with men who have sex with men and anorectal or oropharyngeal infections. The declining cefixime resistance (0·5%, nine of 1932) and negligible ceftriaxone resistance (0·1%, one of 1932) was largely because of the progressive disappearance of NG-STAR CC90 (with mosaic penA allele), which was predominant in 2013. No known resistance determinants for novel antimicrobials (zoliflodacin, gepotidacin, and lefamulin) were found.INTERPRETATION: Azithromycin-resistant clones, mainly with mtrD mosaic or semi-mosaic variants, appear to be stabilising at a relatively high level in the EEA. This mostly low-level azithromycin resistance might threaten the recommended ceftriaxone-azithromycin therapy, but the negligible ceftriaxone resistance is encouraging. The decreased genomic population diversity and increased clonality could be explained in part by the COVID-19 pandemic resulting in lower importation of novel strains into Europe.
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| 4. |
- Golparian, Daniel, 1984-, et al.
(författare)
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Genomic evolution of Neisseria gonorrhoeae since the preantibiotic era (1928-2013) : antimicrobial use/misuse selects for resistance and drives evolution
- 2020
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Ingår i: BMC Genomics. - : BioMed Central. - 1471-2164. ; 21:1
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Multidrug-resistant Neisseria gonorrhoeae strains are prevalent, threatening gonorrhoea treatment globally, and understanding of emergence, evolution, and spread of antimicrobial resistance (AMR) in gonococci remains limited. We describe the genomic evolution of gonococci and their AMR, related to the introduction of antimicrobial therapies, examining isolates from 1928 (preantibiotic era) to 2013 in Denmark. This is, to our knowledge, the oldest gonococcal collection globally.METHODS: Lyophilised isolates were revived and examined using Etest (18 antimicrobials) and whole-genome sequencing (WGS). Quality-assured genome sequences were obtained for 191 viable and 40 non-viable isolates and analysed with multiple phylogenomic approaches.RESULTS: Gonococcal AMR, including an accumulation of multiple AMR determinants, started to emerge particularly in the 1950s-1970s. By the twenty-first century, resistance to most antimicrobials was common. Despite that some AMR determinants affect many physiological functions and fitness, AMR determinants were mainly selected by the use/misuse of gonorrhoea therapeutic antimicrobials. Most AMR developed in strains belonging to one multidrug-resistant (MDR) clade with close to three times higher genomic mutation rate. Modern N. gonorrhoeae was inferred to have emerged in the late-1500s and its genome became increasingly conserved over time.CONCLUSIONS: WGS of gonococci from 1928 to 2013 showed that no AMR determinants, except penB, were in detectable frequency before the introduction of gonorrhoea therapeutic antimicrobials. The modern gonococcus is substantially younger than previously hypothesized and has been evolving into a more clonal species, driven by the use/misuse of antimicrobials. The MDR gonococcal clade should be further investigated for early detection of strains with predispositions to develop and maintain MDR and for initiation of public health interventions.
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| 5. |
- Golparian, Daniel, 1984-, et al.
(författare)
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GyrB in silico mining in 27 151 global gonococcal genomes from 1928-2021 combined with zoliflodacin in vitro testing of 71 international gonococcal isolates with different GyrB, ParC and ParE substitutions confirms high susceptibility
- 2022
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Ingår i: Journal of Antimicrobial Chemotherapy. - : Oxford University Press. - 0305-7453 .- 1460-2091. ; 78:1, s. 150-154
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVES: Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a global threat and novel treatment alternatives are imperative. Herein, susceptibility to the novel antimicrobial zoliflodacin, currently in a global Phase 3 randomized controlled clinical trial for gonorrhoea treatment, was investigated by screening for zoliflodacin GyrB target mutations in publicly available gonococcal genomes and, where feasible, determination of the associated zoliflodacin MIC.METHODS: The European Nucleotide Archive was queried using the search term 'Taxon: 485'. DNA sequences from 27 151 gonococcal isolates were analysed and gyrB, gyrA, parC and parE alleles characterized.RESULTS: GyrB amino acid alterations were rare (97.0% of isolates had a wild-type GyrB sequence). GyrB V470L (2.7% of isolates) was the most prevalent alteration, followed by S467N (0.12%), N. meningitidis GyrB (0.092%), V470I (0.059%), Q468R/P (0.015%), A466T (0.0074%), L425I + L465I (0.0037%), L465I (0.0037%), G482S (0.0037%) and D429V (0.0037%). Only one isolate (0.0037%) carried a substitution in a resistance-associated GyrB codon (D429V), resulting in a zoliflodacin MIC of 8 mg/L. None of the other detected gyrB, gyrA, parC or parE mutations caused a zoliflodacin MIC outside the wild-type MIC distribution.CONCLUSIONS: The zoliflodacin target GyrB was highly conserved among 27 151 global gonococcal isolates cultured in 1928-2021. The single zoliflodacin-resistant clinical isolate (0.0037%) was cultured from a male patient in Japan in 2000. Evidently, this strain has not clonally expanded nor has the gyrB zoliflodacin-resistance mutation disseminated through horizontal gene transfer to other strains. Phenotypic and genomic surveillance, including gyrB mutations, of zoliflodacin susceptibility are imperative.
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| 6. |
- Golparian, Daniel, 1984-, et al.
(författare)
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Neisseria gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR) clonal complexes are consistent with genomic phylogeny and provide simple nomenclature, rapid visualization and antimicrobial resistance (AMR) lineage predictions
- 2021
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Ingår i: Journal of Antimicrobial Chemotherapy. - : Oxford University Press. - 0305-7453 .- 1460-2091. ; 76:4, s. 940-944
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVES: Surveillance of antimicrobial resistance (AMR) in Neisseria gonorrhoeae, supported by molecular typing, ideally through genome sequencing, is imperative. We defined N. gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR) clonal complexes (CCs) and validated their usefulness in gonococcal AMR surveillance.METHODS: All NG-STAR alleles and STs available in the public database (https://ngstar.canada.ca/) were analysed using PHYLOViZ 2.0 to define CCs according to the closest founder ST with ≥5 identical alleles and founding ST with the highest number of links. The published 2013 European gonococcal dataset (n = 1054), the 2016 WHO reference strain panel (n = 14) and N. gonorrhoeae isolates with ceftriaxone resistance determinant penA-60.001 (n = 7) from several countries were used for validation.RESULTS: The majority of the isolates (n = 1063) were designated to 71 CCs. The most common CC was CC90 (n = 194), followed by CC63 (n = 166), CC139 (n = 73), CC158 (n = 73) and CC127 (n = 62). CC90 included isolates belonging to the internationally spread MDR clone N. gonorrhoeae Multi-Antigen Sequence Typing (NG-MAST) G1407 (predominantly MLST ST1901). The ceftriaxone-resistant isolates with penA-60.001 (n = 7) belonged to CC73 or STs linking between CC90 and CC73 (ST233 and ST1133). Phylogenomic analysis revealed that NG-STAR CCs more appropriately correlated to phylogenomic AMR clusters compared with MLST STs, NG-MAST STs, NG-MAST genogroups and NG-STAR STs.CONCLUSIONS: NG-STAR CCs: are consistent with the gonococcal genome phylogeny; allow rapid visualizations with limited computational requirements; provide a simple, reproducible and portable nomenclature (for WGS and conventional Sanger sequencing data); and predict AMR lineages. Phenotypic AMR surveillance, supplemented with WGS, is imperative and NG-STAR CCs can effectively support this.
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| 7. |
- Harris, Simon R., et al.
(författare)
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Public health surveillance of multidrug-resistant clones of Neisseria gonorrhoeae in Europe : a genomic survey
- 2018
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Ingår i: The Lancet - Infectious diseases. - : Elsevier. - 1473-3099 .- 1474-4457. ; 18:7, s. 758-768
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 8. |
- Ma, Kevin C., et al.
(författare)
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Adaptation to the cervical environment is associated with increased antibiotic susceptibility in Neisseria gonorrhoeae
- 2020
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Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Neisseria gonorrhoeae is an urgent public health threat due to rapidly increasing incidence and antibiotic resistance. In contrast with the trend of increasing resistance, clinical isolates that have reverted to susceptibility regularly appear, prompting questions about which pressures compete with antibiotics to shape gonococcal evolution. Here, we used genome-wide association to identify loss-of-function (LOF) mutations in the efflux pump mtrCDE operon as a mechanism of increased antibiotic susceptibility and demonstrate that these mutations are overrepresented in cervical relative to urethral isolates. This enrichment holds true for LOF mutations in another efflux pump, farAB, and in urogenitally-adapted versus typical N. meningitidis, providing evidence for a model in which expression of these pumps in the female urogenital tract incurs a fitness cost for pathogenic Neisseria. Overall, our findings highlight the impact of integrating microbial population genomics with host metadata and demonstrate how host environmental pressures can lead to increased antibiotic susceptibility.
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| 9. |
- Sánchez-Busó, Leonor, et al.
(författare)
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A community-driven resource for genomic epidemiology and antimicrobial resistance prediction of Neisseria gonorrhoeae at Pathogenwatch
- 2021
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Ingår i: Genome Medicine. - : BioMed Central. - 1756-994X. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Antimicrobial-resistant (AMR) Neisseria gonorrhoeae is an urgent threat to public health, as strains resistant to at least one of the two last-line antibiotics used in empiric therapy of gonorrhoea, ceftriaxone and azithromycin, have spread internationally. Whole genome sequencing (WGS) data can be used to identify new AMR clones and transmission networks and inform the development of point-of-care tests for antimicrobial susceptibility, novel antimicrobials and vaccines. Community-driven tools that provide an easy access to and analysis of genomic and epidemiological data is the way forward for public health surveillance.METHODS: Here we present a public health-focussed scheme for genomic epidemiology of N. gonorrhoeae at Pathogenwatch ( https://pathogen.watch/ngonorrhoeae ). An international advisory group of experts in epidemiology, public health, genetics and genomics of N. gonorrhoeae was convened to inform on the utility of current and future analytics in the platform. We implement backwards compatibility with MLST, NG-MAST and NG-STAR typing schemes as well as an exhaustive library of genetic AMR determinants linked to a genotypic prediction of resistance to eight antibiotics. A collection of over 12,000 N. gonorrhoeae genome sequences from public archives has been quality-checked, assembled and made public together with available metadata for contextualization.RESULTS: AMR prediction from genome data revealed specificity values over 99% for azithromycin, ciprofloxacin and ceftriaxone and sensitivity values around 99% for benzylpenicillin and tetracycline. A case study using the Pathogenwatch collection of N. gonorrhoeae public genomes showed the global expansion of an azithromycin-resistant lineage carrying a mosaic mtr over at least the last 10 years, emphasising the power of Pathogenwatch to explore and evaluate genomic epidemiology questions of public health concern.CONCLUSIONS: The N. gonorrhoeae scheme in Pathogenwatch provides customised bioinformatic pipelines guided by expert opinion that can be adapted to public health agencies and departments with little expertise in bioinformatics and lower-resourced settings with internet connection but limited computational infrastructure. The advisory group will assess and identify ongoing public health needs in the field of gonorrhoea, particularly regarding gonococcal AMR, in order to further enhance utility with modified or new analytic methods.
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| 10. |
- Sánchez-Busó, Leonor, et al.
(författare)
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Europe-wide expansion and eradication of multidrug-resistant Neisseria gonorrhoeae lineages : a genomic surveillance study
- 2022
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Ingår i: The Lancet. Microbe. - : Elsevier. - 2666-5247. ; 3:6, s. e452-e463
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Genomic surveillance using quality-assured whole-genome sequencing (WGS) together with epidemiological and antimicrobial resistance (AMR) data is essential to characterise the circulating Neisseria gonorrhoeae lineages and their association to patient groups (defined by demographic and epidemiological factors). In 2013, the European gonococcal population was characterised genomically for the first time. We describe the European gonococcal population in 2018 and identify emerging or vanishing lineages associated with AMR and epidemiological characteristics of patients, to elucidate recent changes in AMR and gonorrhoea epidemiology in Europe.METHODS: We did WGS on 2375 gonococcal isolates from 2018 (mainly Sept 1-Nov 30) in 26 EU and EEA countries. Molecular typing and AMR determinants were extracted from quality-checked genomic data. Association analyses identified links between genomic lineages, AMR, and epidemiological data.FINDINGS: Azithromycin-resistant N gonorrhoeae (8·0% [191/2375] in 2018) is rising in Europe due to the introduction or emergence and subsequent expansion of a novel N gonorrhoeae multi-antigen sequence typing (NG-MAST) genogroup, G12302 (132 [5·6%] of 2375; N gonorrhoeae sequence typing for antimicrobial resistance [NG-STAR] clonal complex [CC]168/63), carrying a mosaic mtrR promoter and mtrD sequence and found in 24 countries in 2018. CC63 was associated with pharyngeal infections in men who have sex with men. Susceptibility to ceftriaxone and cefixime is increasing, as the resistance-associated lineage, NG-MAST G1407 (51 [2·1%] of 2375), is progressively vanishing since 2009-10.INTERPRETATION: Enhanced gonococcal AMR surveillance is imperative worldwide. WGS, linked to epidemiological and AMR data, is essential to elucidate the dynamics in gonorrhoea epidemiology and gonococcal populations as well as to predict AMR. When feasible, WGS should supplement the national and international AMR surveillance programmes to elucidate AMR changes over time. In the EU and EEA, increasing low-level azithromycin resistance could threaten the recommended ceftriaxone-azithromycin dual therapy, and an evidence-based clinical azithromycin resistance breakpoint is needed. Nevertheless, increasing ceftriaxone susceptibility, declining cefixime resistance, and absence of known resistance mutations for new treatments (zoliflodacin, gepotidacin) are promising.
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