Antimicrobial-resistant Neisseria gonorrhoeae in Europe in 2020 compared with in 2013 and 2018: a retrospective genomic surveillance study.

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. FUNDING: European Centre for Disease Prevention and Control and Örebro University Hospital.

A laboratory-based predictive pathway for the development of Neisseria gonorrhoeae high-level resistance to corallopyronin A, an inhibitor of bacterial RNA polymerase.

The continued emergence of Neisseria gonorrhoeae strains that express resistance to multiple antibiotics, including the last drug for empiric monotherapy (ceftriaxone), necessitates the development of new treatment options to cure gonorrheal infections. Toward this goal, we recently reported that corallopyronin A (CorA), which targets the switch region of the ß' subunit (RpoC) of bacterial DNA-dependent RNA polymerase (RNAP), has potent anti-gonococcal activity against a panel of multidrug-resistant clinical strains. Moreover, in that study, CorA could eliminate gonococcal infection of primary human epithelial cells and gonococci in a biofilm state. To determine if N. gonorrhoeae could develop high-level resistance to CorA in a single step, we sought to isolate spontaneous mutants expressing any CorA resistance phenotypes. However, no single-step mutants with high-level CorA resistance were isolated. High-level CorA resistance could only be achieved in this study through a multi-step pathway involving over-expression of the MtrCDE drug efflux pump and single amino acid changes in the ß and ß' subunits (RpoB and RpoC, respectively) of RNAP. Molecular modeling of RpoB and RpoC interacting with CorA was used to deduce how the amino acid changes in RpoB and RpoC could influence gonococcal resistance to CorA. Bioinformatic analyses of whole genome sequences of clinical gonococcal isolates indicated that the CorA resistance determining mutations in RpoB/C, identified herein, are very rare (≤ 0.0029%), suggesting that the proposed pathway for resistance is predictive of how this phenotype could potentially evolve if CorA is used therapeutically to treat gonorrhea in the future. IMPORTANCE: The continued emergence of multi-antibiotic-resistant strains of Neisseria gonorrhoeae necessitates the development of new antibiotics that are effective against this human pathogen. We previously described that the RNA polymerase-targeting antibiotic corallopyronin A (CorA) has potent activity against a large collection of clinical strains that express different antibiotic resistance phenotypes including when such gonococci are in a biofilm state. Herein, we tested whether a CorA-sensitive gonococcal strain could develop spontaneous resistance. Our finding that CorA resistance could only be achieved by a multi-step process involving over-expression of the MtrCDE efflux pump and single amino acid changes in RpoB and RpoC suggests that such resistance may be difficult for gonococci to evolve if this antibiotic is used in the future to treat gonorrheal infections that are refractory to cure by other antibiotics.

Recent dynamics in Neisseria gonorrhoeae genomic epidemiology in Brazil: antimicrobial resistance and genomic lineages in 2017-20 compared to 2015-16.

OBJECTIVES: Regular quality-assured WGS with antimicrobial resistance (AMR) and epidemiological data of patients is imperative to elucidate the shifting gonorrhoea epidemiology, nationally and internationally. We describe the dynamics of the gonococcal population in 11 cities in Brazil between 2017 and 2020 and elucidate emerging and disappearing gonococcal lineages associated with AMR, compare to Brazilian WGS and AMR data from 2015 to 2016, and explain recent changes in gonococcal AMR and gonorrhoea epidemiology. METHODS: WGS was performed using Illumina NextSeq 550 and genomes of 623 gonococcal isolates were used for downstream analysis. Molecular typing and AMR determinants were obtained and links between genomic lineages and AMR (determined by agar dilution/Etest) examined. RESULTS: Azithromycin resistance (15.6%, 97/623) had substantially increased and was mainly explained by clonal expansions of strains with 23S rRNA C2611T (mostly NG-STAR CC124) and mtr mosaics (mostly NG-STAR CC63, MLST ST9363). Resistance to ceftriaxone and cefixime remained at the same levels as in 2015-16, i.e. at 0% and 0.2% (1/623), respectively. Regarding novel gonorrhoea treatments, no known zoliflodacin-resistance gyrB mutations or gepotidacin-resistance gyrA mutations were found. Genomic lineages and sublineages showed a phylogenomic shift from sublineage A5 to sublineages A1-A4, while isolates within lineage B remained diverse in Brazil. CONCLUSIONS: Azithromycin resistance, mainly caused by 23S rRNA C2611T and mtrD mosaics/semi-mosaics, had substantially increased in Brazil. This mostly low-level azithromycin resistance may threaten the recommended ceftriaxone-azithromycin therapy, but the lack of ceftriaxone resistance is encouraging. Enhanced gonococcal AMR surveillance, including WGS, is imperative in Brazil and other Latin American and Caribbean countries.

Pharmacodynamic evaluation of ceftriaxone single-dose therapy (0.125-1†g) to eradicate ceftriaxone-susceptible and ceftriaxone-resistant Neisseria gonorrhoeae strains in a hollow fibre infection model for gonorrhoea.

BACKGROUND: Antimicrobial resistance in Neisseria gonorrhoeae is threatening the gonorrhoea treatment, and optimizations of the current ceftriaxone-treatment regimens are crucial. We evaluated the pharmacodynamics of ceftriaxone single-dose therapy (0.125-1 g) against ceftriaxone-susceptible and ceftriaxone-resistant gonococcal strains, based on EUCAST ceftriaxone-resistance breakpoint (MIC > 0.125 mg/L), in our hollow fibre infection model (HFIM) for gonorrhoea. METHODS: Gonococcal strains examined were WHO F (ceftriaxone-susceptible, MIC < 0.002 mg/L), R (ceftriaxone-resistant, MIC = 0.5 mg/L), Z (ceftriaxone-resistant, MIC = 0.5 mg/L) and X (ceftriaxone-resistant, MIC = 2 mg/L). Dose-range HFIM 7 day experiments simulating ceftriaxone 0.125-1 g single-dose intramuscular regimens were conducted. RESULTS: Ceftriaxone 0.125-1 g single-dose treatments rapidly eradicated WHO F (wild-type ceftriaxone MIC). Ceftriaxone 0.5 and 1 g treatments, based on ceftriaxone human plasma pharmacokinetic parameters, eradicated most ceftriaxone-resistant gonococcal strains (WHO R and Z), but ceftriaxone 0.5 g failed to eradicate WHO X (high-level ceftriaxone resistance). When simulating oropharyngeal gonorrhoea, ceftriaxone 0.5 g failed to eradicate all the ceftriaxone-resistant strains, while ceftriaxone 1 g eradicated WHO R and Z (low-level ceftriaxone resistance) but failed to eradicate WHO X (high-level ceftriaxone resistance). No ceftriaxone-resistant mutants were selected using any ceftriaxone treatments. CONCLUSIONS: Ceftriaxone 1 g single-dose intramuscularly cure most of the anogenital and oropharyngeal gonorrhoea cases caused by the currently internationally spreading ceftriaxone-resistant gonococcal strains, which should be further confirmed clinically. A ceftriaxone 1 g dose (±azithromycin 2 g) should be recommended for first-line empiric gonorrhoea treatment. This will buy countries some time until novel antimicrobials are licensed. Using ceftriaxone 1 g gonorrhoea treatment, the EUCAST ceftriaxone-resistance breakpoint is too low.

Independent clinic-based evaluation of point-of-care testing for the screening of Chlamydia trachomatis, Neisseria gonorrhoea and Trichomonas vaginalis in women-at-risk in Australia, Guatemala, Morocco, and South Africa.

BACKGROUND: In 2018, the World Health Organization commenced a multi-country validation study of the Cepheid GeneXpert for a range of molecular-based point-of-care (POC) tests in primary care settings. One study arm focused on the evaluation of POC tests for screening 'women at risk' for chlamydia (CT), gonorrhoea (NG) and trichomonas (TV) in four countries - Australia, Guatemala, Morocco and South Africa. METHODS: Study participants completed a pre-test questionnaire which included demographics, clinical information and general questions on POC testing (POCT). Two vaginal swab samples (either self-collected or clinician collected) from each patient were tested on the GeneXpert at the POC and at a reference laboratory using quality-assured nucleic acid amplification tests (NAATs). RESULTS: One thousand three hundred and eighty-three women were enrolled: 58.6% from South Africa, 29.2% from Morocco, 6.2% from Guatemala, and 6.0% from Australia. 1296 samples for CT/NG and 1380 samples for TV were tested by the GeneXpert and the reference NAAT. The rate of unsuccessful tests on the GeneXpert was 1.9% for CT, 1.5% for NG and 0.96% for TV. The prevalence of CT, NG and TV was 31%, 13% and 23%, respectively. 1.5% of samples were positive for all three infections; 7.8% were positive for CT and NG; 2.4% were positive for NG and TV; and 7.3% were positive for CT and TV. Compared to reference NAATs, pooled estimates of sensitivity for the GeneXpert tests were 83.7% (95% confidence intervals 69.2-92.1) for CT, 90.5% (85.1-94.1) for NG and 64.7% (58.1-70.7) for TV (although estimates varied considerably between countries). Estimates for specificity were ≥96% for all three tests both within- and between-countries. Pooled positive and negative likelihood ratios were: 32.7 ([CI] 21.2-50.5) and 0.17 (0.08-0.33) for CT; 95.3 (36.9-245.7) and 0.10 (0.06-0.15) for NG; and 56.5 (31.6-101.1) and 0.35 (0.27-0.47) for TV. CONCLUSION: This multi-country evaluation is the first of its kind world-wide. Positive likelihood ratios, as well as specificity estimates, indicate the GeneXpert POC test results for CT, NG and TV were clinically acceptable for ruling in the presence of disease. However, negative likelihood ratios and variable sensitivity estimates from this study were poorer than expected for ruling out these infections, particularly for TV. TRIAL REGISTRATION: Ethics approval to conduct the ProSPeRo study was granted by the WHO Ethics Review Committee, as well as local ethics committees from all participating countries.

Complete reference genomes of two ceftriaxone-resistant Neisseria gonorrhoeae strains identified in routine surveillance in Bangkok, Thailand, using Nanopore Q20+ chemistry, VolTRAX V2b, and Illumina sequencing.

Ceftriaxone-resistant Neisseria gonorrhoeae strains, mostly associated with Asia, threaten gonorrhea treatment. We report the reference genomes of two ceftriaxone-resistant isolates found in routine surveillance in Bangkok, Thailand. The genomes belonged to the more antimicrobial-susceptible genomic lineage B, illustrating that both ceftriaxone-resistant strains and the mosaic penA-60.001 ceftriaxone-resistance determinant are spreading.

Clinic-based evaluation of the dual Xpert CT/NG assay on the GeneXpert System for screening for extragenital chlamydial and gonococcal infections amongst men who have sex with men.

BACKGROUND: Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) infections have increased globally. Asymptomatic infections represent a significant risk of long-term complications. Men who have sex with men (MSM) are disproportionally affected, underscoring the need to offer screening programmes to this population. CT/NG Point of Care Testing (POCT) constitutes a strategic tool to improve the continuum of STI care, however extensive real-life evaluations amongst at risk populations are lacking. The aim of this study is to estimate the GeneXpert CT/NG assay performance and usability for CT and NG at genital and extragenital sites for screening amongst MSM. METHODS: This study was a multi-site sexual health clinic-based evaluation (Italy, Malta and Peru) with consecutive enrolment. A first void urine sample (divided in two aliquots), two oropharyngeal and two anorectal swabs were collected for each study participant. One specimen set (one for each anatomical site) was tested with the dual index test (Cepheid) at the clinics by the healthcare staff, the other set with FDA/CE approved Nucleic Acid Amplification Tests (NAATs) at the laboratory. Clinical sites and reference laboratories participated in an internal and external quality control programme. Sensitivity, specificity, positive and negative likelihood ratios, positive and negative predictive values for each anatomical site were estimated using a meta-analytic approach. RESULTS: One thousand seven hundred two MSM were recruited across all clinical sites for a total of 5049 biological specimens. NG and CT were respectively detected in 274 and 287 of samples. Overall, the NG POCT sensitivity and specificity was 91.43% and 99.75% in urine (LR + 372.80, LR- 0.09), 89.68% and 99.55% in rectal specimens (LR + 197.30, LR- 0.10) and 75.87% and 98.77% at the pharynx respectively (LR + 61.94, LR- 0.24). The CT component of the POCT sensitivity was 84.82% and specificity 99.63% in urine (LR + 228.68, LR- 0.15), 78.07% and 99.19% respectively on rectal site (LR + 96.23, LR-0.22), 67.79% and 99.88% respectively at pharyngeal site (LR + 554.89, LR- 0.32). 95.95% of MSM reported to be willing to wait for POCT results and no provider reported difficulties in terms of performance or interpretation of the results of the Xpert CT/NG. CONCLUSION: Rapid turnaround time, ease of use and high acceptability make the Xpert CT/NG testing system a strategic tool for increasing testing frequency, reaching those not yet tested and offering the possibility of immediate treatment if needed. The assay showed good negative likelihood ratios and confirms its use to rule out CT/NG infections. Sensitivity varied across sites and pathogens. Periodic staff training at the testing sites should be mandatory.

Antimicrobial susceptibility in Neisseria gonorrhoeae and epidemiological data of gonorrhoea patients in five cities across Ethiopia, 2021-22.

Introduction: Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a global public health concern and enhanced global gonococcal AMR surveillance is imperative. As in many African countries, regular, representative and quality-assured gonococcal AMR is lacking in Ethiopia. We describe the AMR in gonococcal isolates from five cities across Ethiopia, 2021-22, and patient epidemiological data. Methods: Urethral discharge from males and cervical discharge from females were collected from October 2021 to September 2022. Epidemiological data were collected using a questionnaire. MIC determination (ETEST; eight antimicrobials) was performed on gonococcal isolates and EUCAST breakpoints (v13.1) were used. Results: From 1142 urogenital swab samples, 299 species-identified gonococcal isolates were identified; 78.3% were from males and 21.7% from females. The median age for males and females was 25 and 23 years, respectively. Most isolates (61.2%) were identified in Addis Ababa, followed by Gondar (11.4%), Adama (10.4%), Bahir Dar (10.0%) and Jimma (7.0%). The resistance level to ciprofloxacin, tetracycline and benzylpenicillin was 97.0%, 97.0% and 87.6%, respectively, and 87.6% of isolates were producing ß-lactamase. All isolates were susceptible to ceftriaxone, cefixime, azithromycin and spectinomycin. Recommended therapy [ceftriaxone (250 mg) plus azithromycin (1 g)] was used for 84.2% of patients. Conclusions: We present the first national quality-assured gonococcal AMR data from Ethiopia. Resistance levels to ciprofloxacin, tetracycline and benzylpenicillin were exceedingly high. However, all isolates were susceptible to ceftriaxone, cefixime, azithromycin and spectinomycin. In Ethiopia, it is essential to strengthen the gonococcal AMR surveillance by including further epidemiological data, more isolates from different cities, and WGS.

pyngoST: fast, simultaneous and accurate multiple sequence typing of Neisseria gonorrhoeae genome collections.

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.

Pharmacodynamics of zoliflodacin plus doxycycline combination therapy against Neisseria gonorrhoeae in a gonococcal hollow-fiber infection model.

Antimicrobial resistance in the sexually transmitted bacterium Neisseria gonorrhoeae is compromising the management and control of gonorrhea globally. Optimized use and enhanced stewardship of current antimicrobials and development of novel antimicrobials are imperative. The first in class zoliflodacin (spiropyrimidinetrione, DNA Gyrase B inhibitor) is a promising novel antimicrobial in late-stage clinical development for gonorrhea treatment, i.e., the phase III randomized controlled clinical trial (ClinicalTrials.gov Identifier: NCT03959527) was recently finalized, and zoliflodacin showed non-inferiority compared to the recommended ceftriaxone plus azithromycin dual therapy. Doxycycline, the first-line treatment for chlamydia and empiric treatment for non-gonococcal urethritis, will be frequently given together with zoliflodacin because gonorrhea and chlamydia coinfections are common. In a previous static in vitro study, it was indicated that doxycycline/tetracycline inhibited the gonococcal killing of zoliflodacin in 6-h time-kill curve analysis. In this study, our dynamic in vitro hollow-fiber infection model (HFIM) was used to investigate combination therapies with zoliflodacin and doxycycline. Dose-range experiments using the three gonococcal strains WHO F (susceptible to relevant therapeutic antimicrobials), WHO X (extensively drug-resistant, including ceftriaxone-resistant; zoliflodacin-susceptible), and SE600/18 (zoliflodacin-susceptible strain with GyrB S467N substitution) were conducted simulating combination therapy with a single oral dose of zoliflodacin 0.5-4 g combined with a doxycycline daily oral dose of 200 mg administered as 100 mg twice a day, for 7 days (standard dose for chlamydia treatment). Comparing combination therapy of zoliflodacin (0.5-4 g single dose) plus doxycycline (200 mg divided into 100 mg twice a day orally, for 7 days) to zoliflodacin monotherapy (0.5-4 g single dose) showed that combination therapy was slightly more effective than monotherapy in the killing of N. gonorrhoeae and suppressing emergence of zoliflodacin resistance. Accordingly, WHO F was eradicated by only 0.5 g single dose of zoliflodacin in combination with doxycycline, and WHO X and SE600/18 were both eradicated by a 2 g single dose of zoliflodacin in combination with doxycycline; no zoliflodacin-resistant populations occurred during the 7-day experiment when using this zoliflodacin dose. When using suboptimal (0.5-1 g) zoliflodacin doses together with doxycycline, gonococcal mutants with increased zoliflodacin MICs, due to GyrB D429N and the novel GyrB T472P, emerged, but both the mutants had an impaired biofitness. The present study shows the high efficacy of zoliflodacin plus doxycycline combination therapy using a dynamic HFIM that more accurately and comprehensively simulate gonococcal infection and their treatment, i.e., compared to static in vitro models, such as short-time checkerboard experiments or time-kill curve analysis. Based on our dynamic in vitro HFIM work, zoliflodacin plus doxycycline for the treatment of both gonorrhea and chlamydia can be an effective combination.

Genomic surveillance and antimicrobial resistance determinants in Neisseria gonorrhoeae isolates from Uganda, Malawi and South Africa, 2015-20.

OBJECTIVES: Global antimicrobial resistance (AMR) surveillance in Neisseria gonorrhoeae is essential. In 2017-18, only five (10.6%) countries in the WHO African Region reported to the WHO Global Gonococcal Antimicrobial Surveillance Programme (WHO GASP). Genomics enhances our understanding of gonococcal populations nationally and internationally, including AMR strain transmission; however, genomic studies from Africa are extremely scarce. We describe the gonococcal genomic lineages/sublineages, including AMR determinants, and baseline genomic diversity among strains in Uganda, Malawi and South Africa, 2015-20, and compare with sequences from Kenya and Burkina Faso. METHODS: Gonococcal isolates cultured in Uganda (n = 433), Malawi (n = 154) and South Africa (n = 99) in 2015-20 were genome-sequenced. MICs were determined using ETEST. Sequences of isolates from Kenya (n = 159), Burkina Faso (n = 52) and the 2016 WHO reference strains (n = 14) were included in the analysis. RESULTS: Resistance to ciprofloxacin was high in all countries (57.1%-100%). All isolates were susceptible to ceftriaxone, cefixime and spectinomycin, and 99.9% were susceptible to azithromycin. AMR determinants for ciprofloxacin, benzylpenicillin and tetracycline were common, but rare for cephalosporins and azithromycin. Most isolates belonged to the more antimicrobial-susceptible lineage B (n = 780) compared with the AMR lineage A (n = 141), and limited geographical phylogenomic signal was observed. CONCLUSIONS: We report the first multi-country gonococcal genomic comparison from Africa, which will support the WHO GASP and WHO enhanced GASP (EGASP). The high prevalence of resistance to ciprofloxacin (and empirical use continues), tetracycline and benzylpenicillin, and the emerging resistance determinants for azithromycin show it is imperative to strengthen the gonococcal AMR surveillance, ideally including genomics, in African countries.

Sustained Transmission of Neisseria gonorrhoeae Strains with High-Level Azithromycin Resistance (MIC ≥ 256 µg/mL) in Argentina, 2018 to 2022.

Azithromycin combined with ceftriaxone is the recommended dual therapy for uncomplicated gonorrhea in many countries. Nevertheless, the increasing prevalence of azithromycin resistance compromises the effectiveness of this treatment strategy. From 2018 to 2022, we collected 13 gonococcal isolates with high-level azithromycin resistance (MIC ≥ 256 µg/mL) across Argentina. Whole-genome sequencing revealed that these isolates were mainly represented by the internationally spreading Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) genogroup G12302, containing the 23S rRNA A2059G mutation (in all four alleles) together with mosaic mtrD and mtrR promoter 2 loci. This information is important to develop targeted public health policies to control the spread of azithromycin-resistant N. gonorrhoeae in Argentina and internationally. IMPORTANCE Azithromycin resistance in Neisseria gonorrhoeae has been increasing in numerous populations worldwide, which is of concern, as azithromycin is part of the recommended dual treatment in many countries. Here, we report 13 N. gonorrhoeae isolates with high-level azithromycin resistance (MIC ≥ 256 µg/mL). This study observed that high-level azithromycin-resistant gonococcal strains have shown sustained transmission in Argentina and are related to the successful international clone NG-MAST G12302. Genomic surveillance together with real-time tracing and data-sharing networks will be crucial in controlling the spread of azithromycin resistance in gonococcus.

High-level in vitro resistance to gentamicin acquired in a stepwise manner in Neisseria gonorrhoeae.

OBJECTIVES: Gentamicin is used in several alternative treatments for gonorrhoea. Verified clinical Neisseria gonorrhoeae isolates with gentamicin resistance are mainly lacking and understanding the mechanisms for gonococcal gentamicin resistance is imperative. We selected gentamicin resistance in gonococci in vitro, identified the novel gentamicin-resistance mutations, and examined the biofitness of a high-level gentamicin-resistant mutant. METHODS: Low- and high-level gentamicin resistance was selected in WHO X (gentamicin MIC = 4 mg/L) on gentamicin-gradient agar plates. Selected mutants were whole-genome sequenced. Potential gentamicin-resistance fusA mutations were transformed into WT strains to verify their impact on gentamicin MICs. The biofitness of high-level gentamicin-resistant mutants was examined using a competitive assay in a hollow-fibre infection model. RESULTS: WHO X mutants with gentamicin MICs of up to 128 mg/L were selected. Primarily selected fusA mutations were further investigated, and fusAR635L and fusAM520I + R635L were particularly interesting. Different mutations in fusA and ubiM were found in low-level gentamicin-resistant mutants, while fusAM520I was associated with high-level gentamicin resistance. Protein structure predictions showed that fusAM520I is located in domain IV of the elongation factor-G (EF-G). The high-level gentamicin-resistant WHO X mutant was outcompeted by the gentamicin-susceptible WHO X parental strain, suggesting lower biofitness. CONCLUSIONS: We describe the first high-level gentamicin-resistant gonococcal isolate (MIC = 128 mg/L), which was selected in vitro through experimental evolution. The most substantial increases of the gentamicin MICs were caused by mutations in fusA (G1560A and G1904T encoding EF-G M520I and R635L, respectively) and ubiM (D186N). The high-level gentamicin-resistant N. gonorrhoeae mutant showed impaired biofitness.

Antimicrobial susceptibility surveillance and antimicrobial resistance in Neisseria gonorrhoeae in Africa from 2001 to 2020: A mini-review.

Antimicrobial resistance (AMR) in Neisseria gonorrhoeae (NG), compromising gonorrhea treatment, is a global public health concern. Improved, quality-assured NG AMR monitoring at the global level is essential. This mini-review examined NG AMR susceptibility surveillance and AMR data from the African continent from 2001 to 2020. Eligible peer-reviewed publications (n = 30) containing NG AMR data for antimicrobials currently recommended for gonorrhea treatment were included. Overall, very limited NG surveillance and AMR data was available. Furthermore, the NG AMR surveillance studies varied greatly regarding surveillance protocols (e.g., populations and samples tested, sample size, antimicrobials examined), methodologies (e.g., antimicrobial susceptibility testing method [agar dilution, minimum inhibitory concentration (MIC) gradient strip test, disc diffusion test] and interpretative criteria), and quality assurance (internal quality controls, external quality assessments [EQA], and verification of AMR detected). Moreover, most studies examined a suboptimal number of NG isolates, i.e., less than the WHO Global Gonococcal Antimicrobial Surveillance Program (GASP) and WHO Enhanced GASP (EGASP) recommendations of ≥100 isolates per setting and year. The notable inter-study variability and frequently small sample sizes make appropriate inter-study and inter-country comparisons of AMR data difficult. In conclusion, it is imperative to establish an enhanced, standardized and quality-assured NG AMR surveillance, ideally including patient metadata and genome sequencing as in WHO EGASP, in Africa, the region with the highest gonorrhea incidence globally. This will enable the monitoring of AMR trends, detection of emerging AMR, and timely refinements of national and international gonorrhea treatment guidelines. To achieve this aim, national and international leadership, political and financial commitments are imperative.

Mycoplasma genitalium prevalence, antimicrobial resistance-associated mutations, and coinfections with non-viral sexually transmitted infections in high-risk populations in Guatemala, Malta, Morocco, Peru and South Africa, 2019-2021.

The prevalence of Mycoplasma genitalium (MG) and MG antimicrobial resistance (AMR) appear to be high internationally, however, prevalence data remain lacking globally. We evaluated the prevalence of MG and MG AMR-associated mutations in men who have sex with men (MSM) in Malta and Peru and women at-risk for sexually transmitted infections in Guatemala, South Africa, and Morocco; five countries in four WHO regions mostly lacking MG prevalence and AMR data, and estimated MG coinfections with Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Trichomonas vaginalis (TV). Male urine and anorectal samples, and vaginal samples were tested for MG, CT, NG, and TV (only vaginal samples) using Aptima assays (Hologic). AMR-associated mutations in the MG 23S rRNA gene and parC gene were identified using ResistancePlus MG kit (SpeeDx) or Sanger sequencing. In total, 1,425 MSM and 1,398 women at-risk were recruited. MG was detected in 14.7% of MSM (10.0% in Malta and 20.0% Peru) and in 19.1% of women at-risk (12.4% in Guatemala, 16.0% Morocco, 22.1% South Africa). The prevalence of 23S rRNA and parC mutations among MSM was 68.1 and 29.0% (Malta), and 65.9 and 5.6% (Peru), respectively. Among women at-risk, 23S rRNA and parC mutations were revealed in 4.8 and 0% (Guatemala), 11.6 and 6.7% (Morocco), and 2.4 and 3.7% (South Africa), respectively. CT was the most frequent single coinfection with MG (in 2.6% of MSM and 4.5% of women at-risk), compared to NG + MG found in 1.3 and 1.0%, respectively, and TV + MG detected in 2.8% of women at-risk. In conclusion, MG is prevalent worldwide and enhanced aetiological MG diagnosis, linked to clinical routine detection of 23S rRNA mutations, in symptomatic patients should be implemented, where feasible. Surveillance of MG AMR and treatment outcome would be exceedingly valuable, nationally and internationally. High levels of AMR in MSM support avoiding screening for and treatment of MG in asymptomatic MSM and general population. Ultimately, novel therapeutic antimicrobials and/or strategies, such as resistance-guided sequential therapy, and ideally an effective MG vaccine are essential.

Multidrug-resistant Neisseria gonorrhoeae isolate SE690: mosaic penA-60.001 gene causing ceftriaxone resistance internationally has spread to the more antimicrobial-susceptible genomic lineage, Sweden, September 2022.

We report a ceftriaxone-resistant, multidrug-resistant urogenital Neisseria gonorrhoeae in a female sex worker in Sweden, September 2022, who was treated with ceftriaxone 1 g, but did not return for test-of-cure. Whole genome sequencing of isolate SE690 identified MLST ST8130, NG-STAR CC1885 (new NG-STAR ST4859) and mosaic penA-60.001. The latter, causing ceftriaxone resistance in the internationally spreading FC428 clone, has now also spread to the more antimicrobial-susceptible genomic lineage B, showing that strains across the gonococcal phylogeny can develop ceftriaxone resistance.

Complete Reference Genome Sequence of the Clinical Neisseria gonorrhoeae Strain H035, with Resistance to the Novel Antimicrobial Zoliflodacin, Identified in Japan in 2000.

Zoliflodacin is a promising novel antimicrobial in clinical development for treatment of gonorrhea; currently, it is in a global phase 3 randomized controlled clinical trial. High activity against global Neisseria gonorrhoeae strains has been shown. We present the complete reference genome of the zoliflodacin-resistant strain H035, which was identified in Japan in 2000.

Pharmacodynamic evaluation of lefamulin in the treatment of gonorrhea using a hollow fiber infection model simulating Neisseria gonorrhoeae infections.

The emergence and spread of antimicrobial resistance in Neisseria gonorrhoeae is seriously threatening the treatment and control of gonorrhea globally. Novel treatment options are essential, coupled with appropriate methods to pharmacodynamically examine the efficacy and resistance emergence of these novel drugs. Herein, we used our dynamic in vitro hollow fiber infection model (HFIM) to evaluate protein-unbound lefamulin, a semisynthetic pleuromutilin, against N. gonorrhoeae. Dose-range and dose-fractionation experiments with N. gonorrhoeae reference strains: WHO F (susceptible to all relevant antimicrobials), WHO X (extensively drug-resistant, including ceftriaxone resistance), and WHO V (high-level azithromycin resistant, and highest gonococcal MIC of lefamulin (2 mg/l) reported), were performed to examine lefamulin gonococcal killing and resistance development during treatment. The dose-range experiments, simulating a single oral dose of lefamulin based on human plasma concentrations, indicated that ≥1.2 g, ≥2.8 g, and ≥9.6 g of lefamulin were required to eradicate WHO F, X, and V, respectively. Dose-fractionation experiments, based on human lefamulin plasma concentrations, showed that WHO X was eradicated with ≥2.8 g per day when administered as q12 h (1.4 g twice a day) and with ≥3.6 g per day when administered as q8 h (1.2 g thrice a day), both for 7 days. However, when simulating the treatment with 5-10 times higher concentrations of free lefamulin in relevant gonorrhea tissues (based on urogenital tissues in a rat model), 600 mg every 12 h for 5 days (approved oral treatment for community-acquired bacterial pneumonia) eradicated all strains, and no lefamulin resistance emerged in the successful treatment arms. In many arms failing single or multiple dose treatments for WHO X, lefamulin-resistant mutants (MIC = 2 mg/l), containing an A132V amino acid substitution in ribosomal protein L3, were selected. Nevertheless, these lefamulin-resistant mutants demonstrated an impaired biofitness. In conclusion, a clinical study is warranted to elucidate the clinical potential of lefamulin as a treatment option for uncomplicated gonorrhea (as well as several other bacterial STIs).

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.

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.

Potent In Vitro and Ex Vivo Anti-Gonococcal Activity of the RpoB Inhibitor Corallopyronin A.

Gonorrhea remains a major global public health problem because of the high incidence of infection (estimated 82 million cases in 2020) and the emergence and spread of Neisseria gonorrhoeae strains resistant to previous and current antibiotics used to treat infections. Given the dearth of new antibiotics that are likely to enter clinical practice in the near future, there is concern that cases of untreatable gonorrhea might emerge. In response to this crisis, the World Health Organization (WHO), in partnership with the Global Antibiotic Research and Development Partnership (GARDP), has made the search for and development of new antibiotics against N. gonorrhoeae a priority. Ideally, these antibiotics should also be active against other sexually transmitted organisms, such as Chlamydia trachomatis and/or Mycoplasma genitalium, which are often found with N. gonorrhoeae as co-infections. Corallopyronin A is a potent antimicrobial that exhibits activity against Chlamydia spp. and inhibits transcription by binding to the RpoB switch region. Accordingly, we tested the effectiveness of corallopyronin A against N. gonorrhoeae. We also examined the mutation frequency and modes of potential resistance against corallopyronin A. We report that corallopyronin A has potent antimicrobial action against antibiotic-susceptible and antibiotic-resistant N. gonorrhoeae strains and could eradicate gonococcal infection of cultured, primary human cervical epithelial cells. Critically, we found that spontaneous corallopyronin A-resistant mutants of N. gonorrhoeae are exceedingly rare (≤10-10) when selected at 4× the MIC. Our results support pre-clinical studies aimed at developing corallopyronin A for gonorrheal treatment regimens. IMPORTANCE The high global incidence of gonorrhea, the lack of a protective vaccine, and the emergence of N. gonorrhoeae strains expressing resistance to currently used antibiotics demand that new treatment options be developed. Accordingly, we investigated whether corallopyronin A, an antibiotic which is effective against other pathogens, including C. trachomatis, which together with gonococci frequently cause co-infections in humans, could exert anti-gonococcal action in vitro and ex vivo, and potential resistance emergence. We propose that corallopyronin A be considered a potential future treatment option for gonorrhea because of its potent activity, low resistance development, and recent advances in scalable production.