ABSTRACT
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 (MICâ>â0.125 mg/L), in our hollow fibre infection model (HFIM) for gonorrhoea. METHODS: Gonococcal strains examined were WHO F (ceftriaxone-susceptible, MICâ<â0.002 mg/L), R (ceftriaxone-resistant, MICâ=â0.5 mg/L), Z (ceftriaxone-resistant, MICâ=â0.5 mg/L) and X (ceftriaxone-resistant, MICâ=â2 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.
Subject(s)
Anti-Bacterial Agents , Ceftriaxone , Gonorrhea , Microbial Sensitivity Tests , Neisseria gonorrhoeae , Ceftriaxone/pharmacokinetics , Ceftriaxone/pharmacology , Ceftriaxone/administration & dosage , Neisseria gonorrhoeae/drug effects , Anti-Bacterial Agents/pharmacokinetics , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/administration & dosage , Gonorrhea/drug therapy , Gonorrhea/microbiology , Humans , Drug Resistance, BacterialABSTRACT
OBJECTIVES: The novel dual-target triazaacenaphthylene, gepotidacin, recently showed promising results in its Phase III randomized controlled trial for the treatment of gonorrhoea. We investigated alterations in the gepotidacin GyrA and ParC targets in gonococci by in silico mining of publicly available global genomes (nâ=â33â213) and determined gepotidacin MICs in isolates with GyrA A92 alterations combined with other GyrA and/or ParC alterations. METHODS: We examined gonococcal gyrA and parC alleles available at the European Nucleotide Archive. MICs were determined using the agar dilution method (gepotidacin) or Etest (four antimicrobials). Models of DNA gyrase and topoisomerase IV were obtained from AlphaFold and used to model gepotidacin in the binding site. RESULTS: GyrA A92 alterations were identified in 0.24% of genomes: GyrA A92P/S/Vâ+âS91Fâ+âD95Y/A/N (0.208%), A92Pâ+âS91F (0.024%) and A92P (0.003%), but no A92T (previously associated with gepotidacin resistance) was found. ParC D86 alterations were found in 10.6% of genomes: ParC D86N/G (10.5%), D86Nâ+âS87I (0.051%), D86Nâ+âS88P (0.012%) and D86Gâ+âE91G (0.003%). One isolate had GyrA A92Pâ+âParC D86N alterations, but remained susceptible to gepotidacin (MICâ=â0.125 mg/L). No GyrA plus ParC alterations resulted in a gepotidacin MICâ>â4 mg/L. Modelling of gepotidacin binding to GyrA A92/A92T/A92P suggested that gepotidacin resistance due to GyrA A92T might be linked to the formation of a new polar contact with DNA. CONCLUSIONS: In silico mining of 33â213 global gonococcal genomes (isolates from 1928 to 2023) showed that A92 is highly conserved in GyrA, while alterations in D86 of ParC are common. No GyrA plus ParC alterations caused gepotidacin resistance. MIC determination and genomic surveillance of potential antimicrobial resistance determinants are imperative.
ABSTRACT
OBJECTIVES: MDR and XDR Neisseria gonorrhoeae strains remain major public health concerns internationally, and quality-assured global gonococcal antimicrobial resistance (AMR) surveillance is imperative. The WHO global Gonococcal Antimicrobial Surveillance Programme (GASP) and WHO Enhanced GASP (EGASP), including metadata and WGS, are expanding internationally. We present the phenotypic, genetic and reference genome characteristics of the 2024 WHO gonococcal reference strains (nâ=â15) for quality assurance worldwide. All superseded WHO gonococcal reference strains (nâ=â14) were identically characterized. MATERIAL AND METHODS: The 2024 WHO reference strains include 11 of the 2016 WHO reference strains, which were further characterized, and four novel strains. The superseded WHO reference strains include 11 WHO reference strains previously unpublished. All strains were characterized phenotypically and genomically (single-molecule PacBio or Oxford Nanopore and Illumina sequencing). RESULTS: The 2024 WHO reference strains represent all available susceptible and resistant phenotypes and genotypes for antimicrobials currently and previously used (nâ=â22), or considered for future use (nâ=â3) in gonorrhoea treatment. The novel WHO strains include internationally spreading ceftriaxone resistance, ceftriaxone resistance due to new penA mutations, ceftriaxone plus high-level azithromycin resistance and azithromycin resistance due to mosaic MtrRCDE efflux pump. AMR, serogroup, prolyliminopeptidase, genetic AMR determinants, plasmid types, molecular epidemiological types and reference genome characteristics are presented for all strains. CONCLUSIONS: The 2024 WHO gonococcal reference strains are recommended for internal and external quality assurance in laboratory examinations, especially in the WHO GASP, EGASP and other GASPs, but also in phenotypic and molecular diagnostics, AMR prediction, pharmacodynamics, epidemiology, research and as complete reference genomes in WGS analysis.
Subject(s)
Anti-Bacterial Agents , Genome, Bacterial , Gonorrhea , Microbial Sensitivity Tests , Neisseria gonorrhoeae , Phenotype , Neisseria gonorrhoeae/genetics , Neisseria gonorrhoeae/drug effects , Humans , Anti-Bacterial Agents/pharmacology , Gonorrhea/microbiology , Microbial Sensitivity Tests/standards , World Health Organization , Whole Genome Sequencing , Genotype , Drug Resistance, Bacterial/genetics , Quality Assurance, Health Care , Reference StandardsABSTRACT
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.
Subject(s)
Anti-Bacterial Agents , Azithromycin , Drug Resistance, Bacterial , Gonorrhea , Microbial Sensitivity Tests , Neisseria gonorrhoeae , Whole Genome Sequencing , Neisseria gonorrhoeae/genetics , Neisseria gonorrhoeae/drug effects , Neisseria gonorrhoeae/classification , Brazil/epidemiology , Humans , Gonorrhea/microbiology , Gonorrhea/epidemiology , Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial/genetics , Azithromycin/pharmacology , Male , Genome, Bacterial , Female , Adult , Molecular Epidemiology , Young Adult , Genomics , RNA, Ribosomal, 23S/genetics , Middle Aged , Ceftriaxone/pharmacology , Adolescent , Multilocus Sequence Typing , Cefixime/pharmacologyABSTRACT
OBJECTIVE: To estimate the prevalence of the curable sexually transmitted infections (STIs) Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis and Treponema pallidum, to identify associated risk factors and to assess ciprofloxacin resistance in N. gonorrhoeae-positive specimens among female sex workers (FSWs) in Guinea-Bissau. METHODS: For this cross-sectional study, FSWs were recruited from October 2014 to May 2019. A questionnaire on STI risk factors was completed by the study participants, and the women were asked to provide a vaginal swab for nucleic acid amplification tests for C. trachomatis, N. gonorrhoeae, M. genitalium, T. vaginalis (Aptima, Hologica), as well as a blood sample for T. pallidum serological testing and discriminatory HIV-testing. The prevalence of STIs was determined, and multivariate logistic regression was used to identify STI risk factors. RESULTS: The study included 467 women. The prevalence of current infection with any curable STI was 46.7%, and the most common pathogen was T. vaginalis (26.3%), followed by M. genitalium (21.9%), C. trachomatis (11.8%), N. gonorrhoeae (10.1%) and T. pallidum (2.8%). The proportion of asymptomatic infections among the diagnosed STIs was 61.8%, 61.5%, 55.3%, 55.3% and 52.2% for C. trachomatis, T. pallidum, N. gonorrhoeae, T. vaginalis and M. genitalium, respectively. The prevalence of the gyrA S91F mutation conferring ciprofloxacin resistance in N. gonorrhoeae-positive specimens was 84.0%. Significant risk factors for having a curable STI were age and HIV-1 infection, while use of female condoms was a protective factor. CONCLUSION: This study demonstrated that the prevalence of curable STIs was high among FSWs in Guinea-Bissau during the study period, indicating an unmet need for STI services. Moreover, the results indicated that symptomatic treatment might be insufficient, highlighting a need for periodic aetiological testing to facilitate detection of asymptomatic as well as symptomatic STIs to stop ongoing transmission.
ABSTRACT
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.
Subject(s)
Chlamydia Infections , Gonorrhea , Sexual and Gender Minorities , Male , Humans , Homosexuality, Male , Neisseria gonorrhoeae/genetics , Chlamydia Infections/diagnosis , Chlamydia Infections/epidemiology , Gonorrhea/diagnosis , Gonorrhea/epidemiology , Chlamydia trachomatis/genetics , Nucleic Acid Amplification Techniques , Tomography, X-Ray ComputedABSTRACT
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.
Subject(s)
Gonorrhea , Trichomonas vaginalis , Female , Humans , Trichomonas vaginalis/genetics , Chlamydia trachomatis/genetics , Gonorrhea/diagnosis , Gonorrhea/epidemiology , Guatemala/epidemiology , Morocco/epidemiology , South Africa/epidemiology , Neisseria gonorrhoeae/genetics , Australia , Point-of-Care TestingABSTRACT
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 MICâ=â4 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 fusAM520Iâ+âR635L 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 (MICâ=â128 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.
Subject(s)
Gonorrhea , Neisseria gonorrhoeae , Humans , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Gentamicins/pharmacology , Peptide Elongation Factor G , Gonorrhea/drug therapy , Microbial Sensitivity Tests , Drug Resistance, BacterialABSTRACT
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 (nâ=â433), Malawi (nâ=â154) and South Africa (nâ=â99) in 2015-20 were genome-sequenced. MICs were determined using ETEST. Sequences of isolates from Kenya (nâ=â159), Burkina Faso (nâ=â52) and the 2016 WHO reference strains (nâ=â14) 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 (nâ=â780) compared with the AMR lineage A (nâ=â141), 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.
Subject(s)
Anti-Bacterial Agents , Gonorrhea , Humans , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Neisseria gonorrhoeae , Azithromycin/pharmacology , Malawi , South Africa , Uganda/epidemiology , Drug Resistance, Bacterial , Gonorrhea/epidemiology , Gonorrhea/drug therapy , Ciprofloxacin/pharmacology , Microbial Sensitivity Tests , Tetracycline/pharmacology , GenomicsABSTRACT
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.
Subject(s)
Anti-Infective Agents , Gonorrhea , Sex Workers , Female , Humans , Ceftriaxone/pharmacology , Neisseria gonorrhoeae , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Multilocus Sequence Typing , Sweden , Microbial Sensitivity Tests , Gonorrhea/drug therapy , Genomics , Drug Resistance, Bacterial/geneticsABSTRACT
The continued emergence of Neisseria gonorrhoeae isolates which are resistant to first-line antibiotics has reinvigorated interest in alternative therapies such as expanded use of gentamicin (Gen). We hypothesized that expanded use of Gen promotes emergence of gonococci with clinical resistance to this aminoglycoside. To understand how decreased susceptibility of gonococci to Gen might develop, we selected spontaneous low-level Gen-resistant (GenR) mutants (Gen MIC = 32 µg/mL) of the Gen-susceptible strain FA19. Consequently, we identified a novel missense mutation in fusA, which encodes elongation factor G (EF-G), causing an alanine (A) to valine (V) substitution at amino acid position 563 in domain IV of EF-G; the mutant allele was termed fusA2. Transformation analysis showed that fusA2 could increase the Gen MIC by 4-fold. While possession of fusA2 did not impair either in vitro gonococcal growth or protein synthesis, it did result in a fitness defect during experimental infection of the lower genital tract in female mice. Through bioinformatic analysis of whole-genome sequences of 10,634 international gonococcal clinical isolates, other fusA alleles were frequently detected, but genetic studies revealed that they could not decrease Gen susceptibility in a similar manner to fusA2. In contrast to these diverse international fusA alleles, the fusA2-encoded A563V substitution was detected in only a single gonococcal clinical isolate. We hypothesize that the rare occurrence of fusA2 in N. gonorrhoeae clinical isolates is likely due to a fitness cost during infection, but compensatory mutations which alleviate this fitness cost could emerge and promote GenR in global strains.
Subject(s)
Gonorrhea , Neisseria gonorrhoeae , Amino Acid Substitution , Animals , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Drug Resistance, Bacterial/genetics , Female , Gentamicins/pharmacology , Gentamicins/therapeutic use , Gonorrhea/drug therapy , Mice , Microbial Sensitivity Tests , Peptide Elongation Factor GABSTRACT
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 27â151 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%), L425Iâ+âL465I (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 8â 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 27â151 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.
Subject(s)
Anti-Infective Agents , Gonorrhea , Male , Humans , Anti-Bacterial Agents/pharmacology , DNA Gyrase/genetics , DNA Topoisomerase IV/genetics , Anti-Infective Agents/pharmacology , Neisseria gonorrhoeae/genetics , Mutation , Microbial Sensitivity Tests , Drug Resistance, Bacterial/geneticsABSTRACT
OBJECTIVES: Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a substantial global public health problem. Gonococcal infections acquired in or from Asia represent most verified ceftriaxone treatment failures, and several ceftriaxone-resistant strains have emerged in Asia and subsequently spread globally. Additionally, in Thailand the gonorrhoea incidence remains high. Herein, we investigate the genomic diversity, AMR and AMR determinants in gonococcal isolates cultured in 2018 in Bangkok, Thailand. METHODS: Gonococcal isolates from males (nâ=â37) and females (nâ=â62) were examined by Etest and WGS. AMR determinants and molecular epidemiological STs were characterized. For phylogenomic comparison, raw sequence data were included from China (432 isolates), Japan (nâ=â270), Vietnam (nâ=â229), Thailand (nâ=â3), a global dataset (nâ=â12â440) and the 2016 WHO reference strains plus WHO Q (nâ=â15). RESULTS: In total, 88, 66 and 41 different NG-MAST, NG-STAR and MLST STs, respectively, and 31 different NG-STAR clonal complexes were found. A remarkably high frequency (88%) of ß-lactamase TEM genes was detected and two novel TEM alleles were found. The phylogenomic analysis divided the isolates into the previously described lineages A and B, with a large proportion of Thai isolates belonging to the novel sublineage A3. CONCLUSIONS: We describe the first molecular epidemiological study using WGS on gonococcal isolates from Thailand. The high prevalence of AMR and AMR determinants for ciprofloxacin, tetracycline and benzylpenicillin, and some strains belonging to clones/clades especially in sublineage A2 that are prone to develop resistance to extended-spectrum cephalosporins (ESCs) and azithromycin, should prompt continued and strengthened AMR surveillance, including WGS, of N. gonorrhoeae in Thailand.
Subject(s)
Gonorrhea , Neisseria gonorrhoeae , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Ceftriaxone/pharmacology , Drug Resistance, Bacterial , Female , Genomics , Gonorrhea/drug therapy , Gonorrhea/epidemiology , Humans , Male , Microbial Sensitivity Tests , Multilocus Sequence Typing , Thailand/epidemiologyABSTRACT
BACKGROUND: Gentamicin has been used for the treatment of gonorrhea in Malawi since 1993. However, declining clinical cure rates have been suspected. We evaluated current Neisseria gonorrhoeae susceptibility to gentamicin in vitro and clinically. METHODS: Men with acute urethritis were recruited at the Bwaila District Hospital STI Clinic in Lilongwe, Malawi, between January 2017 and August 2019. All men provided urethral swabs for etiological testing at enrollment and test of cure (TOC), 1 week later, using Gram-stained microscopy and culture. We used Etest to determine minimum inhibitory concentrations (MICs) of gentamicin, azithromycin, cefixime, ceftriaxone, ciprofloxacin, and spectinomycin; disc diffusion for tetracycline susceptibility; and whole-genome sequencing (WGS) to verify/refute treatment failure. RESULTS: Among 183 N. gonorrhoeae culture-positive men enrolled, 151 (82.5%) had a swab taken for TOC. Of these 151 men, 16 (10.6%) had a positive culture at TOC. One hundred forty-one baseline isolates were tested for gentamicin susceptibility using Etest: 2 (1.4%), MIC = 2 µg/mL; 111 (78.7%), MIC = 4 µg/mL; and 28 (19.9%), MIC = 8 µg/mL. All isolates were susceptible to azithromycin, cefixime, ceftriaxone, and spectinomycin, whereas 63.1% had intermediate susceptibility or resistance to ciprofloxacin. Almost all (96.1%) isolates were resistant to tetracycline. All examined isolates cultured at TOC (n = 13) had gentamicin MICs ≤8 µg/mL. Ten men had pretreatment and posttreatment isolates examined by whole-genome sequencing; 2 (20%) were verified new infections (4119 and 1272 single-nucleotide polymorphisms), whereas 8 (80%) were confirmed treatment failures (0-1 single-nucleotide polymorphism). CONCLUSIONS: Gentamicin MICs poorly predict gonorrhea treatment outcome with gentamicin, and treatment failures are verified with gonococcal strains with in vitro susceptibility to gentamicin. The first-line treatment of gonorrhea in Malawi should be reassessed.
Subject(s)
Gonorrhea , Neisseria gonorrhoeae , Female , Humans , Male , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Azithromycin/pharmacology , Azithromycin/therapeutic use , Cefixime/therapeutic use , Ceftriaxone/pharmacology , Ceftriaxone/therapeutic use , Ciprofloxacin/pharmacology , Ciprofloxacin/therapeutic use , Gentamicins/pharmacology , Gentamicins/therapeutic use , Gonorrhea/drug therapy , Gonorrhea/epidemiology , Malawi/epidemiology , Microbial Sensitivity Tests , Spectinomycin/pharmacology , Spectinomycin/therapeutic use , Tetracycline/pharmacology , Tetracycline/therapeutic use , Treatment Outcome , Polymorphism, Single NucleotideABSTRACT
We describe a gonorrhoea case with ceftriaxone plus high-level azithromycin resistance. In April 2022, an Austrian heterosexual male was diagnosed with gonorrhoea after sexual intercourse with a female sex worker in Cambodia. Recommended treatment with ceftriaxone (1 g) plus azithromycin (1.5 g) possibly failed. Worryingly, this is the second strain in an Asian Neisseria gonorrhoeae genomic sublineage including high-level azithromycin-resistant strains that developed ceftriaxone resistance by acquisition of mosaic penA-60.001. Enhanced resistance surveillance and actions are imperative to prevent spread.
Subject(s)
Gonorrhea , Sex Workers , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Austria , Azithromycin/pharmacology , Azithromycin/therapeutic use , Ceftriaxone/pharmacology , Ceftriaxone/therapeutic use , Drug Resistance, Bacterial/genetics , Female , Gonorrhea/diagnosis , Gonorrhea/drug therapy , Gonorrhea/epidemiology , Humans , Male , Microbial Sensitivity Tests , Neisseria gonorrhoeae/genetics , Treatment FailureABSTRACT
We report a ceftriaxone-resistant, multidrug-resistant urogenital gonorrhoea case in a heterosexual woman in France, June 2022. The woman was successfully treated with azithromycin 2â¯g. She had unprotected sex with her regular partner, who developed urethritis following travel to Vietnam and Switzerland. Whole genome sequencing of the gonococcal isolate (F92) identified MLST ST1901, NG-STAR CC-199, and the novel mosaic penA-237.001, which caused ceftriaxone resistance. penA-237.001 is 98.7% identical to penA-60.001, reported in various ceftriaxone-resistant strains, including the internationally spreading FC428 clone.
Subject(s)
Ceftriaxone , Gonorrhea , Humans , Female , Ceftriaxone/pharmacology , Ceftriaxone/therapeutic use , Neisseria gonorrhoeae , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Multilocus Sequence Typing , Gonorrhea/diagnosis , Gonorrhea/drug therapy , Microbial Sensitivity Tests , Drug Resistance, Bacterial/geneticsABSTRACT
Azithromycin-nonsusceptible Neisseria gonorrhoeae strains are an emerging global public health threat. During 2015-2018, the prevalence of azithromycin-nonsusceptible gonococcal infection increased significantly in Argentina. To investigate the genomic epidemiology and resistance mechanisms of these strains, we sequenced 96 nonsusceptible isolates collected in Argentina during 2005-2019. Phylogenomic analysis revealed 2 main clades, which were characterized by a limited geographic distribution, circulating during January 2015-November 2019. These clades included the internationally spreading multilocus sequence types (STs) 1580 and 9363. The ST1580 isolates, which had MICs of 2-4 µg/mL, had mutations in the 23S rRNA. The ST9363 isolates, which had MICs of 2-4 or >256 µg/mL, had mutations in the 23S rRNA, a mosaic mtr locus, or both. Identifying the geographic dissemination and characteristics of these predominant clones will guide public health policies to control the spread of azithromycin-nonsusceptible N. gonorrhoeae in Argentina.
Subject(s)
Azithromycin , Neisseria gonorrhoeae , Anti-Bacterial Agents/pharmacology , Argentina/epidemiology , Azithromycin/pharmacology , Drug Resistance, Bacterial , Genomics , Neisseria gonorrhoeae/geneticsABSTRACT
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.
Subject(s)
Gonorrhea , Neisseria gonorrhoeae , Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial , Genomics , Gonorrhea/epidemiology , Humans , Microbial Sensitivity Tests , Multilocus Sequence Typing , Neisseria gonorrhoeae/genetics , PhylogenyABSTRACT
OBJECTIVES: Novel antimicrobials for treatment of gonorrhoea are imperative. The first-in-class spiropyrimidinetrione zoliflodacin is promising and currently in an international Phase 3 randomized controlled clinical trial (RCT) for treatment of uncomplicated gonorrhoea. We evaluated the in vitro activity of and the genetic conservation of the target (GyrB) and other potential zoliflodacin resistance determinants among 1209 consecutive clinical Neisseria gonorrhoeae isolates obtained from 25 EU/European Economic Area (EEA) countries in 2018 and compared the activity of zoliflodacin with that of therapeutic antimicrobials currently used. METHODS: MICs of zoliflodacin, ceftriaxone, cefixime, azithromycin and ciprofloxacin were determined using an agar dilution technique for zoliflodacin or using MIC gradient strip tests or an agar dilution technique for the other antimicrobials. Genome sequences were available for 96.1% of isolates. RESULTS: Zoliflodacin modal MIC, MIC50, MIC90 and MIC range were 0.125, 0.125, 0.125 and ≤0.004-0.5 mg/L, respectively. The resistance was 49.9%, 6.7%, 1.6% and 0.2% to ciprofloxacin, azithromycin, cefixime and ceftriaxone, respectively. Zoliflodacin did not show any cross-resistance to other tested antimicrobials. GyrB was highly conserved and no zoliflodacin gyrB resistance mutations were found. No fluoroquinolone target GyrA or ParC resistance mutations or mutations causing overexpression of the MtrCDE efflux pump substantially affected the MICs of zoliflodacin. CONCLUSIONS: The in vitro susceptibility to zoliflodacin was high and the zoliflodacin target GyrB was conserved among EU/EEA gonococcal isolates in 2018. This study supports further clinical development of zoliflodacin. However, additional zoliflodacin data regarding particularly the treatment of pharyngeal gonorrhoea, pharmacokinetics/pharmacodynamics and resistance selection, including suppression, would be valuable.
Subject(s)
Gonorrhea , Neisseria gonorrhoeae , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Azithromycin/pharmacology , Barbiturates , Ceftriaxone/pharmacology , Ciprofloxacin/pharmacology , Drug Resistance, Bacterial , Europe , Gonorrhea/drug therapy , Humans , Isoxazoles , Microbial Sensitivity Tests , Morpholines , Neisseria gonorrhoeae/genetics , Oxazolidinones , Spiro CompoundsABSTRACT
BACKGROUND: Accurate molecular assays for prediction of antimicrobial resistance (AMR)/susceptibility in Neisseria gonorrhoeae (Ng) can offer individualized treatment of gonorrhoea and enhanced AMR surveillance. OBJECTIVES: We evaluated the new ResistancePlus® GC assay and the GC 23S 2611 (beta) assay (SpeeDx), for prediction of resistance/susceptibility to ciprofloxacin and azithromycin, respectively. METHODS: Nine hundred and sixty-seven whole-genome-sequenced Ng isolates from 20 European countries, 143 Ng-positive (37 with paired Ng isolates) and 167 Ng-negative clinical Aptima Combo 2 (AC2) samples, and 143 non-gonococcal Neisseria isolates and closely related species were examined with both SpeeDx assays. RESULTS: The sensitivity and specificity of the ResistancePlus® GC assay to detect Ng in AC2 samples were 98.6% and 100%, respectively. ResistancePlus® GC showed 100% sensitivity and specificity for GyrA S91 WT/S91F detection and 99.8% sensitivity and specificity in predicting phenotypic ciprofloxacin resistance. The sensitivity and specificity of the GC 23S 2611 (beta) assay for Ng detection in AC2 samples were 95.8% and 100%, respectively. GC 23S 2611 (beta) showed 100% sensitivity and 99.9% specificity for 23S rRNA C2611 WT/C2611T detection and 64.3% sensitivity and 99.9% specificity for predicting phenotypic azithromycin resistance. Cross-reactions with non-gonococcal Neisseria species were observed with both assays, but the analysis software solved most cross-reactions. CONCLUSIONS: The new SpeeDx ResistancePlus® GC assay performed well in the detection of Ng and AMR determinants, especially in urogenital samples. The GC 23S 2611 (beta) assay performed relatively well, but its sensitivity, especially for predicting phenotypic azithromycin resistance, was suboptimal and further optimizations are required, including detection of additional macrolide resistance determinant(s).