Gonorrhoeae Multi-antigen Sequence Typing Research Articles

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.

Antimicrobial Resistance and Molecular Typing of Neisseria gonorrhoeae Isolates From the Eastern Cape Province in South Africa.

There is a paucity of Neisseria gonorrhoeae antimicrobial resistance data from resource-constrained settings because of the lack of diagnostic testing and limited scale of surveillance programs. This study aimed to determine the antimicrobial resistance profile of N. gonorrhoeae in the rural Eastern Cape province of South Africa. Specimens for N. gonorrhoeae culture were obtained from men with urethral discharge and women with vaginal discharge attending primary health care facilities. Direct inoculation of the agar plates was performed followed by culture and drug susceptibility testing using the Etest at the laboratory. Whole-genome sequencing of the isolates was performed to identify resistance-determining variants. One hundred N. gonorrhoeae isolates were obtained. Most strains were nonsusceptible to ciprofloxacin (76%), tetracycline (75%), and penicillin G (72%). The gyrA S91F mutation was present in 68 of 72 ciprofloxacin-resistant isolates (94%), with concurrent parC mutations in 47 of 68 (69%); gyrA I250M was the only mutation in 4 other resistant strains. One azithromycin-resistant isolate was identified with a minimal inhibitory concentration (MIC) of 8.0 mg/L and the 23S rDNA gene mutation C2597T. The median MIC of cefixime was 0.016 mg/L (range, 0.016-0.064 mg/L), and that of ceftriaxone was 0.016 mg/L (range, 0.016 mg/L). Whole-genome sequencing showed 58 sequence types as revealed in N. gonorrhoeae sequence typing for antimicrobial resistance and 70 sequence types in N. gonorrhoeae multiantigen sequence typing. This study confirmed high rates of N. gonorrhoeae antimicrobial resistance to ciprofloxacin, penicillin G, and tetracycline in our setting. The MICs of cephalosporins are reassuring for ceftriaxone use in syndromic treatment regimens, but the identification of azithromycin resistance warrants further attention.

Antimicrobial susceptibilities of Neisseria gonorrhoeae in Canada, 2021.

In Canada, gonorrhea is the second most prevalent bacterial sexually transmitted infection. The Gonococcal Antimicrobial Surveillance Programme (GASP - Canada), a passive surveillance system monitoring antimicrobial resistance in Neisseria gonorrhoeae in Canada since 1985, is the source for this summary of demographics, antimicrobial resistance and N. gonorrhoeae multi-antigen sequence typing (NG-MAST) of gonococcal isolates collected in Canada in 2021. Provincial and territorial public health laboratories submitted N. gonorrhoeae cultures and data to the National Microbiology Laboratory in Winnipeg as part of the surveillance system. The antimicrobial resistance and molecular type of each isolate received were determined. In total, 3,439 N. gonorrhoeae cultures were received from laboratories across Canada in 2021, a 9.9% increase since 2020 (n=3,130). Decreased susceptibility to cefixime increased significantly (p<0.001) in 2021 (1.5%) compared to 2017 (0.6%). No significant change in decreased susceptibility to ceftriaxone was detected between 2017 and 2021 (0.6%) (p>0.001); however, one ceftriaxone-resistant isolate was identified. Azithromycin resistance decreased significantly (p<0.001) in 2021 (7.6%) compared to 2017 (11.7%); however, there was a significant increase (p<0.001) in the proportion of cultures with an azithromycin minimum inhibitory concentration of at least 1 mg/L (2017=22.2% to 2021=28.1%). In 2021, NG-MAST-19875 (15.3%) was the most prevalent sequence type in Canada; 20.3% of isolates with this sequence type were resistant to azithromycin. The spread of antimicrobial-resistant gonorrhea is a significant public health concern. The continued regional and national surveillance of antimicrobial resistance in N. gonorrhoeae is essential in ensuring effective treatment therapies are recommended.

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.

Increase in Multidrug Resistant Neisseria gonorrhoeae FC428-Like Isolates Harboring the Mosaic penA 60.001 Gene, in Nanjing, China (2017-2020).

Since the first Chinese report of the ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone in 2016, additional FC428-like, penA 60.001 isolates have been identified in China. To document the rise in penA 60.001 isolates in Nanjing, China, and characterize their molecular and epidemiological features. N. gonorrhoeae minimum inhibitory concentrations (MICs, mg/L) for ceftriaxone, cefixime, penicillin, tetracycline, ciprofloxacin, azithromycin, spectinomycin, gentamicin and zoliflodacin were determined by agar dilution. MICs for ertapenem were measured by E-test. N. gonorrhoeae antimicrobial sequence typing (NG-STAR) of seven loci (penA, mtrR, porB, ponA, gyrA, parC and 23S rRNA) was analyzed together with N. gonorrhoeae multiantigen sequence typing (NG-MAST) and multilocus sequence typing (MLST). Phylogenetic analysis was also performed using whole genomic sequencing (WGS). Fourteen FC428-related penA 60.001 N. gonorrhoeae infections were identified out of 677 infections from 2017 to 2020, in Nanjing, representing an incremental yearly rise in the percentage of the city's N. gonorrhoeae isolates that were FC428-related. Seven FC428-related N. gonorrhoeae infections were acquired in Nanjing, proper; four others in eastern Chinese cities and three from unknown locations. All FC428-related isolates were resistant to ceftriaxone, cefixime, ciprofloxacin, tetracycline and penicillin but susceptible to spectinomycin, gentamicin, ertapenem and zoliflodacin; three strains were resistant to azithromycin. penA 60.001 isolates displayed closely related MLST types and NG-STAR types but relatively distant NG-MAST types. WGS showed a phylogenetic analysis that intermingled with other international isolates. penA 60.001 N. gonorrhoeae isolates emerged in Nanjing, China, beginning in 2017, and have continued to rise.

Surveillance of the Antimicrobial Susceptibility and Molecular Characteristics of Neisseria gonorrhoeae Isolates Collected in Changsha, China from 2016 to 2021.

Antibiotic treatment is critical for individuals infected with gonorrhea and preventing disease transmission. This study aimed to analyze the antimicrobial susceptibility and molecular epidemiological characteristics of Neisseria gonorrhoeae isolates in Changsha, China. A total of 271 N.gonorrhoeae isolates collected from the clinical laboratories of two hospitals between 2016 and 2021 were analyzed for antimicrobial susceptibility using the agar dilution method. N. gonorrhoeae multi-antigen sequence typing (NG-MAST) was conducted for genotyping, and phylogenetic analysis was performed using the porB and tbpB sequences. The results showed that antimicrobial resistance against ciprofloxacin, tetracycline, and penicillin was high, and these drugs are no longer recommended for the treatment of gonorrhea. All isolates were susceptible to spectinomycin. However, in 2016-2021, a total of 15 (5.5%) ceftriaxone (CRO)-resistant strains and 31 (11.4%) isolates with decreased susceptibility to CRO were found, and the resistance rate to azithromycin had reached 7.1% in 2016-2017. Epidemiologically, the mosaic penA allele was identified in all CRO-resistant isolates. Based on NG-MAST, ST5061 was the most prevalent ST. Phylogenetic analysis suggested that the resistant isolates did not cluster independently. Despite focus on the local situation, this study raises the need for better gonorrhea medication and highlights that CRO may not be adequate as first-line treatment for gonorrhea in Changsha.

Molecular Epidemiology of Penicillinase-Producing Neisseria gonorrhoeae Isolates and Their blaTEM-135 Gene Variant in Bangkok, Thailand, 2015-2017.

Penicillinase-producing Neisseria gonorrhoeae (PPNG) possessing blaTEM-135 is a serious public health threat. With only a single change in the amino acid sequence, blaTEM-135 could evolve into a TEM-type extended-spectrum beta-lactamase (ESBL), which hydrolyzes extended-spectrum cephalosporins, including ceftriaxone and cefixime. We investigated the molecular epidemiological characteristics, types of plasmids in PPNG isolates, and prevalence of PPNG clinical isolates producing TEM-135 beta-lactamases. N. gonorrhoeae multi-antigen sequence typing (NG-MAST) was used to determine the molecular epidemiological characteristics of 99 PPNG isolates collected from 2015 to 2017. A mismatch amplification mutation assay was used to examine the blaTEM-135 gene prevalence. Of the 89 identified NG-MAST sequence types, 65 (73.0%) were novel. Only 17.7% (43/243) of PPNG isolates belonged to 16 genogroups. The most frequent plasmid was African, followed by Rio/Toronto, and Asian. The blaTEM-135 allele was found in Rio/Toronto plasmids. The blaTEM-135 allele was present in 23.2% (23/99) of the PPNG isolates. PPNG isolates expressing TEM-135 beta-lactamase exhibited significantly higher penicillin MIC (minimum inhibitory concentration) values than TEM-1 PPNG isolates. The PPNG isolates showed high genetic diversity and a high proportion of blaTEM-135 alleles. Mutation of the blaTEM-135 allele is worrisome as only one mutation could cause TEM-1 to evolve into an ESBL variant that degrades ceftriaxone. Ongoing surveillance of blaTEM-135 and new PPNG isolates is imperative.

Seven Years of Culture Collection of Neisseria gonorrhoeae: Antimicrobial Resistance and Molecular Epidemiology.

The emergence of Neisseria gonorrhoeae isolates displaying resistance to antimicrobials, in particular to ceftriaxone monotherapy or ceftriaxone plus azithromycin, represents a global public health concern. This study aimed to analyze the trend of antimicrobial resistance in a 7-year isolate collection retrospective analysis in Italy. Molecular typing on a subsample of gonococci was also included. A total of 1,810 culture-positive gonorrhea cases, collected from 2013 to 2019, were investigated by antimicrobial susceptibility, using gradient diffusion method, and by the N. gonorrhoeae multiantigen sequence typing (NG-MAST). The majority of infections occurred among men with urogenital infections and 57.9% of male patients were men who have sex with men. Overall, the cefixime resistance remained stable during the time. An increase of azithromycin resistance was observed until 2018 (26.5%) with a slight decrease in the last year. In 2019, gonococci showing azithromycin minimum inhibitory concentration above the EUCAST epidemiological cutoff value (ECOFF) accounted for 9.9%. Ciprofloxacin resistance and penicillinase-producing N. gonorrhoeae (PPNG) percentages increased reaching 79.1% and 18.7% in 2019, respectively. The most common sequence types identified were 5,441, 1,407, 6,360, and 5,624. The predominant genogroup (G) was the 1,407; moreover, a new genogroup G13070 was also detected. A variation in the antimicrobial resistance rates and high genetic variability were observed in this study. The main phenotypic and genotypic characteristics of N. gonorrhoeae isolates were described to monitor the spread of drug-resistant gonorrhea.

Fourier transform infrared spectroscopy; can it be used as a rapid typing method of Neisseria gonorrhoeae?

BackgroundTyping of Neisseria gonorrhoeae is necessary for epidemiologic surveillance, while time consuming and resource intensive. Fourier transform infrared (FTIR) spectroscopy has shown promising results when typing several bacterial species. This study investigates whether FTIR spectroscopy can be used as a rapid method for typing clinical N. gonorrhoeae isolates, comparing FTIR spectroscopy to multi locus sequence typing (MLST), N. gonorrhoeae multi antigen sequence typing (NG-MAST) and whole genome sequencing (WGS). MethodsSixty consecutive isolates from a venereology clinic and three isolates from an outbreak were included. Isolates were analysed with FTIR spectroscopy on the IR Biotyper system (Bruker Daltonik) with the IR Biotyper software (version 2.1) with default analysis settings (spectral range 1300–800 cm−1). Four technical replicates of each isolate were analysed in three different runs. The output was a hierarchical cluster analysis (HCA) presented as a dendrogram; a tree-like overview of how closely different isolates are related. FTIR spectroscopy was compared to MLST, NG-MAST and WGS to see if the FTIR spectroscopy-dendrogram grouped the isolates in the same clusters. ResultsFifty-one out of 60 isolates, and the three outbreak isolates, produced at least one spectrum in each run and were included. No agreement between FTIR spectroscopy and MLST or NG-MAST or WGS was shown. The FTIR spectroscopy-dendrogram failed to cluster the outbreak isolates. ConclusionFTIR spectroscopy (spectral range 1300–800 cm−1) is not yet suitable for epidemiologic typing of N. gonorrhoeae. Absence of a capsule as well as phase- and antigenic variation of carbohydrate surface structures of the gonococcal cell wall may contribute to our findings. Future studies should include analysis of a wider range of the spectrum recorded (4000-500 cm−1), and should also explore further mathematical analytic approaches of the similarity between spectra.

Emergence of high-level azithromycin-resistant Neisseria gonorrhoeae causing male urethritis in Johannesburg, South Africa, 2021.

In South Africa, Neisseria gonorrhoeae , which is the predominant cause of male urethritis, is treated syndromically using dual ceftriaxone and azithromycin therapy. We determined antimicrobial susceptibilities of N. gonorrhoeae isolates from urethral discharge specimens, and genetically characterised those with elevated minimum inhibitory concentrations (MICs) for first-line antimicrobials. Routine antimicrobial susceptibility testing (AST) of N. gonorrhoeae isolates included E-test for ceftriaxone, cefixime and gentamicin and agar dilution for azithromycin and spectinomycin. Neisseria gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR) was performed for isolates with elevated MICs to identify antimicrobial resistance (AMR) determinants, and Neisseria gonorrhoeae Multi-Antigen Sequence Typing (NG-MAST) was used to determine strain relatedness. N. gonorrhoeae was cultured from urethral discharge swab specimens obtained from 196 of 238 (82.4%) men presenting to a primary healthcare facility in Johannesburg in 2021. All viable isolates were susceptible to extended-spectrum cephalosporins. Four isolates had high azithromycin MICs ranging from 32mg/L to >256mg/L and grouped into two novel NG-MAST and NG-STAR groups. Two isolates from Group 1 (NG-MAST ST20366, NG-STAR ST4322) contained mutated mtrR (G45D) and 23S rRNA (A2059G) alleles, while the two isolates from Group 2 (NG-MAST ST20367, NG-STAR ST4323) had different mutations in mtrR (A39T) and 23S rRNA (C2611T). We report the first cases of high-level azithromycin resistance in N. gonorrhoeae from South Africa. Continued AMR surveillance is critical to detect increasing azithromycin resistance prevalence in N. gonorrhoeae , which may justify future modifications to the STI syndromic management guidelines.

Antimicrobial susceptibilities of Neisseria gonorrhoeae in Canada, 2020.

The Gonococcal Antimicrobial Surveillance Programme is a passive surveillance system that has monitored antimicrobial resistance in Neisseria gonorrhoeae in Canada since the 1980s. This article summarizes the demographics, antimicrobial resistances and NG-MAST (N. gonorrhoeae multiantigen sequence typing) for cultures collected in 2020. The National Microbiology Laboratory (NML) in Winnipeg received resistant N. gonorrhoeae cultures from provincial and territorial public health laboratories. Agar dilution was used to determine the minimum inhibitory concentrations to ten antimicrobials for all cultures received at NML, according to Clinical and Laboratory Standards Institute guidelines. The NG-MAST typing was also determined for each culture. A total of 3,130 N. gonorrhoeae cases were cultured across Canada in 2020; a 36% decrease from 2019 (n=4,859). The level of decreased susceptibility to cefixime increased significantly between 2016 and 2020 to 2.8% (p=0.0054). Decreased susceptibility to ceftriaxone declined significantly between 2016 (1.8%) and 2020 to 0.9% (p=0.001), and there was no significant change with azithromycin between 2016 (7.2%) and 2020 (6.1%). The proportion of cultures with an azithromycin minimum inhibitory concentrations of ≥1 mg/L increased significantly from 11.6% in 2016 to 15.3% in 2020 (p=0.0017). The most common NG-MAST type in Canada for 2020 was sequence type (ST)-11461, while ST-12302 was most commonly associated with azithromycin resistance and ST-16639 with cephalosporin decreased susceptibility. Antimicrobial resistance in N. gonorrhoeae remains an important public health concern and continued surveillance is imperative to monitor trends to ensure the recommended therapies will be the most effective.

Molecular characterisation of Neisseria gonorrhoeae associated with disseminated gonococcal infections in Queensland, Australia: a retrospective surveillance study

ObjectivesGonorrhoea caused by Neisseria gonorrhoeae is the second most notified sexually transmitted infection (STI) in Australia and the case numbers for this STI have been increasing globally. Progressive gonococcal infection...

High prevalence of blaTEM-135 and genetic epidemiology of blaTEM-135-carrying Neisseria gonorrhoeae isolates in Shandong, China, 2017-19.

Only one additional specific SNP in blaTEM-135 in penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates is required for TEM-135 to evolve into an ESBL that is capable of degrading all extended-spectrum cephalosporins (ESCs). Identification of the blaTEM-135 gene was achieved by performing a mismatch amplification mutation assay PCR. PPNG isolates were then genotyped using MLST and N. gonorrhoeae multiantigen sequence typing (NG-MAST) techniques. Finally, phylogenetic analyses based on MLST and NG-MAST were performed on all of the PPNG isolates, and genogroups were classified according to the phylogenetic relatedness on the basis of each MLST and NG-MAST gene typing and the blaTEM allele varieties. Among the 197 PPNG isolates, one (0.5%) isolate possessed the blaTEM-220 allele, 90 (45.7%) possessed the blaTEM-135 allele, and 106 (53.8%) possessed the blaTEM-1 allele. Among the blaTEM-135-carrying PPNG isolates, MLST ST8109 (n = 30) and NG-MAST ST12199 (n = 5), ST12754 (n = 5) and ST15073 (n = 5) were the most prevalent in the Shandong province of China. The phylogenetic analyses revealed that MLST ST8109, ST8140 and ST14417, mainly carrying the blaTEM-135 allele, belonged to the same genogroup, G1, while NG-MAST ST12199 and ST15073, mainly carrying the blaTEM-135 allele, belonged to the same genogroup, Ga. This study has shown that the molecular epidemiology of PPNG isolates carrying the blaTEM-135 allele in Shandong has a dynamically changing status. Therefore, it is very pressing to continuously monitor the prevalence and mutation of the blaTEM-135 allele and the genetic epidemiology of PPNG isolates carrying the blaTEM-135 allele in this district.

Europe-wide expansion and eradication of multidrug-resistant Neisseria gonorrhoeae lineages: a genomic surveillance study

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. 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. 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. 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. European Centre for Disease Prevention and Control, Centre for Genomic Pathogen Surveillance, Örebro University Hospital, Wellcome.

Comparative Whole-Genome Analysis of Neisseria gonorrhoeae Isolates Revealed Changes in the Gonococcal Genetic Island and Specific Genes as a Link to Antimicrobial Resistance.

Comparative whole-genome analysis was performed for Neisseria gonorrhoeae isolates belonging to the Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST) types predominant worldwide — 225, 1407, 2400, 2992, and 4186 — and to genogroup 807, the most common genogroup in the Russian Federation. Here, for the first time, the complete genomes of 25 N. gonorrhoeae isolates from genogroup 807 were obtained. For NG-MAST types 225, 1407, 2400, 2992, and 4186, genomes from the Pathogenwatch database were used. The phylogenetic network constructed for 150 genomes showed that the clustering according to NG-MAST type corresponded to the clustering according to genome. Comparisons of genomes of the six sequence types revealed 8-20 genes specific to each sequence type, including the loci for phase variations and genetic components of the gonococcal genetic island (GGI). NG-MAST type 2992 and 4186 isolates either lacked the GGI or carried critical mutations in genes essential for DNA secretion. In all analyzed genogroup 807 isolates, substitution of the essential atlA gene with the eppA gene was found, accompanied by a change in the traG allele, replacement of the ych gene with ych1, and the absence of the exp1 gene, which is likely to result in loss of GGI functionality. For the NG-MAST type 225, 1407 and 2400 isolates, no premature stop codons or reading frameshifts were found in the genes essential for GGI function. A relationship between isolate susceptibility to ciprofloxacin, penicillin, tetracycline and the presence of lesions in GGI genes necessary for DNA secretion was established. The N. gonorrhoeae evolutionary pathways, which allow a particular sequence type to maintain long-term predominance in the population, may include changes in genes responsible for adhesion and virulence, changes in the GGI structure, preservation of genes carrying drug resistance determinants, and changes in genes associated with host adaptation or encoding enzymes of biochemical pathways.

Increasing Azithromycin Resistance in Neisseria gonorrhoeae Due to NG-MAST 12302 Clonal Spread in Canada, 2015 to 2018.

ABSTRACTAzithromycin-resistant (AZIR) gonorrhea has been steadily increasing in Canada over the past decade, which is cause for alarm, as azithromycin (AZI) has been part of the combination therapy recommended by the Canadian Guidelines on Sexually Transmitted Infections (CGSTI) since 2012. Neisseria gonorrhoeae with AZI MICs ≥1 mg/L collected between 2015 and 2018 as part of the Gonococcal Antimicrobial Surveillance Program-Canada underwent antimicrobial susceptibility testing, molecular typing, and whole-genome sequencing. Regional, demographic, and clinical isolation site comparisons were made to aid in our understanding of AZI susceptibility trending. We identified 3,447 N. gonorrhoeae with AZI MICs of ≥1 mg/L in Canada, which increased from 6.3% in 2015 to 26.5% of isolates in 2018. Central Canada had the highest proportion, rising from 9.2% in 2015 to 31.2% in 2018. We identified 273 different N. gonorrhoeae multiantigen sequence types (NG-MAST) among these isolates, with ST-12302 the most prevalent (50.9%). Whole-genome sequencing identified the Neisseria lactamica-like mosaic mtr locus as the mechanism of AZIR in isolates of ST-12302 and isolates genetically similar (differing by ≤5 bp), designated the ST-12302 genogroup, accounting for 65.2% of study isolates which were originally identified in central Canada but spread to other regions by 2018. Genomic analysis indicated that AZIR in Canadian N. gonorrhoeae expanded rapidly due to clonal spread of the ST-12302 genogroup. The rapid expansion of this AZIR clonal group in all regions of Canada is of concern. CGSTI are currently under review to address the increase in AZIR in Canada.

Antimicrobial resistance in Neisseria gonorrhoeae and Mycoplasma genitalium isolates from the private healthcare sector in South Africa: A pilot study.

Reports have emerged globally of antimicrobial resistance (AMR) in Neisseria gonorrhoeae and Mycoplasma genitalium infections. In South Africa (SA), there are substantial differences between private and public healthcare with regard to antimicrobial drug prescribing practice, which could affect AMR patterns of private and public healthcare patients. To perform a pilot study to determine the frequency of AMR of N. gonorrhoeae and M. genitalium in patients accessing SA's private healthcare sector. In this cross-sectional study, N. gonorrhoeae-positive cultures and M. genitalium DNA samples were collected from a private healthcare reference laboratory from August 2018 to August 2019. In N. gonorrhoeae-positive cultures, antimicrobial susceptibility testing was performed, followed by N. gonorrhoeae multiantigen sequence typing (NG-MAST) to determine genetic relatedness of the isolates. To determine macrolide and fluoroquinolone resistance rates, M. genitalium-positive samples were analysed by sequencing the 23S rRNA, gyrA and parC genes. Twenty-one N. gonorrhoeae- and 27 M. genitalium-positive specimens were included in this analysis. High rates of resistance were detected among gonococcal isolates, with 90% resistance to tetracycline, 86% to penicillin and 62% to ciprofloxacin, but no resistance to azithromycin, cefixime and ceftriaxone. NG-MAST revealed genetically diverse isolates with 83% novel NG-MAST sequence types. Macrolide and fluoroquinolone resistance-associated mutations were detected in 18.5% (n=5/27) and 7.4% (n=2/27) of M. genitalium strains, respectively. We observed high frequencies of ciprofloxacin, penicillin and tetracycline resistance in N. gonorrhoeae and macrolide resistance-associated mutations in M. genitalium in private healthcare sector patients in SA. This finding highlights the need to use diagnostics for sexually transmitted infections and to include the private healthcare sector in antimicrobial surveillance and stewardship programmes.

Large Increase in Azithromycin-Resistant Neisseria gonorrhoeae in Northern Spain

The aim of this study was to characterize the evolution of gonorrhea in the general population by correlating epidemiological, genotypic, and antimicrobial resistance data of Neisseria gonorrhoeae isolates collected in northern Spain from 2014 to 2018. One hundred ninety-four strains underwent antimicrobial susceptibility testing and were genetically analyzed by N. gonorrhoeae multiantigen sequence typing. Increasing cases of gonococcal infections have been observed after 2015. Most occurred in male with urethritis. Sequence type (ST)-9972 and ST-1576, the predominant genotypes identified, have not been previously described as epidemic clones. Of great concern was the significant increase in azithromycin-resistant N. gonorrhoeae. More than 30% of these isolates were obtained from men who have sex with men (MSM). ST-12302 was the most prevalent clone among the azithromycin-resistant strains, and was also resistant to penicillin, ciprofloxacin, and tetracycline. This multidrug-resistant clone was exclusively isolated from MSM during 2018. The incidence rates of gonorrhea and azithromycin-resistant N. gonorrhoeae have significantly increased due to the emergence of new clones. ST-12302 has recently been recognized as an epidemic clone; therefore, its surveillance could be the key in controlling further dissemination of azithromycin resistance. These data highlight the need to perform local studies to update treatment guidelines and reinforce preventive measures against gonorrhea.

Molecular characteristics and antimicrobial susceptibility of penicillinase-producing Neisseria gonorrhoeae isolates in Fukuoka, Japan, 1996–2018

The objective of this study was to investigate the molecular characteristics and antimicrobial susceptibility of penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates collected in Fukuoka, Japan, from 1996-2018. Antimicrobial susceptibility to seven antibiotics was determined by the agar dilution method. Molecular characteristics were determined by Sanger sequencing of the blaTEM allele, plasmid typing and N. gonorrhoeae multiantigen sequence typing (NG-MAST). Furthermore, full sequences of the penA gene, encoding penicillin-binding protein 2 (PBP2), of PPNG isolates with reduced susceptibility to cefixime were analysed. Among 50 PPNG isolates, 17 and 33 were collected during 1996-2006 and 2007-2018, respectively. In 1996-2006, blaTEM-1 in African plasmid was most frequent (64.7%), followed by blaTEM-1 in Asian plasmid (29.4%) and blaTEM-135 in Toronto/Rio plasmid (5.9%). In 2007-2018, blaTEM-135 in Toronto/Rio plasmid was predominant (54.5%), followed by blaTEM-1 in African plasmid (36.4%) and blaTEM-135 in Asian plasmid (6.1%). Among isolates with the blaTEM-135-carrying Toronto/Rio plasmid in 2007-2018, a novel genogroup G15576 was predominant (66.7%). Isolates with the TEM-135 β-lactamase were more resistant to ciprofloxacin but were more susceptible to ceftriaxone and tetracycline than isolates with TEM-1. Seven PPNG isolates less susceptible to cefixime possessed the plasmidic blaTEM-1 allele and had mosaic or non-mosaic alterations within PBP2. The proportion of PPNG with the blaTEM135-carrying Toronto/Rio plasmid increased during the last 12 years. The increase in PPNG carrying the blaTEM-135 allele is of particular concern as it is considered a possible direct precursor of an extended-spectrum β-lactamase (ESBL).

Genomic surveillance of Neisseria gonorrhoeae in the Philippines, 2013-2014.

Antimicrobial-resistant Neisseria gonorrhoeae is a major threat to public health and is of particular concern in the Western Pacific Region, where the incidence of gonorrhoea is high. The Antimicrobial Resistance Surveillance Program (ARSP) has been capturing information on resistant gonorrhoea since 1996, but genomic epidemiology studies on this pathogen are lacking in the Philippines.We sequenced the whole genomes of 21 N. gonorrhoeae isolates collected in 2013–2014 by ARSP. The multilocus sequence type, multiantigen sequence type, presence of determinants of antimicrobial resistance and relatedness among the isolates were all derived from the sequence data. The concordance between phenotypic and genotypic resistance was also determined.Ten of 21 isolates were resistant to penicillin, ciprofloxacin and tetracycline, due mainly to the presence of the blaTEM gene, the S91F mutation in the gyrA gene and the tetM gene, respectively. None of the isolates was resistant to ceftriaxone or cefixime. The concordance between phenotypic and genotypic resistance was 92.38% overall for five antibiotics in four classes. Despite the small number of isolates studied, they were genetically diverse, as shown by the sequence types, the N. gonorrhoeae multiantigen sequence typing types and the tree. Comparison with global genomesplaced the Philippine genomes within global lineage A and led to the identification of an international transmission route.This first genomic survey of N. gonorrhoeae isolates collected by ARSP will be used to contextualize prospective surveillance. It highlights the importance of genomic surveillance in the Western Pacific and other endemic regions for understanding the spread of drug-resistant gonorrhoea worldwide.