Neisseria Gonorrhoeae Multi-antigen Sequence Typing Research Articles

Prevalence and Molecular Characterization of Pharyngeal Gonorrhea in Korean Men With Urethritis.

Pharyngeal infection is more difficult to diagnose and treat than genital or rectal infection and can act as a reservoir for gonococcal infection. We determined the prevalence of pharyngeal gonorrhea in Korean men with urethritis and analyzed the molecular characteristics and antimicrobial susceptibility of the isolates. Seventy-two male patients with symptoms of urethritis who visited a urology clinic in Wonju, Korea, between September 2016 and March 2018 were included. Urethral and pharyngeal gonococcal cultures, antimicrobial susceptibility testing, Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST), and multiplex real-time PCR (mRT-PCR) were performed. Among the 72 patients, 59 tested positive for gonococcus by mRT-PCR. Of these 59 patients, 18 (30.5%) tested positive in both the pharynx and urethra, whereas 41 tested positive only in the urethra. NG-MAST was feasible in 16 out of 18 patients and revealed that 14 patients had the same sequence types in both urethral and pharyngeal specimens, whereas two patients exhibited different sequence types between the urethra and pharynx. Of the 72 patients, 33 tested culture-positive. All patients tested positive only in urethral specimens, except for one patient who tested positive in both. All culture-positive specimens also tested positive by mRT-PCR. All isolates were susceptible to azithromycin and spectinomycin, but resistance rates to ceftriaxone and cefixime were 2.9% and 14.7%, respectively. The prevalence of pharyngeal gonorrhea in Korean men with gonococcal urethritis is as high as 30.5%, highlighting the need for pharyngeal screening in high-risk groups. Ceftriaxone is the recommended treatment for pharyngeal gonorrhea.

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.

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.

Antibiotic susceptibility and genotypic characterization of Neisseria gonorrhoeae isolates in the Comunidad Valenciana (Spain): GONOvig project

IntroductionThe increase in sexually transmitted infections (STI) caused by Neisseria gonorrhoeae (NG) worldwide, together with the decrease in antibiotic susceptibility, forced us to understand the epidemiology of gonococcal infection. MethodsThe GONOvig project analyzed, comparatively following CLSI and EUCAST criteria, the antibiotic susceptibility of 227 NG strains collected in thirteen representative hospitals of the Valencia Community (CV) between 2013 and 2018. Additionally, molecular typing of 175 strains using the NG multi-antigen sequence typing technique (NG-MAST) was performed. ResultsHigh rates of resistance to tetracycline (38.2% by CLSI and 50.9% by EUCAST) and ciprofloxacin (49.1% CLSI and 54% EUCAST), and low percentages of resistance to spectinomycin (0%), cefixime (0.5% CLSI but 5.9% EUCAST), and ceftriaxone (1.5% CLSI and 2.4% EUCAST) were detected. Azithromycin resistance was 6% (both CLSI and EUCAST). Molecular analysis revealed the presence of 86 different sequence types (ST), highlighting ST2992 (7.4%), ST3378 (6.9%), ST2400 (4.6%) and ST13288 (6.9%), which was associated with resistance to cefixime (P=.031). The main genogroups (G) were G1407 (13.1%), G2992 (10.3%), G2400 (6.3%) and G387 (3.4%). G1407 and G2400 were associated with resistance to ciprofloxacin (P<.03). ConclusionLow resistance to ceftriaxone, a worrying resistance to azithromycin and a wide variety of circulating sequence types have been detected, some of which show correlation with certain resistance profiles.

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.

The changing epidemiology of Neisseria gonorrhoeae genogroups and antimicrobial resistance in Queensland, Australia, 2010-15: a case series analysis of unique Neisseria gonorrhoeae isolates.

Neisseria gonorrhoeae (NG) can lead to serious reproductive and sexual health outcomes, and the annual number of NG notifications in Australia increased steadily from 10329 in 2010 to 29549 by 2020. Australian populations most affected are urban men who have sex with men and First Nations peoples living in remote areas, and a resurgence in urban heterosexuals has been observed since 2012. A case series analysis of Queensland NG isolates (2010-15) exploring temporal trends and antimicrobial resistance by demographic and geographic distribution and genotype was performed. Proportions describe age, sex, strain, genogroup (NG multi-antigen sequence typing), region, swab site, antimicrobial sensitivity and isolate rates per 100000 population. Dominant genogroups were identified. Among 3953 isolates, the median age was 25years (IQR 20-34years) and most (n =2871/3915, 73%) were men. Brisbane city (68.8) and Far North Queensland (54.1) excluding Cairns showed the highest rates. Forty-six genogroups were documented, seven (G2992, G6876, G1415, G4186, G5, G1407 and G6937) comprised half of all isolates. The predominant male genogroup was G2992 (16%), and G6876 (20%) for females; G5 was predominantly male from 2010 to 2011, but equal in both sexes from 2012 to 2015. Considerable temporal, geographical and demographical diversity was observed in Queensland NG isolates, which has public health implications. Certain genogroups are more transient than others, and evidence suggests bridging from male-dominant networks to heterosexual networks. Molecular surveillance can enhance tracking the epidemiology and movement of NG in Australia, highlighting the necessity of genotyping to expose potentially prevalent strains circulating in undetected or underrepresented networks by current screening methods.

Biochemical Atypia in Russian Neisseria gonorrhoeae Clinical Isolates Belonging to the G807 NG-MAST Genogroup/ST1594 MLST.

Many current gonococcal clinical isolates in Russia show atypical taxonomically significant biochemical activity, which leads to species misidentification. Molecular typing of such cultures according Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST) and multilocus sequence typing (MLST) protocols assigned them to the G807 NG-MAST GENOGROUP/ST1594 MLST that has been predominant in Russia in recent years. The goal of the study was to analyze the molecular mechanisms of biochemical atypia in N. gonorrhoeae clinical isolates characterized as the members of G807 NG-MAST GENOGROUP/ST1594 MLST. Sixteen isolates of this genogroup were included in the study, eight showed defective amino acid metabolism or loss of D-glucose fermentation. Comparative bioinformatic analysis based on WGS data divided these isolates into two clusters strictly associated with typical or atypical biochemical activity. Cultures with defective amino acid metabolism had a 5-nucleotide insertion in the pip-gene that caused a stop codon and led to synthesis of the non-functional enzyme. Comparison of the sequenced genomes with publicly available N. gonorrhoeae genomes showed the rarity of this insertion. In the global N. gonorrhoeae phylogenetic tree the G807 NG-MAST GENOGROUP/ST1594 MLST forms a distinct branch characterized by 170 SNPs, most of which are non-synonymous. We hypothesized a unique strategy for G807 NG-MAST GENOGROUP/ST1594 MLST clone persistence in the global N. gonorrhoeae population via escape of antimicrobial therapy due to diagnostic misidentification.

Culture-Independent Genotyping Improves Surveillance of Neisseria gonorrhoeae, Especially in Oropharyngeal Samples, the Netherlands, 2017 to 2018.

It is important i to monitor the transmission and antimicrobial resistance of Neisseria gonorrhoeae (NG). Current surveillance relies on culturing, which frequently fails. Previously, a culture-independent genotyping method was developed based on NG multi-antigen sequence typing (NG-MAST). To determine whether crucial sequence types (STs) are missed during culture-dependent surveillance, NG-positive NAAT samples were genotyped, and the results of the culture-positive and culture-negative samples were compared. In total, 196 NG-positive NAAT samples, from January 2017 until August 2018, which were also routinely cultured, were retrospectively included. Genotyping was successful in 152 NAAT samples (77.0%), 33 NAAT samples failed, and 11 NAAT samples showed possible mixed strain infections. Oropharyngeal samples (n = 16) showed the largest increase in typing rate from 6.3% (1/16) success in culture-dependent genotyping to 81.3% (13/16) in culture-independent genotyping. Nine genogroups (n ≥ 5 samples) were found; all included both culture-positive and culture-negative NG. However, culture-independent surveillance revealed 14 additional STs in the culture-negative samples. Overall, culture-dependent surveillance could detect all genogroups, indicating that major trends could be identified with culture-dependent surveillance. However, culture-independent surveillance provides more STs, mixed infections and more oropharyngeal samples, giving a more detailed view and could result in an earlier detection of outbreaks and transmission.

Susceptibilidad antibiótica y caracterización genotípica de aislados de Neisseria gonorrhoeae circulantes en la Comunidad Valenciana (España): proyecto GONOvig

IntroductionThe increase in sexually transmitted infections (STI) caused by Neisseria gonorrhoeae (NG) worldwide, together with the decrease in antibiotic susceptibility, forced us to understand the epidemiology of gonococcal infection. MethodsThe GONOvig project analyzed, comparatively following CLSI and EUCAST criteria, the antibiotic susceptibility of 227 NG strains collected in thirteen representative hospitals of the Valencia Community (CV) between 2013 and 2018. Additionally, molecular typing of 175 strains using the NG multi-antigen sequence typing technique (NG-MAST) was performed. ResultsHigh rates of resistance to tetracycline (38.2% by CLSI and 50.9% by EUCAST) and ciprofloxacin (49.1% CLSI and 54% EUCAST), and low percentages of resistance to spectinomycin (0%), cefixime (0.5% CLSI but 5.9% EUCAST), and ceftriaxone (1.5% CLSI and 2.4% EUCAST) were detected. Azithromycin resistance was 6% (both CLSI and EUCAST). Molecular analysis revealed the presence of 86 different sequence types (ST), highlighting ST2992 (7.4%), ST3378 (6.9%), ST2400 (4.6%) and ST13288 (6.9%), which was associated with resistance to cefixime (p = 0.031). The main genogroups (G) were G1407 (13.1%), G2992 (10.3%), G2400 (6.3%) and G387 (3.4%). G1407 and G2400 were associated with resistance to ciprofloxacin (p < 0.03). ConclusiónLow resistance to ceftriaxone, a worrying resistance to azithromycin and a wide variety of circulating sequence types have been detected, some of which show correlation with certain resistance profiles.

Disseminated Gonococcal Infections in Manitoba, Canada: 2013 to 2020.

Gonorrhea, when left untreated, can enter the blood and cause disseminated gonococcal infections (DGIs). Disseminated gonococcal infections, which can include dermatitis, tenosynovitis, migratory polyarthralgia, and arthritis, have been increasing in Manitoba (MB), Canada, since 2013. Endocarditis, a rare DGI, was identified in 3 MB patients in 2018 and 2019. Antimicrobial resistance, molecular types, and resistance-associated mutations were determined for MB DGI isolates (n = 103) identified from 2013 to 2020 using phenotypic and genotypic methods. Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST) of residual nucleic acid amplification testing samples (n = 13) from 2019 and 2020 were also determined. The increase in DGI in MB in 2019 and 2020 was due to the NG-MAST 11508 molecular type with porB -2206, a persistent PorB protein structure type "A" allele. These isolates had low-level resistance to erythromycin and tetracycline. Molecular surveillance of gonorrhea and, in particular, gonococcal strains resulting in DGI is imperative to monitor clonal transmission within populations. These data can be used to alert public health of emerging issues and support public health interventions.

Molecular characteristics of cephalosporins resistant Neisseria gonorrhoeae strains isolated from Gonococcal Antimicrobial Resistance Surveillance Program in China, 2015-2016

Abstract Background: An increasing level of antimicrobial resistance to extended-spectrum cephalosporins (ESCs), has been reported by the China Gonococcal Antimicrobial Resistance Surveillance Program (China-GRSP). Methods: 2038 Neisseria gonorrhoeae (NG) isolates were collected from China-GRSP between 2015 and 2016. NG multiantigen sequence typing (NG-MAST), MLST (multi-locus sequence typing) and NG sequence typing for antimicrobial resistance (NG-STAR) were used to determine characteristics and resistant determinants of cephalosporins resistant isolates. Results: 74 (3.6%) cephalosporins resistant isolates were confirmed. MLSTST7363 was the most prevalent MLST among cefixime resistant (CFX-R) isolates, while MLSTST7365 (n = 8) in ceftriaxone resistant (CRO-R) isolates. And CFX-R strains mainly associated with penA-X(n = 27,44.3%), penA-XIII (n = 6,9.8%), penA-XVIII (n = 5, 8.2%), and CRO-R strains mainly related to penA-XVIII (n = 10,25.6%), penA-43(n = 9,23.1%), penA-XIII(n = 7,17.9%). Conclusion: The main conclusion of this study is that the predominant cephalosporin-resistant clones in China include locally emerged and imported linages. The CFX-R and CRO-R strains are primarily related to penA mutations and their predominant genotypes are different. With the increase of international travel, tracking high-risk resistant clones and identifying outbreaks and their association with epidemiological characteristics are critical to improve public health.

Antimicrobial susceptibility testing and molecular characterization of Neisseria gonorrhoeae in Tehran, Iran.

Gonorrhea is a sexually transmitted infection occurring worldwide. Antimicrobial resistance (AMR) surveillance in Neisseria gonorrhoeae and associated molecular epidemiological studies are crucial to ascertain the spread of antibiotic-resistant and developing the local treatment guidelines. This study was performed to determine the antimicrobial susceptibility testing (AST) and molecular epidemiology of N. gonorrhoeae isolates in Tehran, Iran. During 1 July 2018-30 July 2020, a total of 500 urogenital (468 endocervical, 32 urethral) swabs were collected from patients with signs and symptoms of genitourinary infections presenting to two women's hospitals and one health center located center and south of Tehran. Specimens were cultured and examined for the presence of N. gonorrhoeae isolates by biochemical tests. MIC Test Strip determined the MICs of ceftriaxone, azithromycin, and ciprofloxacin. Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST) was also performed. A total of 38N. gonorrhoeae isolates were identified. The proportions of resistant N. gonorrhoeae isolates were as follows: ceftriaxone (MIC ≥0.125μg/mL) 10.5% (4/38), azithromycin (MIC >1μg/mL) 34% (13/38), and ciprofloxacin (MIC ≥1μg/mL) 31.5% (12/38). In total, 25 different NG-MAST STs were identified. The STs comprised 1-4 isolates each, and the predominant ST was ST266 (n = 4). Our study demonstrates a diverse gonococcal population with high rates of resistance to azithromycin and evidence of resistance to ceftriaxone. The results have potential implications for antibiotic choice for the gonococcal treatment and highlight the need to broaden gonococcal AMR monitoring in Iran.

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.

Emergence of a Neisseria gonorrhoeae clone with reduced cephalosporin susceptibility between 2014 and 2019 in Amsterdam, The Netherlands, revealed by genomic population analysis.

BackgroundEmerging resistance to cephalosporins in Neisseria gonorrhoeae (Ng) is a major public health threat, since these are considered antibiotics of last resort. Continuous surveillance is needed to monitor the circulation of resistant strains and those with reduced susceptibility.ObjectivesFor the purpose of epidemiological surveillance, genomic population analysis was performed on Ng isolates from Amsterdam with a focus on isolates with reduced susceptibility to ceftriaxone.MethodsWGS data were obtained from 318 isolates from Amsterdam, the Netherlands between 2014 and 2019. Isolates were typed according to MLST, Ng Multi-Antigen Sequence Typing (NG-MAST) and Ng Sequence Typing for Antimicrobial Resistance (NG-STAR) schemes and additional resistance markers were identified. Phylogenetic trees were created to identify genetic clusters and to compare Dutch and non-Dutch MLST7827 isolates.ResultsMLST7363 and MLST1901 were the predominant strains having reduced susceptibility to ceftriaxone during 2014–16; MLST7827 emerged and dominated during 2017–19. NG-STAR38 and NG-MAST2318/10386 were predominant among MLST7827 isolates. MLST7827 reduced susceptibility isolates carried a non-mosaic 13.001 penA allele with an A501V mutation and porB1b G120K/A121D mutations, which were lacking in susceptible MLST7827 isolates. Phylogenetic analysis of all publicly available MLST7827 isolates showed strong genetic clustering of Dutch and other European MLST7827 isolates.ConclusionsMLST7827 isolates with reduced ceftriaxone susceptibility have emerged during recent years in Amsterdam. Co-occurrence of penA A501V and porB1b G120K/A121D mutations was strongly associated with reduced susceptibility to ceftriaxone. Genetic clustering of Dutch and other European MLST7827 isolates indicates extensive circulation of this strain in Europe. Close monitoring of the spread of this strain having an alarming susceptibility profile is needed.

The Mosaic mtr Locus as Major Genetic Determinant of Azithromycin Resistance of Neisseria gonorrhoeae-Germany, 2018.

Within the German Gonococcal Resistance Network's (GORENET) Neisseria gonorrhoeae (NG) sample collection, azithromycin-resistant NG isolates increased from 4.3% in 2016 to 9.2% in 2018. We aim to understand this observed increase using whole genome sequencing of NG isolates combined with epidemiological and clinical data. Reduced susceptibility to azithromycin in 2018 was predominately clonal (NG multiantigen sequence typing G12302) and could mainly be attributed to the recently described mosaic-like mtr locus. Our data suggest that, together with horizontal gene transfer of resistance determinants and well-established point mutations, international spread of resistant lineages plays a major role regarding azithromycin resistance in Germany.

Culture-Independent Genotyping Revealed 3 Strain Clusters in a Potential Neisseria gonorrhoeae Outbreak in Young Heterosexuals (

Investigation was undertaken to determine the genetic relatedness of Neisseria gonorrhoeae (NG) isolates of young (<25 years) heterosexuals of a potential outbreak from October 2017 to March 2019 in South-Limburg, the Netherlands. Data and residual sample material of routine diagnostics were retrieved for outbreak cases (78/81), young heterosexuals at baseline (January 2016 to September 2017, n = 30), and men who have sex with men (2018, n = 47). Total DNA was isolated, and NG was genotyped using culture-free NG multiantigen sequence typing. Sanger sequence data were used to construct a phylogenetic tree. Cases of outbreak clusters were geographically mapped, and descriptive analyses were performed on patient characteristics, comparing these clusters. Outbreak investigation showed 81 cases of young heterosexuals between October 2017 and March 2019 (4.5 per month) compared with 30 between January 2016 and September 2017 (1.4 per month), which was considered as baseline. Culture-independent genotyping of NG was performed to assess the genetic relatedness, as only 21 outbreak cases were culture confirmed. This revealed 3 independent outbreak clusters G2 (n = 18), G13113 (n = 11), and GNewST (n = 24). None of the clusters were geographically linked or introduced by bridging with men who have sex with men networks. Number of sex partners reported by men and Chlamydia trachomatis coinfection were associated with clusters G2 and GNewST, respectively. Culture-independent typing proved to be essential to identify the 3 outbreak clusters. However, targeted interventions were difficult because information on sex partners was limited. Therefore, prospective culture-independent typing could be used for early outbreak detection and aid in transmission prevention.

Emergence and dissemination of three mild outbreaks of Neisseria gonorrhoeae with high-level resistance to azithromycin in Barcelona, 2016-18.

Neisseria gonorrhoeae (NG) isolates with high-level azithromycin resistance (HL-AziR) have emerged worldwide in recent decades, threatening the sustainability of current dual-antimicrobial therapy. This study aimed to characterize the first 16 NG isolates with HL-AziR in Barcelona between 2016 and 2018. WGS was used to identify the mechanisms of antimicrobial resistance, to establish the MLST ST, NG multiantigen sequence typing (NG-MAST) ST and NG sequence typing for antimicrobial resistance (NG-STAR) ST and to identify the clonal relatedness of the isolates with other closely related NG previously described in other countries based on a whole-genome SNP analysis approach. The sociodemographic characteristics of the patients included in the study were collected by comprehensive review of their medical records. Twelve out of 16 HL-AziR isolates belonged to the MLST ST7823/NG-MAST ST5309 genotype and 4 to MLST ST9363/NG-MAST ST3935. All presented the A2059G mutation in all four alleles of the 23S rRNA gene. MLST ST7823/NG-MAST ST5309 isolates were only identified in men who have sex with women and MLST ST9363/NG-MAST ST3935 were found in MSM. Phylogenomic analysis revealed the presence of three transmission clusters of three different NG strains independently associated with sexual behaviour. Our findings support the first appearance of three mild outbreaks of NG with HL-AziR in Spain. These results highlight the continuous capacity of NG to develop antimicrobial resistance and spread among sexual networks. The enhanced resolution of WGS provides valuable information for outbreak investigation, complementing the implementation of public health measures focused on the prevention and dissemination of MDR NG.

Molecular Typing of Neisseria gonorrhoeae Clinical Isolates in Russia, 2018-2019: A Link Between penA Alleles and NG-MAST Types.

This work aimed to study penA gene polymorphisms in clinical isolates of Neisseria gonorrhoeae collected in Russia in 2018–2019 and the contribution of the penA allele type to susceptibility to β-lactam antibiotics. A total of 182 isolates were analyzed. penA allele types were determined by sequencing, and the minimum inhibitory concentrations (MICs) of benzylpenicillin and ceftriaxone were measured. The influence of genetic factors on MICs was evaluated by regression analysis. All isolates were susceptible to ceftriaxone, and 40.1% of isolates were susceptible to penicillin. Eleven penA allele types were identified. The mosaic type XXXIV penA allele and the Gly120Lys substitution in PorB made the greatest contributions to increasing the ceftriaxone MIC; the presence of the blaTEM plasmid, Gly120Asp, Ala121Gly/Asn substitutions in PorB, and the adenine deletion in the promoter region of the mtrR gene caused an increase in the penicillin MIC. Among 61 NG-MAST types identified, the most frequent were types 228, 807, 9486, 1993, and 6226. A link between penA alleles and Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) types was established. Resistance to two groups of β-lactam antibiotics was associated with non-identical changes in penA alleles. To prevent the emergence of ceftriaxone resistance in Russia, NG-MAST genotyping must be supplemented with penA allele analysis.

Comparative phylogenetic analysis of Neisseria gonorrhoeae clinical isolates in Russia, European Union, and Japan

Surveillance of multidrug-resistant infections is a priority task for contemporary epidemiology. The aim of this study was to genotype modern clinical isolates of N. gonorrhoeae using the NG-MAST technique (Neisseria gonorrhoeae multi-antigen sequence typing) and to compare the phylogeny of the gonococcal pathogens coming from Russia, European Union and Japan. We studied a total of 822 isolates collected in Russia from 2013 through 2018. We also used NG-MAST data from the following databases: PathogenWatch (European Union, 1,071 isolates) and PubMLST (Japan, 206 isolates). Russian isolates represented 301 different NG-MAST types. The most common were types 807, 228, 1993, 5714, and 9476 (8.3%, 3.3%, 3.2%, 3.2%, and 2.7%, respectively). There were only 3 isolates (0.4%) from Russia that represented the epidemiologically significant sequence type 1407 prevailing in many countries and characterized by multiple determinants of antimicrobial resistance. A phylogenetic tree for the NG-MAST types found in Russia and European countries was constructed. The cluster analysis of the proportion of isolates belonging to unique sequence types and the country population size allowed us to identify 2 clusters (significance level — 0.01): the first cluster included Russia and Japan, the second, European countries. A distribution pattern was identified for unique sequence types: the greater is the population size, the higher is their proportion. The phylogenetic analysis demonstrated a genetic distance between the most common Russian, European and Japanese sequence types, suggesting that the Russian population of N. gonorrhoeae has been evolving relatively locally.

Epidemiological surveillance study of gonococcal infection in Northern Spain

BackgroundTreatment of gonorrhoea is threatened by antimicrobial resistance, and decreased susceptibility to recommended therapies is emerging. Thus, gonococcal infection (GI) is becoming a public health problem. The objectives of the present study were to monitor the antimicrobial sensitivity in Neisseria gonorrhoeae (NG) during 2011–2015 and to study their genogroups. MethodsAntimicrobial susceptibility was studied by disc diffusion, in addition to the agar dilution method for cefixime and ceftriaxone and the Etest® for azithromycin. Genotyping was performed by the NG multi-antigen sequence typing (NG-MAST) method. Genogroups of closely related sequence types (STs) were defined. ResultsAll the strains were susceptible to cefixime, ceftriaxone and gentamicin and 1.8% of the strains were resistant to azithromycin. A total of 531 STs and 6 genotypes (Gs) were identified during 2012–2015 period. G2992 was the largest and was associated with resistance to azithromycin, and with men who have sex with men (MSM), alongside G2400. G1407 and G2400 strains were related to high minimum inhibitory concentration (MICs) to cefixime and G1407 also to ceftriaxone. For the first time, G1861 and G2018 were described and associated with ciprofloxacin resistance and G2018 also with high MICs to ceftriaxone. ConclusionMolecular typing is a useful tool to predict antimicrobial resistance. These results show the need to develop novel antimicrobials or to design new antimicrobial therapies based on drugs that show their efficacy against GI. This also highlights the importance of developing sexually transmitted infection (STI) surveillance in homosexual populations.