Gonorrhoeae Multi-antigen Sequence Typing Research Articles

Culture-free genotyping of Neisseria gonorrhoeae revealed distinct strains at different anatomical sites in a quarter of patients, the Netherlands, 2012 to 2016.

BackgroundGenotyping of Neisseria gonorrhoeae (NG) is essential for surveillance to monitor NG transmission and dissemination of resistant strains. Current genotyping methods rely on bacterial culture which frequently fails.AimOur aim was to develop a culture-free genotyping method that is compatible with the widely used N. gonorrhoeae multi-antigen sequence typing (NG-MAST) database, which facilitates genotyping of NG detected at separate anatomical sites in individual patients.MethodsSpecific primers for both PCR targets porB and tbpB were designed and technically validated by assessing the analytical sensitivity, cross-reactivity with 32 non-gonoccocal Neisseria species, and concordance with NG-MAST. Clinical application was assessed on 205 paired samples from concurrent NG infections at different anatomical sites of 98 patients (81 men who have sex with men and 17 women) visiting our sexually transmitted infections clinic.ResultsTyping could be consistently performed on samples with a PCR quantification cycle (Cq) value <35. Furthermore, the method showed no cross-reactivity and was concordant with NG-MAST. Culture-free NG-MAST improved the typing rate from 62% (59/95) for cultured samples to 94% (89/95) compared with culture-dependent NG-MAST. Paired samples of 80 of 98 patients were genotyped, revealing distinct NG strains in separate anatomical sites in 25% (20/80) of the patients.ConclusionsThis NG-specific genotyping method can improve NG surveillance as it facilitates genotyping of non-culturable and extra-genital samples. Furthermore, 25% of patients were infected with multiple NG strains, which is missed in current culture-dependent surveillance. Including non-culturable and concurrent NG infections in surveillance informs actions on dissemination of multidrug-resistant NG strains.

1489. Trends in Antimicrobial Resistance in N. gonorrhoeae Isolated in Korea During 2015–2017

BackgroundThe trends of antimicrobial resistance (AMR) and the molecular epidemiology of gonococcal strains were analyzed in Korean during 2015 to 2017.MethodsThe susceptibility tests of 187 N. gonorrhoeae isolates were investigated. The minimum inhibitory concentration (MIC) of N. gonorrhoeae isolates were determined by the agar dilution method. Penicillinase-producing N. gonorrhoeae (PPNG) and plasmid-mediated tetracycline-resistant N. gonorrhoeae (TRNG) were evaluated. Isolates were tested for mutations in penA and also genotyped using N. gonorrhoeae multi-antigen sequence typing (NG-MAST) to determine the molecular epidemiological characteristics of the isolates.ResultsThe rates of resistance to ceftriaxone (CRO) increased from 6.5% in 2015 to 13.4% in 2017. Azithromycin resistance was documented only in 1.1%, but 53.5% of isolates showed intermediate susceptibility. All isolates were susceptible to spectinomycin. However, none of isolates was susceptible to ciprofloxacin and penicillin (PEN) and 4.3% of isolates were susceptible to tetracycline. The PPNG and TRNG increased from 25.7% and 39.4% in 2015 to 47.8% and 81.5% in 2017, respectively. 74.7% of the extended-spectrum cephalosporins (ESCs)-resistant isolates contained the mosaic PBP2 X allele, which has been previously associated with ESC resistance including treatment failures. penA mosaic alleles X were found in 83.8% of all isolates that harbored decreased susceptibility to cefixime. Among the 16 N. gonorrhoeae isolates with decreased susceptibility to CRO, penA mosaic X allele was identified in the 11 isolates. The 189 N. gonorrhoeae isolates were assigned to 112 different NG-MASTs; seven sequence types (STs) were novel. The most common ST was 10,668 (16.1%), followed by ST15024 (7.1%). Two ST1407 strains have been isolated in 2015. Although ST1407 was known to display decreased susceptibility to ESCs or full resistance, they were susceptible for CRO (MIC = 0.06 μg/mL) and cefixime (MIC = 0.12 μg/mL).ConclusionThe recent emergence of ESC-resistant N. gonorrhoeae strains which was often associated with mutations in the penA gene is a major concern and enhanced AMR surveillance is necessary to prevent transmission of these strains.Disclosures All authors: No reported disclosures.

Increasing prevalence of Neisseria gonorrhoeae with decreased susceptibility to ceftriaxone and resistance to azithromycin in Hangzhou, China (2015-17).

Development of resistance in Neisseria gonorrhoeae to ceftriaxone monotherapy or ceftriaxone plus azithromycin dual therapy is a global public health concern. The aim of this study was to analyse the trend in antimicrobial resistance in Hangzhou, China, over the period 2015-17. In total, 379 clinical isolates were collected from seven hospitals and antimicrobial susceptibility was determined using the agar dilution method. Isolates showing resistance to ceftriaxone, azithromycin or cefixime were analysed for the presence of resistance determinants. STs were determined with the N. gonorrhoeae multiantigen sequence typing (NG-MAST) method and phylogenetic analysis and strain clustering was determined using porB and tbpB sequences. Ceftriaxone resistance, decreased susceptibility to ceftriaxone and azithromycin resistance were observed in 3%, 17% and 21% of the isolates, respectively. This resulted in 5% of the isolates showing both decreased susceptibility to ceftriaxone and azithromycin resistance. Importantly, resistance levels to ceftriaxone and azithromycin increased over the study period, resulting in 5% ceftriaxone resistance, 27% decreased susceptibility to ceftriaxone and 35% azithromycin resistance in 2017 and 11% of the isolates showing both decreased susceptibility to ceftriaxone and azithromycin resistance. Phylogenetic and cluster analysis showed the emergence and expansion in 2017 of a clonally related cluster containing strains with high abundance of decreased susceptibility to ceftriaxone and/or cefixime, which was related to the presence of the mosaic penA allele X. Co-resistance to azithromycin was also observed in this cluster. Our findings have major implications for the future reliability of ceftriaxone monotherapy and ceftriaxone plus azithromycin dual therapy in China.

Association of Neisseria gonorrhoeae genogroups and specific PBP2/MtrR/PorB mutation patterns with susceptibility to penicillin in a susceptible gonococcal population.

To ascertain whether the antimicrobial susceptibility of Neisseria gonorrhoeae isolates with differing susceptibilities to penicillin is associated with genogroups (GGs) and combined mutation patterns in PBP2 (penA), the multiple transfer resistance repressor (MtrR; mtrR) and porin B (PorB; porB). The susceptibility of 146 clinical N. gonorrhoeae isolates to penicillin was determined using the agar dilution method and the interpretation criteria of CLSI. The DNA sequences of penA, mtrR and porB in isolates were compared with WT sequences and mutation patterns were determined. Isolates were typed by N. gonorrhoeae multi-antigen sequence typing (NG-MAST) and STs were grouped into specific GGs. The isolates tested carried 9 mutation patterns in PBP2 and 12 mutation patterns in each of MtrR and PorB. Of the 146 isolates, 121 (82.9%) were grouped into 13 different GGs. Isolates with penicillin MICs of 0.03-0.06 mg/L were significantly associated with GG25 (P < 0.05) and PBP2/MtrR/PorB mutation pattern I/WT/WT (P < 0.01). Isolates with a penicillin MIC of 1.0 mg/L were associated (P < 0.05) with: (i) GG3655 and mutation pattern XXII/A-;G45D/G120K;A121N; (ii) GG921 and mutation pattern IX/G45D/G120D;A121N; and (iii) GG1109 and mutation pattern IX/G45D/WT. Sixty percent (9/15) of penicillin-resistant isolates (MIC ≥2 mg/L) were GG3654 (P < 0.0001) and carried mutation pattern IX/G45D/G120K;A121D or IX/G45D/G120D;A121D (P < 0.05). Specific mutation patterns in PBP2/MtrR/PorB were associated with specific GGs and penicillin susceptibility. This approach of typing strains and resistance patterns is ideal for predicting antimicrobial resistance and should be used in instances in which gonococcal culture is not available but DNA can be obtained from clinical specimens.

Genotype Diversity in the Local Arkhangelsk (Russia) Neisseria gonorrhoeae Population: Emergence Mechanisms and Relation with Antimicrobial Resistance

132 clinical isolates of N. gonorrhoeae collected in Arkhangelsk region during 2014–2016 were analyzed in accordance with the NG-MAST protocol (Neisseria gonorrhoeae Multi Antigen Sequence Typing). A diversity of the analyzed strains was represented by 53 NG-MAST sequence-types; 30 of them were new registered due to earlier unknown either porB and tbpB sequences or their combinations. Three pathways of the diversity formation are supposed: long-term circulation of some NG-MAST-types with sequential transmission via the “sexual chains” (40.1% of the total population); introduction of new molecular types (27.3%); appearance of point mutations in porB and tbpB genes with the emergence of previously unknown genotypic variants of N. gonorrhoeae (32.6%). Emerged NG-MAST types inherited the mutational profile in penA, ponA, rpsJ, gyrA, and parC genes followed by conservation of antimicrobial resistance in the local N. gonorrhoeae population, whereas these determinants do not play a significant role in contemporary gonococci molecular evolution.

Public health surveillance of multidrug-resistant clones of Neisseria gonorrhoeae in Europe: a genomic survey

SummaryBackgroundTraditional methods for molecular epidemiology of Neisseria gonorrhoeae are suboptimal. Whole-genome sequencing (WGS) offers ideal resolution to describe population dynamics and to predict and infer transmission of antimicrobial resistance, and can enhance infection control through linkage with epidemiological data. We used WGS, in conjunction with linked epidemiological and phenotypic data, to describe the gonococcal population in 20 European countries. We aimed to detail changes in phenotypic antimicrobial resistance levels (and the reasons for these changes) and strain distribution (with a focus on antimicrobial resistance strains in risk groups), and to predict antimicrobial resistance from WGS data.MethodsWe carried out an observational study, in which we sequenced isolates taken from patients with gonorrhoea from the European Gonococcal Antimicrobial Surveillance Programme in 20 countries from September to November, 2013. We also developed a web platform that we used for automated antimicrobial resistance prediction, molecular typing (N gonorrhoeae multi-antigen sequence typing [NG-MAST] and multilocus sequence typing), and phylogenetic clustering in conjunction with epidemiological and phenotypic data.FindingsThe multidrug-resistant NG-MAST genogroup G1407 was predominant and accounted for the most cephalosporin resistance, but the prevalence of this genogroup decreased from 248 (23%) of 1066 isolates in a previous study from 2009–10 to 174 (17%) of 1054 isolates in this survey in 2013. This genogroup previously showed an association with men who have sex with men, but changed to an association with heterosexual people (odds ratio=4·29). WGS provided substantially improved resolution and accuracy over NG-MAST and multilocus sequence typing, predicted antimicrobial resistance relatively well, and identified discrepant isolates, mixed infections or contaminants, and multidrug-resistant clades linked to risk groups.InterpretationTo our knowledge, we provide the first use of joint analysis of WGS and epidemiological data in an international programme for regional surveillance of sexually transmitted infections. WGS provided enhanced understanding of the distribution of antimicrobial resistance clones, including replacement with clones that were more susceptible to antimicrobials, in several risk groups nationally and regionally. We provide a framework for genomic surveillance of gonococci through standardised sampling, use of WGS, and a shared information architecture for interpretation and dissemination by use of open access software.FundingThe European Centre for Disease Prevention and Control, The Centre for Genomic Pathogen Surveillance, Örebro University Hospital, and Wellcome.

Genomic epidemiology and population structure of Neisseria gonorrhoeae from remote highly endemic Western Australian populations

BackgroundNeisseria gonorrhoeae causes gonorrhoea, the second most commonly notified sexually transmitted infection in Australia. One of the highest notification rates of gonorrhoea is found in the remote regions of Western Australia (WA). Unlike isolates from the major Australian population centres, the remote community isolates have low rates of antimicrobial resistance (AMR).Population structure and whole-genome comparison of 59 isolates from the Western Australian N. gonorrhoeae collection were used to investigate relatedness of isolates cultured in the metropolitan and remote areas. Core genome phylogeny, multilocus sequencing typing (MLST), N. gonorrhoeae multi-antigen sequence typing (NG-MAST) and N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) in addition to hierarchical clustering of sequences were used to characterize the isolates.ResultsPopulation structure analysis of the 59 isolates together with 72 isolates from an international collection, revealed six population groups suggesting that N. gonorrhoeae is a weakly clonal species. Two distinct population groups, Aus1 and Aus2, represented 63% of WA isolates and were mostly composed of the remote community isolates that carried no chromosomal AMR genotypes. In contrast, the Western Australian metropolitan isolates were frequently multi-drug resistant and belonged to population groups found in the international database, suggesting international transmission of the isolates.ConclusionsOur study suggests that the population structure of N. gonorrhoeae is distinct between the communities in remote and metropolitan WA. Given the high rate of AMR in metropolitan regions, ongoing surveillance is essential to ensure the enduring efficacy of the empiric gonorrhoea treatment in remote WA.

Antibiotic resistance and NG-MAST sequence types of Neisseria gonorrhoeae isolates in Poland compared to the world.

Gonorrhoea is one of the most common sexually transmitted infections and in 2012, the World Health Organization estimated about 78 million of new global urogenital cases among adults per year. The main concern during the latest decade has been the emergence and spread of multidrug-resistant strains of Neisseria gonorrhoeae. Resistance has emerged internationally to the extended-spectrum cephalosporins, ceftriaxone and cefixime, which are the last remaining options for empiric first-line monotherapy of gonorrhoea. In Poland, the levels of resistance to ciprofloxacin, benzylpenicillin and tetracycline are high, and the prevalence of azithromycin resistance has increased. However, no resistance to ceftriaxone has been identified. The currently spread multidrug-resistant strains frequently represent epidemic clones. The present paper reviews and describes the antimicrobial resistance and N. gonorrhoeae multiantigen sequence typing (NG-MAST) sequence types of N. gonorrhoeae strains spreading in Poland compared to the world.

Emergence and Spread of Neisseria gonorrhoeae Isolates With Decreased Susceptibility to Extended-Spectrum Cephalosporins in Argentina, 2009 to 2013.

The emergence of Neisseria gonorrhoeae strains with decreased susceptibility to cephalosporins represents a major concern globally. The aim of this study was to examine the phenotypic and molecular characteristics of N. gonorrhoeae isolates with decreased susceptibility to ceftriaxone and cefixime in Argentina. A total of 1987 isolates were collected during 2009 and 2013. The susceptibility to penicillin G, tetracycline, ciprofloxacin, cefixime, ceftriaxone, and azithromycin was determined using the agar dilution method. The major extended-spectrum cephalosporin resistance determinants (penA, mtrR, and porB1b) were sequenced in 42 N. gonorrhoeae isolates that showed decreased susceptibility to ceftriaxone (minimum inhibitory concentration [MIC], 0.06-0.125 mg/L) and cefixime (MIC, 0.125-0.25 mg/L). Genotyping by N. gonorrhoeae multiantigen sequence typing (NG-MAST) was performed. Between 2009 and 2013, there was a shift in the modal MICs for ceftriaxone. Among the 42 isolates exhibiting decreased susceptibility to ceftriaxone and cefixime, 95.2% were resistant to penicillin G, 95.2% to tetracycline, 97.6% to ciprofloxacin, and 33.3% to azithromycin. Thirty-five (83.3%) of the 42 isolates had a mosaic penA allele XXXIV, which has been previously associated with resistance to ceftriaxone and cefixime as well as treatment failures. The isolates that contained the mosaic penicillin-binding protein 2 (PBP2) XXXIV were associated with NG-MAST ST1407 or closely related genotypes. In Argentina, N. gonorrhoeae isolates with decreased susceptibility to cefixime and ceftriaxone have now emerged, mostly due to the introduction of the internationally spread multidrug-resistant NG-MAST ST1407.

Antimicrobial susceptibility of Neisseria gonorrhoeae isolates from Hefei (2014\u20132015): genetic characteristics of antimicrobial resistance

BackgroundAntimicrobial resistance (AMR) and genetic determinants of resistance of N. gonorrhoeae isolates from Hefei, China, were characterized adding a breadth of information to the molecular epidemiology of gonococcal resistance in China.Methods126 N. gonorrhoeae isolates from a hospital clinic in Hefei, were collected between January, 2014, and November, 2015. The minimum inhibitory concentration (MIC) of N. gonorrhoeae isolates for seven antimicrobials were determined by the agar dilution method. Isolates were tested for mutations in penA and mtrR genes and 23S rRNA, and also genotyped using N. gonorrhoeae multi-antigen sequence typing (NG-MAST).ResultsAll N. gonorrhoeae isolates were resistant to ciprofloxacin; 81.7% (103/126) to tetracycline and 73.8% (93/126) to penicillin. 39.7% (50/126) of isolates were penicillinase producing N. gonorrhoeae (PPNG), 31.7% (40/126) were tetracycline resistant N. gonorrhoeae (TRNG) and 28.6% (36/126) were resistant to azithromycin. While not fully resistant to extended spectrum cephalosporins (ESCs), a total of 14 isolates (11.1%) displayed decreased susceptibility to ceftriaxone (MIC ≥ 0.125 mg/L, n = 10), cefixime (MIC ≥ 0. 25 mg/L, n = 1) or to both ESCs (n = 3). penA mosaic alleles XXXV were found in all isolates that harbored decreased susceptibility to cefixime, except for one. Four mutations were found in mtrR genes and mutations A2143G and C2599T were identified in 23S rRNA. No isolates were resistant to spectinomycin. Gonococcal isolates were distributed into diverse NG-MAST sequence types (STs); 86 separate STs were identified.ConclusionsN. gonorrhoeae isolates from Hefei during 2014–2015, displayed high levels of resistance to antimicrobials that had been recommended previously for treatment of gonorrhea, e.g., penicillin, tetracycline and ciprofloxacin. The prevalence of resistance to azithromycin was also high (28.6%). No isolates were found to be fully resistant to spectinomycin, ceftriaxone or cefixime; however, 11.1% isolates, overall, had decreased susceptibility to ESCs.

A Case-Control Study of Molecular Epidemiology in Relation to Azithromycin Resistance in Neisseria gonorrhoeae Isolates Collected in Amsterdam, the Netherlands, between 2008 and 2015.

Neisseria gonorrhoeae resistance to ceftriaxone and azithromycin is increasing, which threatens the recommended dual therapy. We used molecular epidemiology to identify N. gonorrhoeae clusters and associations with azithromycin resistance in Amsterdam, the Netherlands. N. gonorrhoeae isolates (n = 143) were selected from patients visiting the Amsterdam STI Outpatient Clinic from January 2008 through September 2015. We included all 69 azithromycin-resistant isolates (MIC ≥ 2.0 mg/liter) and 74 frequency-matched susceptible controls (MIC ≤ 0.25 mg/liter). The methods used were 23S rRNA and mtrR sequencing, N. gonorrhoeae multiantigen sequence typing (NG-MAST), N. gonorrhoeae multilocus variable-number tandem-repeat analysis (NG-MLVA), and a specific PCR to detect mosaic penA genes. A hierarchical cluster analysis of NG-MLVA related to resistance and epidemiological characteristics was performed. Azithromycin-resistant isolates had C2611T mutations in 23S rRNA (n = 62, 89.9%, P < 0.001) and were NG-MAST genogroup G2992 (P < 0.001), G5108 (P < 0.001), or G359 (P = 0.02) significantly more often than susceptible isolates and were more often part of NG-MLVA clusters (P < 0.001). Two resistant isolates (2.9%) had A2059G mutations, and five (7.3%) had wild-type 23S rRNA. No association between mtrR mutations and azithromycin resistance was found. Twenty-four isolates, including 10 azithromycin-resistant isolates, showed reduced susceptibility to extended-spectrum cephalosporins. Of these, five contained a penA mosaic gene. Four of the five NG-MLVA clusters contained resistant and susceptible isolates. Two clusters consisting mainly of resistant isolates included strains from men who have sex with men and from heterosexual males and females. The co-occurrence of resistant and susceptible strains in NG-MLVA clusters and the frequent occurrence of resistant strains outside of clusters suggest that azithromycin resistance develops independently from the background genome.

Ceftriaxone susceptibility and molecular characteristics of Neisseria gonorrhoeae isolates in Changsha, China.

A total of 128 Neisseria gonorrhoeae isolates were consecutively obtained in Changsha, China, between April 2015 and June 2016. The minimum inhibitory concentrations (MICs) of ceftriaxone were measured by agar dilution method for each isolate. Ceftriaxone resistance determinants involving penA, mtrR, porB and ponA were amplified by PCR, and the PCR products were sequenced. N.gonorrhoeae multi-antigen sequence typing (NG-MAST) was conducted for genotyping. The results exhibited 11% (14/128) of isolates were reduced susceptibility or resistant to ceftriaxone. One isolate with a susceptibility MIC of 0.03mg/L harbored a penA mosaic allele. PBP2 A501V/T or P551S and the PorB1b G120K/A121D mutations were predominant in the 14 decreased susceptible or resistant isolates. Among the 128 isolates, 31 sequence types (STs) were identified, and the most prevalent STs were STnew1 (n=40), ST5061 (n=25) and ST9176 (n=18). The 14 decreased susceptible or resistant isolates were resolved into 9 STs, displaying considerable diversity. The results of ceftriaxone susceptibility testing indicated that ceftriaxone can continue to be recommended as the first-line drug for gonorrhea treatment in Changsha; however, it is important to maintain promptly surveillance for antimicrobial resistance (AMR). Non-mosaic penA alleles with A501V/T, P551S, and porB1b mutations may contribute to ceftriaxone resistance or decreased susceptibility among N.gonorrhoeae in this area, and heterogenous STs in these isolates excluded the clonal expansion for a particular subtype.

Antibiotic resistant mechanism and epidemiological characteristics of azithromycin-resistant Neisseria gonorrhoeae strains in Shenzhen

Objective To investigate the prevalence, molecular mechanism and genetic characteristics of azithromycin-resistant Neisseria gonorrhoeae (N.gonorrhoeae) strains isolated in Shenzhen. Methods N. gonorrhoeae strains were collected in Shenzhen from 2011 to 2015. Agar dilution method and E-test were used to detect the minimum inhibitory concentrations (MIC) of these strains to azithromycin. All azithromycin-resistant (AZM-R) strains (MIC≥2 μg/ml) and some azithromycin-sensitive strains (MIC≤0.25 μg/ml) which were randomly selected as the control group were screened for mutations in 23S rRNA, mtrR and erm genes and genotyped by using N. gonorrhoeae multi-antigen sequence typing (NG-MAST). Results A total of 788 N. gonorrhoeae strains were collected, 148 (18.8%) of which were AZM-R strains (MIC≥1 μg/ml). Eighteen out of 21 high-level AZM-R (AZM-HLR) strains had A2143G mutations in the four copies of the 23S rRNA gene. Twelve out of 29 middle-level AZM-R (AZ-MLR) strains had missense mutations, among which C2611T mutations in the four copies of the 23S rRNA were detected in 10 strains. Incidence of G45D/Y105H mutation in AZM-HLR strains was higher than that in AZM-MLR (χ2=12.702, P=0.000) or AZ-S (χ2=4.462, P=0.035) strains according to the analysis of the promoter and coding region of mtrR gene. PCR analysis revealed that only one strain carried ermB gene (MIC=2 μg/ml). The 788 N. gonorrhoeae strains were typed into 81 sequence types (STs) by NG-MAST, most of which were represented by one strain only. STs of ST3356 and ST1866 that were identified in the AZ-R strains in the current study had been noted in a previous report of emerging AZM-R N. gonorrhoeae strains in Nanjing, Chongqing and Guangzhou. Neighbor-joining (NJ) phylogenetic tree showed that the resistant strains did not form a separate cluster. Conclusion Currently, it is not suitable to use azithromycin as a monotherapy for gonorrhea in Shenzhen. Mutations of A2059G and C2611T in 23S rRNA of N. gonorrhoeae were respectively responsible for high-level and middle-level resistance to azithromycin. Repeated emergence of ST1866 and ST3356 will help us monitor and analyze the epidemiological characteristics of N. gonorrhoeae strains resistant to azithromycin in Shenzhen. Key words: Neisseria gonorrhoeae; Azithromycin; Resistance gene; NG-MAST

In vitro activity of tigecycline alone and antimicrobial combinations against clinical Neisseria gonorrhoeae isolates

In this study, we determined the in vitro activity of various combinations of antimicrobial agents against 54 Neisseria gonorrhoeae isolates. The combined activity of ceftriaxone (CRO) and azithromycin (AZM), CRO and doxycycline (DOX), CRO and spectinomycin (SPT), cefixime (CFX) and AZM, CFX and DOX, and CFX and SPT was determined using a checkerboard method. The fractional inhibitory concentration index (FICI) values for all combinations were either additive or indifferent, and no synergistic or antagonistic effects were found. The FICI comparison in each combination did not show any difference according to the N.gonorrhoeae-resistant phenotypes and genotypic characteristics, including penicillinase-producing N. gonorrhoeae, tetracycline-resistant N. gonorrhoeae, stratified MIC of all antibiotics, and N. gonorrhoeae multiantigen sequence typing. MIC50 and MIC90 of tigecycline by agar dilution were 0.5 mg/L and 0.5 mg/L, respectively, which were lower than that of tetracycline and DOX. Additive/indifference results could suggest that combinations that include CRO may be used safely without a significant likelihood of generating resistance.

NG-MAST GENOTYPING OF NEISSERIA GONORRHOEAE STRAINS ISOLATED IN RUSSIAN FEDERATION IN 2012 - 2015

Aim. Characteristics of contemporary strains of N. gonorrhoeae using NG-MAST genotyping (.Neisseria gonorrhoeae multi-antigen sequence typing), isolation of dominating variants and analysis of their territorial distribution. Materials and methods. 440 strains of N. gonorrhoeae isolated in 2012 - 2015 in 19 subjects of the Russian Federation were used in the study. Genotyping was carried out based on sequencing of variable regions of porB and tbpB genes. Identification of alleles and sequence-types was carried out according to http://www.ng-mast.net. Results. 172 NG-MAST genotypes were identified, 100 of which -were described for the first time. 807,1152, 1544,5714 and 5941 were predominating sequence-types, typical for several subjects of the Russian Federation and some neighboring countries, as well as sequence-types 8583 and 9476 described for the first time, that were exclusively distributed in the regions of their emergence. Sequence-types 225, 1407 and 2992, that are epidemically significant in distant countries, were represented by single isolates in this study. Conclusion. A peculiar and significant genetic diversity of N. gonorrhoeae strains, circulating in the Russian Federation, with characteristic bond of several NG-MAST genotypes to one or several regions, was shown.

Molecular epidemiology of drug-resistant Neisseria gonorrhoeae in Russia (Current Status, 2015).

BackgroundThe widespread distribution of Neisseria gonorrhoeae strains that are resistant to previously used and clinically implemented antibiotics is a significant global public health problem. In line with WHO standards, the national Gonococcal Antimicrobial Surveillance Programme (RU-GASP) has been in existence in Russia since 2004; herein, the current status (2015) is described, including associations between N. gonorrhoeae antimicrobial susceptibility, primary genetic resistance determinants and specific strain sequence types.MethodsA total of 124 N. gonorrhoeae strains obtained from 9 regions in Russia in 2015 were examined using N. gonorrhoeae Multi-Antigen Sequence Typing (NG-MAST), an antimicrobial susceptibility test according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria and an oligonucleotide microarray for the identification of mutations in the penA, ponA, rpsJ, gyrA and parC genes responsible for penicillin G, tetracycline, and fluoroquinolone resistance. Genogroup (G) isolates were evaluated based on their porB and tbpB sequence types (STs).ResultsNG-MAST analysis showed a diversified population of N. gonorrhoeae in Russia with 58 sequence types, 35 of which were described for the first time. The STs 807, 1544, 1993, 5714, 9476 and 12531, which were typical for some Russian Federation regions and several countries of the former Soviet Union, were represented by five or more isolates. The internationally widespread ST 1407 was represented by a single strain in the present study. Division into genogroups facilitated an exploration of the associations between N. gonorrhoeae sequence type, antimicrobial resistance spectra and genetic resistance determinant contents. Preliminarily susceptible (G-807, G-12531) and resistant (G-5714, G-9476) genogroups were revealed. The variability in the most frequently observed STs and genogroups in each participating region indicated geographically restricted antimicrobial susceptibility in N. gonorrhoeae populations.ConclusionsResistance or intermediate susceptibility to previously recommended antimicrobials, such as penicillin G (60.5 %), ciprofloxacin (41.1 %) and tetracycline (25 %), is common in the N. gonorrhoeae population. Based on previous reports and current data, ceftriaxone and spectinomycin should be recommended for first-line empiric antimicrobial monotherapy for gonorrhoea in Russia.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-016-1688-7) contains supplementary material, which is available to authorized users.

Rapid spread of Neisseria gonorrhoeae ciprofloxacin resistance due to a newly introduced resistant strain in Nuuk, Greenland, 2012–2015: a community-based prospective cohort study

ObjectivesTo determine the antimicrobial susceptibility and genotype distribution of Neisseria gonorrhoeae strains isolated from a cohort of patients in Nuuk, Greenland in order to assess the risk of rapid spread...

Antimicrobial resistance and Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST) genotypes in N. gonorrhoeae during 2012–2014 in Karachi, Pakistan

BackgroundGlobally, increasing antimicrobial resistance (AMR) in Neisseria gonorrhoea has led to decreased treatment options for gonorrhoea. Continuous monitoring of resistance is crucial to determine evolving resistance trends in Neisseria gonorrhoea and to suggest treatment recommendations. Quality assured gonococcal AMR data from Pakistan are mainly lacking. This study was performed to determine prevalence and trends of gonococcal AMR and molecular epidemiology of local strains during 2012–2014 in Karachi, Pakistan.MethodsGonococcal isolates (n = 100) were obtained from urogenital specimens submitted to the Aga Khan University Laboratory, Karachi, Pakistan. Antimicrobial susceptibility was determined using Etest and molecular epidemiology was assessed by N. gonorrhoeae multiantigen sequence typing (NG-MAST). Quality control was performed using N. gonorrhoeae WHO reference strains C, F, G, K, L, M, N, O, and P, and ATCC 49226.ResultsSusceptibility to spectinomycin, ceftriaxone and cefixime was 100 % and to azithromycin was 99 %. All isolates had low ceftriaxone MICs, i.e., ≤0.032 mg/L. Resistance to ciprofloxacin, tetracycline and penicillin G were 86 %, 51 % and 43 %, respectively. NG-MAST analysis identified 74 different sequence types (STs).ConclusionsA highly diversified gonococcal population, 74 NG-MAST STs (62 novel STs) with an increased resistance to penicillin G, ciprofloxacin and tetracycline circulated in Karachi, Pakistan. Fortunately, no resistance to ceftriaxone was detected. Accordingly, ceftriaxone can continuously be recommended as the treatment of choice. However it is recommended to increase the dose of ceftriaxone from 125 mg intramuscularly to 250 mg intramuscularly due to ceftriaxone MIC creep and emerging resistance reported in the region. Furthermore, due to the high level of resistance to ciprofloxacin (86 %) it is essential to exclude ciprofloxacin from the recommended first-line therapy. It is imperative to significantly broaden the gonococcal AMR monitoring with participation from other laboratories and cities in Pakistan.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-016-1673-1) contains supplementary material, which is available to authorized users.

WGS analysis and molecular resistance mechanisms of azithromycin-resistant (MIC >2 mg/L) Neisseria gonorrhoeae isolates in Europe from 2009 to 2014.

To elucidate the genome-based epidemiology and phylogenomics of azithromycin-resistant (MIC >2 mg/L) Neisseria gonorrhoeae strains collected in 2009-14 in Europe and clarify the azithromycin resistance mechanisms. Seventy-five azithromycin-resistant (MIC 4 to >256 mg/L) N. gonorrhoeae isolates collected in 17 European countries during 2009-14 were examined using antimicrobial susceptibility testing and WGS. Thirty-six N. gonorrhoeae multi-antigen sequence typing STs and five phylogenomic clades, including 4-22 isolates from several countries per clade, were identified. The azithromycin target mutation A2059G (Escherichia coli numbering) was found in all four alleles of the 23S rRNA gene in all isolates with high-level azithromycin resistance (n = 4; MIC ≥256 mg/L). The C2611T mutation was identified in two to four alleles of the 23S rRNA gene in the remaining 71 isolates. Mutations in mtrR and its promoter were identified in 43 isolates, comprising isolates within the whole azithromycin MIC range. No mutations associated with azithromycin resistance were found in the rplD gene or the rplV gene and none of the macrolide resistance-associated genes [mef(A/E), ere(A), ere(B), erm(A), erm(B), erm(C) and erm(F)] were identified in any isolate. Clonal spread of relatively few N. gonorrhoeae strains accounts for the majority of the azithromycin resistance (MIC >2 mg/L) in Europe. The four isolates with high-level resistance to azithromycin (MIC ≥256 mg/L) were widely separated in the phylogenomic tree and did not belong to any of the main clades. The main azithromycin resistance mechanisms were the A2059G mutation (high-level resistance) and the C2611T mutation (low- and moderate-level resistance) in the 23S rRNA gene.

Neisseria gonorrhoeae antimicrobial susceptibility in Barcelona: penA, ponA, mtrR, and porB mutations and NG-MAST sequence types associated with decreased susceptibility to cephalosporins.

The aims of this study were to determine the antimicrobial susceptibility of Neisseria gonorrhoeae (NG) in our area, to analyze the molecular mechanisms involved in cephalosporins resistance, and to undertake molecular typing of our NG strains. Antimicrobial susceptibility was determined using the Etest. The genes penA, mtrR, penB, and ponA were studied. Molecular typing was performed by N. gonorrhoeae multiantigen sequence typing. Of 329 strains analyzed in 2013, none showed high-level cephalosporin resistance, but 8.2% had resistance to cefixime [minimum inhibitory concentration (MIC) > 0.125μg/mL] and 0.6% to ceftriaxone (MIC > 0.125μg/mL). Azithromycin resistance was documented in 4.3% and ciprofloxacin resistance in 49.2%. Among 48 strains with an MIC ≥ 0.125μg/mL to cefixime, 58.3% showed the penA mosaic pattern XXXIV, 98% a Leu → Pro substitution at position 421 of the ponA gene, 100% amino acid changes at positions 101 and 102 of the PorB1b porin, and 87.5% of strains an adenine deletion in the promoter region of the MtrC-D-E efflux pump. A significant difference between strains with and without decreased cephalosporin susceptibility (MIC ≥ 0.125μg/mL) was observed for these four genes. Of the 48 strains with an MIC ≥ 0.125μg/mL to cefixime, 43.8% belonged to the genogroup G1407 and 27.1% belonged to the genogroup G2400. A significant association of G1407 with decreased susceptibility (MIC ≥ 0.125μg/mL) and G2992 with susceptibility was found, and also between G1407 and mosaic pattern XXXIV and between G2400 and A501T substitution in penA. The NG resistance rate in our area is higher than the median of Europe. We have detected the emergence of G2400, which may be a source of antimicrobial resistance.