N. Gonorrhoeae Multiantigen Sequence Typing Research Articles

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

Emerging azithromycin-resistance among the Neisseria gonorrhoeae strains isolated in Hungary.

BackgroundIn the 1990s, azithromycin became the drug of choice for many infectious diseases but emerging resistance to the drug has only been reported in the last decade. In the last 5 years, the National Neisseria gonorrhoeae Reference Laboratory of Hungary (NNGRLH) has also observed an increased number of N. gonorrhoeae strains resistant to azithromycin. The aim of this study was to determine the most frequent sequence types (ST) of N. gonorrhoeae related to elevated levels of azithromycin MIC (minimal inhibitory concentration). Previously and currently isolated azithromycin-resistant strains have been investigated for the existence of molecular relationship.MethodsMaldi-Tof technic was applied for the identification of the strains isolated from outpatients attending the reference laboratory. Testing antibiotic susceptibility of azithromycin, cefixime, ceftriaxone, tetracycline, spectinomycin and ciprofloxacin was carried out for all the identified strains, using MIC strip test Liofilchem®. N. gonorrhoeae multiantigen sequence typing (NG-MAST) was performed exclusively on azithromycin-resistant isolates. A phylogenetic tree was drawn using MEGA6 (Molecular Evolutionary Genetics Analysis Version 6.0) Neighbour-Joining method.ResultsOut of 192 N. gonorrhoeae isolates, 30.0 % (58/192) proved resistant to azithromycin (MIC > 0.5 mg/L). Of the azithromycin-resistant isolates, ST1407, ST4995 and ST11064 were the most prevalent. Based on the phylogenetic analysis, the latter two STs are closely related.ConclusionsIn contrast to West-European countries, in our region, resistance to azithromycin has increased up to 30 % in the last 5 years, so the recommendation of the European Guideline −500 mg of ceftriaxone combined with 2 g of azithromycin as first choice therapy against N. gonorrhoeae- should be seriously considered in case of Hungary.Electronic supplementary materialThe online version of this article (doi:10.1186/s12941-016-0166-9) contains supplementary material, which is available to authorized users.

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...

Molecular epidemiology and mechanisms of resistance of azithromycin-resistant Neisseria gonorrhoeae isolated in France during 2013-14.

The objective of this study was to determine the prevalence and mechanisms of azithromycin resistance of Neisseria gonorrhoeae French isolates from 2013 to 2014. N. gonorrhoeae samples isolated in a network of laboratories were tested for susceptibility to azithromycin between April 2013 and March 2014. Fifty-four isolates that were non-susceptible to azithromycin and 18 susceptible isolates were characterized for molecular mechanisms of resistance by PCR/sequencing and genotyped using N. gonorrhoeae multiantigen sequence typing (NG-MAST). Among the 970 N. gonorrhoeae isolates, 54 (5.56%) were non-susceptible to azithromycin, 9 (1%) were resistant and 45 (4.6%) showed intermediate resistance. Azithromycin-non-susceptible isolates harboured a C2599T mutation in the rrl gene encoding the 23S rRNA alleles (5.5%), a C substitution in the mtrR promoter (5.5%), an A deletion in the mtrR promoter (53.7%) and mutations in the L4 ribosomal protein (14.8%) and in the MtrR repressor (25.9%). No isolates showed an L22 mutation or carried an erm, ere, mef(A)/(E) or mphA gene. Thirty different STs were highlighted using the NG-MAST technique. The predominant genogroups non-susceptible to azithromycin were G21 (31%), G1407 (20%) and G2400 (15%). Genogroup G2400 (15%) was revealed to be a novel cluster prevalent in the south of France and resistant to azithromycin, ciprofloxacin and tetracycline. Our study highlights that the prevalence of resistance of N. gonorrhoeae to azithromycin in France is low and essentially due to multiple genetic mutations. Its dissemination occurs through three major genogroups including a novel one in France (G2400).

Genetic characteristics of Neisseria gonorrhoeae isolates from Guangzhou city and relationship between their multiantigen sequence typing sequence types and ciprofloxacin resistance

Objective To investigate genetic characteristics of Neisseria gonorrhoeae (N. gonorrhoeae)isolates from Guangzhou city in 2014, and to analyze the relationship of N. gonorrhoeae multi-antigen sequence typing (NG-MAST)sequence types(STs)with ciprofloxacin resistance. Methods An agar dilution method was used to determine the minimal inhibitory concentration(MIC)of ciprofloxacin in 97 N. gonorrhoeae isolates from Guangzhou city. PCR was performed to amplify the gyrA, parC, porB and tbpB genes from these isolates, followed by gene sequencing and determination of NG-MAST STs. Results Of the 97 N. gonorrhoeae isolates, 95 (97.9%)were resistant to ciprofloxacin, including 41 high-level (MIC ≥ 16 mg/L)and 54 low-level (1 mg/L ≤ MIC < 16 mg/L)resistant strains. Mutations were detected at codons 91 and 95 encoding serine in the gyrA gene of all the 95 ciprofloxacinresistant strains, and in the parC gene of 93 resistant strains. The frequency of the mutation at codon 87 in the parC gene was 85.4% (35/41)in high-level resistant strains, significantly higher than that in low-level resistant strains (59.3%[32/54], χ2 = 7.64, P < 0.05). MAST STs were successfully determined for all the 97 N. gonorrhoeae isolates except 1 isolate with incorrect PCR amplicons. Of the 96 genotyped isolates, 50 were assigned to 35 known STs by using the NG-MAST website (www. ng-mast.net), among which, 10 STs each contained 2 to 4 isolates. The most common ST was ST5309. Phylogenetic tree analysis revealed that the 96 genotyped N. gonorrhoeae isolates could be classified into 2 groups, and the proportion of isolates with MIC ≥ 16 mg/L is 46.4% (39/84)in group 1, but only 1/12 in group 2 (χ2 = 6.27, P = 0.012). Conclusions High-level resistance of N. gonorrhoeae to ciprofloxacin may be mainly associated with the mutation at codon 87 in the parC gene. NG-MAST STs may be related to the degree of ciprofloxacin resistance. Key words: Neisseria gonorrhoeae; Ciprofloxacin; Drug resistance, bacterial; NG-MAST

Azithromycin-resistant Neisseria gonorrhoeae isolates in Guangzhou, China (2009-2013): coevolution with decreased susceptibilities to ceftriaxone and genetic characteristics.

BackgroundThe recent emergence of azithromycin-resistant (AZM-R) N. gonorrhoeae isolates that have coevolved decreased susceptibility to extended-spectrum cephalosporins has caused great concern. Here we investigated the prevalence of decreased susceptibility to ceftriaxone (CROD) in AZM-R isolates and genetically characterized AZM-R isolates in Guangzhou, China from 2009 to 2013.MethodsThe minimum inhibitory concentration (MIC) of AZM and ceftriaxone was determined using an agar-dilution method. All AZM-R isolates were screened for mutations in 23S rRNA, mtrR and penA genes and genotyped using N. gonorrhoeae multi-antigen sequence typing (NG-MAST).ResultsOf the 485 identified N. gonorrhoeae isolates, 445 (91.8 %) were isolated from male urethritis subjects, and 77 (15.9 %) were AZM-R (MIC ≥ 1 mg/L), including 33 (6.8 %) with AZM low-level resistant (AZM-LLR, MIC = 1 mg/L) and 44 (9.1 %) with AZM middle-level resistant (AZM-MLR, MIC ≥ 2 mg/L). Significantly more CROD (MIC ≥ 0.125 mg/L) showed in AZM-MLR isolates (43.2 %, 19/44) as compared with that in AZM-LLR isolates (18.2 %, 6/33) (p < 0.05). For the 23S rRNA, mtrR, penA or combined 23S rRNA/MtrR/penA mutations, no significant difference was found between AZM-LLR isolates and AZM-MLR isolates (P > 0.05); similar results were detected between combined AZM-LLR/CROD isolates and combined AZM-MLR/CROD isolates (P > 0.05). No mutation A2059G or AZM high-level resistant (AZM-HLR, MIC ≥ 256 mg/L) isolate was detected. Among 77 AZM-R isolates, 67 sequence types (STs) were identified by NG-MAST, of which 30 were novel. Most STs were represented by a single isolate.ConclusionsThe AZM-R together CROD isolates are now present in Guangzhou, China, which deserve continuous surveillance and the mechanism of concurrent resistance needs further study.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-016-1469-3) contains supplementary material, which is available to authorized users.

First nationwide study regarding ceftriaxone resistance and molecular epidemiology of Neisseria gonorrhoeae in China.

Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a major public health concern worldwide. This is the first nationwide study, performed within the China Gonococcal Antimicrobial Susceptibility Programme (China-GASP), regarding AMR, including ceftriaxone genetic resistance determinants, and molecular epidemiology of gonococci in China. Gonococcal isolates (n = 1257) from consecutive patients were collected at 11 sentinel sites distributed across China during 2012-13. Susceptibility to ceftriaxone, spectinomycin, ciprofloxacin and tetracycline was determined using the agar dilution method. Ceftriaxone resistance determinants penA and penB were examined using sequencing. N. gonorrhoeae multiantigen sequence typing (NG-MAST) was performed for molecular epidemiology. Among isolates, 0.2% were resistant to spectinomycin, 4.4% to ceftriaxone, 42.9% to tetracyclines (high-level resistance) and 99.8% to ciprofloxacin. Among 890 sequenced isolates, 16 (1.8%) possessed a penA mosaic allele; 4 of these isolates belonged to the MDR internationally spread NG-MAST genogroup G1407 (first description in China). Non-mosaic penA alleles with an A501T mutation and an A102D alteration in porB1b were statistically associated with decreased susceptibility/resistance to ceftriaxone. NG-MAST G10339, G1424 and G1053 were associated with decreased susceptibility/resistance to ceftriaxone. In China, ceftriaxone and spectinomycin can continue to be recommended for gonorrhoea treatment, with the possible exception of Hainan and Sichuan provinces where ceftriaxone resistance exceeded 5% and AMR surveillance needs to be strengthened. Molecular approaches including genotyping and AMR determinant analysis can be valuable to supplement and enhance conventional surveillance of gonococcal AMR in China.

Emergence and evolution of internationally disseminated cephalosporin-resistant Neisseria gonorrhoeae clones from 1995 to 2005 in Japan.

BackgroundNeisseria gonorrhoeae strains with resistance to extended-spectrum cephalosporins (ESCs), last options for first-line monotherapy of gonorrhoea, likely emerged and initially disseminated in Japan, followed by international transmission. In recent years, multi-locus sequence typing (MLST) ST1901 and N. gonorrhoeae multiantigen sequence typing (NG-MAST) ST1407 isolates with the mosaic penicillin-binding protein (PBP) 2 XXXIV have accounted for most ESC resistance globally. Our aim was to elucidate the initial emergence and transmission of ESC-resistant strains by detailed examination of N. gonorrhoeae isolates from 1995 to 2005 in Kanagawa, Japan.MethodsN. gonorrhoeae isolates were examined phenotypically (n = 690) and genetically (n = 372) by agar dilution method (cefixime, ceftriaxone and ciprofloxacin), penA gene sequencing, MLST and NG-MAST.ResultsAlready in 1995, one cefixime-resistant (CFM-R) isolate was found, which is the first CFM-R isolate described globally. After 1996, the prevalence of CFM-R and CFM-decreased susceptibility (CFM-DS) isolates significantly increased, with the peak resistance level in 2002 (57.1 % CFM-R). In 1997–2002, the CFM-R MLST ST7363 strain type with the mosaic PBP 2 X was predominant among CFM-R/DS isolates. The first CFM-R/DS MLST ST1901 clone(s), which became the predominant CFM-R/DS strain type(s) already in 2003–2005, possessed the mosaic PBP 2 X, which was possibly originally transferred from the MLST ST7363 strains, and subsequently acquired the mosaic PBP 2 XXXIV. The first MLST ST1901 and NG-MAST ST1407 isolate was identified in Kanagawa already in 2003.ConclusionsThe two main internationally spread cefixime-resistant gonococcal clones, MLST ST7363 and ST1901 (NG-MAST ST1407 most frequent internationally) that also have shown their capacity to develop high-level ceftriaxone resistance (superbugs H041 and F89), likely emerged, evolved and started to disseminate in the metropolitan area, including Kanagawa, in Japan, which was followed by global transmission.

P05.09 Phenotypic, genetic and genomic characterisation of the 2015 whoneisseria gonorrhoeaereference strains for quality assurance of laboratory investigations globally

Introduction Gonorrhoea and antimicrobial resistance (AMR) in Neisseria gonorrhoeae are major public health concerns globally. Resistance to all antimicrobials available for treatment of gonorrhoea has now been reported in N. gonorrhoeae . Enhanced quality assured gonococcal AMR surveillance is crucial worldwide and the WHO Global Gonococcal Antimicrobial Surveillance Programme (GASP) was revitalised in 2009. To obtain reliable and comparable AMR data internationally, appropriate and well-characterised N. gonorrhoeae reference strains are essential for quality assurance. The phenotypic and genetic characteristics of the 2008 WHO N. gonorrhoeae reference strains (n = 8) were previously published. Here, we describe the phenotypic, genetic, and genomic characteristics of the 2015 WHO N. gonorrhoeae reference strains. Methods In the 2015 WHO N. gonorrhoeae reference strain panel (n = 14), six additional strains have been selected to include representation of high-level cephalosporin and azithromycin resistance and porA mutant strain. These strains were phenotypically characterised by antibiogram, serovar, and prolyliminopeptidase (PIP) screening; and genetically in regards of resistance plasmid types, polymorphisms in divergent genetic resistance-mediating loci (n = 14), porB sequencing, N. gonorrhoeae multiantigen sequence typing (NG-MAST), and multi-locus sequence typing (MLST). Fully characterised finished reference genomes for all the 2015 WHO N. gonorrhoeae reference strains were produced using PacBio and Illumina sequencing technologies. Results The 2015 WHO reference strains represent all available main phenotypes of resistance and susceptibility to antimicrobials previously and currently used for treatment of gonorrhoea, as well as several considered for future use. All corresponding resistance genotypes and molecular epidemiological types were also elucidated. Finally, references genomes of each strain were obtained and characterised in detail. Conclusion The 2015 WHO N. gonorrhoeae reference strains are intended for internal and external quality assurance in all types of laboratory investigations, i.e. particularly in GASP, but also for phenotypic (e.g. culture) and molecular diagnostics, species determination, genetic AMR detection, molecular epidemiology, and genome sequencing as well as other novel molecular technologies. Disclosure of interest statement This work was funded by the Orebro County Council Research Committee and the Foundation for Medical Research at Orebro University Hospital, Sweden, the WHO, and The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridgeshire, United Kingdom.

Genetic diversity of blaTEM alleles, antimicrobial susceptibility and molecular epidemiological characteristics of penicillinase-producing Neisseria gonorrhoeae from England and Wales.

The objective of this study was to investigate the genetic diversity of blaTEM alleles, antimicrobial susceptibility and molecular epidemiological characteristics of penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates collected in 2012 from England and Wales. PPNG isolates were from the 2012 Gonococcal Resistance to Antimicrobial Surveillance Programme (GRASP). Their susceptibility to seven antimicrobials was determined using agar dilution methodology. β-Lactamase production was detected using a nitrocefin test. β-Lactamase plasmid types were determined and blaTEM genes were sequenced. Isolates were also typed by N. gonorrhoeae multi-antigen sequence typing (NG-MAST). Seventy-three PPNG isolates were identified in the 2012 GRASP collection (4.6%, 73/1603). Three different blaTEM alleles were identified, encoding three TEM amino acid sequences: TEM-1 (53%), TEM-1 with a P14S substitution (19%) and TEM-135 (27%). The blaTEM-135 allele was present in nine different NG-MAST types and was found mostly on Asian (60%) and Toronto/Rio (35%) plasmids. By contrast, most TEM-1-encoding plasmids were African (98%). All the TEM-135 isolates displayed high-level ciprofloxacin and tetracycline resistance. The high proportion of blaTEM-135 alleles (27%) demonstrates that this variant is circulating within several gonococcal lineages. Only a single specific mutation near the β-lactamase active site could result in TEM-135 evolving into an ESBL. This is concerning particularly because the TEM-135 isolates were associated with high-level ciprofloxacin and tetracycline resistance. It is encouraging that no further TEM alleles were detected in this gonococcal population; however, vigilance is vital as an ESBL in N. gonorrhoeae would render the last remaining option for monotherapy, ceftriaxone, useless.

Penicillinase-producing Neisseria gonorrhoeae and its blaTEM-135 gene variants at several gonococcal antimicrobial surveillance sites in China: an epidemiological study

Objective To determine the prevalence of penicillinase-producing Neisseria gonorrhoeae(PPNG) and the distribution of blaTEM-135 gene variants in PPNG at several gonococcal antimicrobial surveillance sites in China, to compare N. gonorrhoeae multi-antigen sequence typing (NG-MAST) types of PPNG and its blaTEM-135 gene variants, and to assess the difference and association in NG-MAST types of blaTEM-135 gene variants among different regions. Methods A total of 572 N. gonorrhoeae isolates were collected at 6 gonococcal antimicrobial surveillance sites from Jiangsu, Shanghai, Zhejiang, Tianjin, Guangdong and Guangxi in 2012. After isolation, purification, and identification, cefalotin paper discs were used for detection of PPNG. DNA was extracted by QIAxtractor DX kits after cultivation of the PPNG strains. Then, mismatch amplification mutation assay (MAMA) PCR was performed to identify blaTEM-135variants, and NG-MAST analysis to determine N. gonorrhoeae genotypes. Results Among the 572 N. gonorrhoeae strains, 38.1%(218/572) were identified as PPNG, and of the PPNG strains, 52.3%(114/218) were blaTEM-135 variants. The detection rate of PPNG at these surveillance sites from high to low was as follows: 51.7%(45/87, Zhejiang), 45.6%(36/79, Shanghai), 38.0%(78/205, Guangdong), 37.5%(12/32, Guangxi), 31.2%(24/77, Jiangsu) and 25.0%(23/92, Tianjin), and that of blaTEM-135 variants was as follows: 68.9%(31/45, Zhejiang), 58.3%(14/24, Jiangsu), 50.0%(39/78, Guangdong), 47.2%(17/36, Shanghai), 39.1%(9/23, Tianjin) and 33.3%(4/12, Guangxi). NG-MAST analysis showed that the ST2318, ST1768, ST1866, ST1053 and ST8726 types predominated among these blaTEM-135 variants, and a strong correlation was found between blaTEM-135 variants and some NG-MAST types, such as ST1768, ST1053 and ST8726 types. The distribution of NG-MAST types was significantly different between the surveillance site in Tianjin (in the Northern part of China) and the other sites (in the Southern part of China), but highly similar among the surveillance sites in Jiangsu, Zhejiang and Shanghai regions. Conclusions There is a high prevalence of PPNG and its blaTEM-135 variants at several gonococcal antimicrobial surveillance sites in China, with significant differences in NG-MAST genotype distribution of PPNG and its blaTEM-135 variants among different regions. Key words: Neisseria gonorrhoeae; beta-Lactamases; Epidemiology; blaTEM-135 Gene variant; Neisseria gonorrhoeae multi-antigen sequence typing

Antimicrobial susceptibility/resistance and NG-MAST characterisation of Neisseria gonorrhoeae in Belarus, Eastern Europe, 2010–2013

BackgroundGonorrhoea and widely spread antimicrobial resistance (AMR) in its etiological agent Neisseria gonorrhoeae are major public health concerns worldwide. Gonococcal AMR surveillance nationally and internationally, to identify emerging resistance and inform treatment guidelines, is imperative for public health purposes. In 2009, AMR surveillance was initiated in Belarus, Eastern Europe because no gonococcal AMR data had been available for at least two decades. Herein, the prevalence and trends of gonococcal AMR and molecular epidemiological characteristics of N. gonorrhoeae strains from 2010 to 2013 in Belarus, are described.MethodsN. gonorrhoeae isolates (n=193) obtained in the Mogilev (n=142), Minsk (n=36) and Vitebsk (n=15) regions of Belarus in 2010 (n=72), 2011 (n=6), 2012 (n=75) and 2013 (n=40) were analyzed in regards to AMR using the Etest method and for molecular epidemiology with N. gonorrhoeae multi-antigen sequence typing (NG-MAST).ResultsDuring 2010–2013, the proportions of resistant N. gonorrhoeae isolates were as follows: tetracycline 36%, ciprofloxacin 28%, penicillin G 9%, azithromycin 5%, and cefixime 0.5%. Only one (0.5%) β-lactamase producing isolate was detected. No isolates resistant to ceftriaxone and spectinomycin were identified. Overall, the resistance levels to tetracycline, ciprofloxacin and penicillin G were relatively stable. Interestingly, the level of resistance to azithromycin declined from 12% in 2010 to 0% in 2013 (P < 0.05). In total, 70 NG-MAST STs were identified. The predominant STs were ST1993 (n=53), ST807 (n=13), ST285 (n=8) and ST9735 (n=8). Many novel STs (n=43, 61%), representing 41% of all isolates, were found.ConclusionsDuring 2010–2013, the N. gonorrhoeae population in Belarus displayed high and relatively stable resistance levels to tetracycline, ciprofloxacin, and penicillin G, while the resistance to azithromycin declined. One isolate was resistant to cefixime, but no resistance to ceftriaxone or spectinomycin was found. The results of the present surveillance initiated in 2009 were also used to replace penicillin G with ceftriaxone (1 g single dose intramuscularly) as the first-line drug for empiric treatment of gonorrhoea in the national treatment guidelines in Belarus in late 2009. It is essential to further strengthen the surveillance of gonococcal AMR and ideally survey also treatment failures and molecular epidemiological genotypes in Belarus.

Estimating the prevalence of mixed-type gonococcal infections in Queensland, Australia

Background Mixed gonococcal infections within the one anatomical site have been recognised but questions remain over how often they occur. In this study, the aim was to estimate the prevalence of mixed gonococcal infections using novel real-time polymerase chain reaction (PCR) methods that were developed and validated, targeting the gonococcal porB gene. Neisseria gonorrhoeae strains were categorised into three different porB groups, based on sequence data derived from N. gonorrhoeae multi-antigen sequence typing (NG-MAST) analyses of local isolates. Specific PCR methods for each group were then developed and these PCR methods were used to test clinical samples (n=350) that were positive for gonorrhoea as determined by nucleic acid amplification test (NAAT) diagnostic screening. Initial validation using isolates showed the group PCR methods proved 100% sensitive and 100% specific for their respective porB groups. When applied to the clinical specimens, 298/350 (85%) provided positive results by the group PCR methods. Of these, four specimens showed evidence of mixed infections, supported by subsequent DNA sequencing of the PCR products. The data provide further evidence of mixed gonococcal infections at the same anatomical site, but show that such infections may be relatively infrequent (1.3%; 95% confidence interval 0.01-2.6%) in a general screening population.

Antimicrobial susceptibility and genotyping analysis of Hungarian Neisseria gonorrhoeae strains in 2013.

Emergence and spread of antimicrobial resistance in Neisseria gonorrhoeae is a major public health concern worldwide. The current study aims to determine the antimicrobial resistance in N. gonorrhoeae and associated molecular typing to enhance gonococcal antimicrobial surveillance in Hungary. In the National N. gonorrhoeae Reference Laboratory of Hungary 187 N. gonorrhoeae infections were detected in 2013, antibiograms were determined for all the isolated strains, and 52 (one index strain from every sexually contact related group) of them were also analysed by the N. gonorrhoeae multi-antigen sequence typing (NG-MAST) method. Twenty-two different NG-MAST sequence types (STs) were identified, of which 8 STs had not been previously described. In Hungary, the highly diversified gonococcal population displayed high resistance to penicillin, ciprofloxacin and tetracycline (the antimicrobials previously recommended for gonorrhoea treatment). Resistance to the currently recommended extended spectrum cephalosporines were rare: only two of the expected strains, an ST 1407 and an ST 210, had cefixime MIC above the resistance breakpoint. By the revision of our National Treatment Guideline, it must be considered, that the azithromycin resistance is about 60% among the four most frequently isolated STs in Hungary.

Characterisation of blaTEM genes and types of β-lactamase plasmids in Neisseria gonorrhoeae - the prevalent and conserved blaTEM-135 has not recently evolved and existed in the Toronto plasmid from the origin.

BackgroundAntimicrobial resistance (AMR) in Neisseria gonorrhoeae is a major concern worldwide. It has been recently feared that the blaTEM-1 gene is, via blaTEM-135, evolving into an extended-spectrum β-lactamase (ESBL), which could degrade all cephalosporins including ceftriaxone. The aims of the present study were to characterize the blaTEM genes, types of β-lactamase plasmids, the degradation of ampicillin by TEM-135 compared to TEM-1, and to perform molecular epidemiological typing of β-lactamase-producing N. gonorrhoeae strains internationally.Methodsβ-lactamase producing N. gonorrhoeae isolates (n = 139) cultured from 2000 to 2011 in 15 countries were examined using antibiograms, blaTEM gene sequencing, β-lactamase plasmid typing, and N. gonorrhoeae multiantigen sequence typing (NG-MAST). Furthermore, the blaTEM gene was sequenced in the first described Toronto plasmid (pJD7), one of the first Asian plasmids (pJD4) and African plasmids (pJD5) isolated in Canada. The degradation of ampicillin by TEM-135 compared to TEM-1 was examined using a MALDI-TOF MS hydrolysis assay.ResultsSix different blaTEM sequences were identified (among isolates with 125 different NG-MAST STs), i.e. blaTEM-1 (in 104 isolates), blaTEM-135 (in 30 isolates), and four novel blaTEM sequences (in 5 isolates). The blaTEM-1 allele was only found in the African and Asian plasmids, while all Rio/Toronto plasmids possessed the blaTEM-135 allele. Most interesting, the first described gonococcal Toronto plasmid (pJD7), identified in 1984, also possessed the highly conserved blaTEM-135 allele. The degradation of ampicillin by TEM-135 compared to TEM-1 was indistinguishable in the MALDI-TOF MS hydrolysis assay.ConclusionsblaTEM-135, encoding TEM-135, is predominantly and originally associated with the Rio/Toronto plasmid and prevalent among the β-lactamase producing gonococcal strains circulating globally. blaTEM-135 does not appear, as previously hypothesized, to have recently evolved due to some evolutionary selective pressure, for example, by the extensive use of extended-spectrum cephalosporins worldwide. On the contrary, the present study shows that blaTEM-135 existed in the Toronto plasmid from its discovery and that blaTEM-135 is highly conserved (not further evolved in the past >30 years). Nevertheless, international studies for monitoring the presence of different blaTEM alleles, the possible evolution of the blaTEM-135 allele, and the types of β-lactamase producing plasmids, remain imperative.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2334-14-454) contains supplementary material, which is available to authorized users.

Antimicrobial resistance, genetic resistance determinants for ceftriaxone and molecular epidemiology of Neisseria gonorrhoeae isolates in Nanjing, China.

Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a major problem worldwide. This study investigated the AMR, genetic ceftriaxone resistance determinants and molecular epidemiology of N. gonorrhoeae in Nanjing, China. N. gonorrhoeae isolates were collected in 2007 (n = 198) and 2012 (n = 80). The susceptibility to ceftriaxone, spectinomycin, ciprofloxacin and tetracycline was determined using an agar-dilution method. The ceftriaxone resistance determinants penA, mtrR and penB were examined using sequencing. N. gonorrhoeae multi-antigen sequence typing (NG-MAST) was performed for molecular epidemiology. All isolates were resistant to ciprofloxacin, 42.4% produced β-lactamase and 34.9% showed high-level resistance to tetracycline (MIC ≥16 mg/L). In total, 5.4% of isolates were resistant to ceftriaxone; however, all of these isolates were obtained in 2007 and the susceptibility to ceftriaxone appeared to have increased. All isolates were susceptible to spectinomycin. No penA mosaic alleles were found. Non-mosaic penA alleles with A501T and G542S alterations, an H105Y alteration in mtrR and an A102D/N alteration in porB1b were statistically associated with decreased susceptibility or resistance to ceftriaxone. The most prevalent NG-MAST sequence types (STs) were ST568 (n = 13), ST270 (n = 9) and ST421 (n = 7). ST270 was the most common ST in isolates with decreased susceptibility or resistance to ceftriaxone. Ceftriaxone, ideally 500 mg and together with azithromycin (1-2 g), should be recommended for treatment of gonorrhoea in Nanjing, China. However, N. gonorrhoeae strains with resistance to ceftriaxone have been found in Nanjing. NG-MAST and ceftriaxone resistance determinant analysis can be valuable to supplement the antimicrobial resistance surveillance in China, which needs to be further strengthened.