N. Gonorrhoeae Multiantigen Sequence Typing Sequence Types Research Articles

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

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

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

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

Molecular epidemiological typing of Neisseria gonorrhoeae isolates identifies a novel association between genogroup G10557 (G7072) and decreased susceptibility to cefixime, Germany, 2014 to 2017.

BackgroundEmerging antimicrobial resistance (AMR) challenges gonorrhoea treatment and requires surveillance.AimThis observational study describes the genetic diversity of Neisseria gonorrhoeae isolates in Germany from 2014 to 2017 and identifies N. gonorrhoeae multi-antigen sequence typing (NG-MAST) genogroups associated with AMR or some patient demographics.Methods1,220 gonococcal isolates underwent AMR testing and NG-MAST. Associations between genogroups and AMR or sex/age of patients were statistically assessed.ResultsPatients’ median age was 32 years (interquartile range: 25–44); 1,078 isolates (88.4%) originated from men. In total, 432 NG-MAST sequence types including 156 novel ones were identified, resulting in 17 major genogroups covering 59.1% (721/1,220) of all isolates. Genogroups G1407 and G10557 (G7072) were significantly associated with decreased susceptibility to cefixime (Kruskal–Wallis chi-squared: 549.3442, df: 16, p < 0.001). Their prevalences appeared to decline during the study period from 14.2% (15/106) to 6.2% (30/481) and from 6.6% (7/106) to 3.1% (15/481) respectively. Meanwhile, several cefixime susceptible genogroups’ prevalence seemed to increase. Proportions of isolates from men differed among genogroups (Fisher’s exact test, p < 0.001), being e.g. lower for G25 (G51) and G387, and higher for G5441 and G2992. Some genogroups differed relative to each other in affected patients’ median age (Kruskal–Wallis chi-squared: 47.5358, df: 16, p < 0.001), with e.g. G25 (G51) and G387 more frequent among ≤ 30 year olds and G359 and G17420 among ≥ 40 year olds.ConclusionAMR monitoring with molecular typing is important. Dual therapy (ceftriaxone plus azithromycin) recommended in 2014 in Germany, or only the ceftriaxone dose of this therapy, might have contributed to cefixime-resistant genogroups decreasing.

Antimicrobial resistance (AMR) and molecular characterization of Neisseria gonorrhoeae in Ghana, 2012-2015

Neisseria gonorrhoeae antimicrobial resistance (AMR) surveillance is essential for tracking the emergence and spread of AMR strains in local, national and international populations. This is crucial for developing or refining treatment guidelines. N. gonorrhoeae multiantigen sequence typing (NG-MAST) is beneficial for describing the molecular epidemiology of gonococci at national and international levels. Elucidation of AMR determinants to β-lactam drugs, is a means of monitoring the development of resistance. In Ghana, little is known about the current gonococcal AMR prevalence and no characterization of gonococcal isolates has been previously performed. In this study, gonococcal isolates (n = 44) collected from five health facilities in Ghana from 2012 to 2015, were examined using AMR testing, NG-MAST and sequencing of penA. High rates of resistance were identified to tetracycline (100%), benzylpenicillin (90.9%), and ciprofloxacin (81.8%). One isolate had a high cefixime MIC (0.75 μg/ml). Twenty-eight NG-MAST sequence types (STs) were identified, seventeen of which were novel. The isolate with the high cefixime MIC contained a mosaic penA-34 allele and belonged to NG-MAST ST1407, an internationally spreading multidrug-resistant clone that has accounted for most cefixime resistance in many countries. In conclusion, AMR testing, NG-MAST, and sequencing of the AMR determinant penA, revealed high rates of resistance to tetracycline, benzylpenicillin, and ciprofloxacin; as well as a highly diverse population of N. gonorrhoeae in Ghana. It is imperative to continue with enhanced AMR surveillance and to understand the molecular epidemiology of gonococcal strains circulating in Ghana and other African countries.

Increasing Resistance to Azithromycin in Neisseria gonorrhoeae in Eastern Chinese Cities: Resistance Mechanisms and Genetic Diversity among Isolates from Nanjing.

ABSTRACTAzithromycin resistance (AZM-R) of Neisseria gonorrhoeae is emerging as a clinical and public health challenge. We determined molecular characteristics of recent AZM-R Nanjing gonococcal isolates and tracked the emergence of AZM-R isolates in eastern Chinese cities in recent years. A total of 384 N. gonorrhoeae isolates from Nanjing collected from 2013 to 2014 were tested for susceptibility to AZM and six additional antibiotics; all AZM-R strains were characterized genetically for resistance determinants by sequencing and were genotyped using N. gonorrhoeae multiantigen sequence typing (NG-MAST). Among the 384 isolates, 124 (32.3%) were AZM-R. High-level resistance (MIC, ≥256 mg/liter) was present in 10.4% (40/384) of isolates, all of which possessed the A2143G mutation in all four 23S rRNA alleles. Low- to mid-level resistance (MIC, 1 to 64 mg/liter) was present in 21.9% (84/384) of isolates, 59.5% of which possessed the C2599T mutation in all four 23S rRNA alleles. The 124 AZM-R isolates were distributed in 71 different NG-MAST sequence types (STs). ST1866 was the most prevalent type in high-level AZM-R (HL-AZM-R) isolates (45% [18/40]). This study, together with previous reports, revealed that the prevalence of AZM-R in N. gonorrhoeae isolates in certain eastern Chinese cities has risen >4-fold (7% to 32%) from 2008 to 2014. The principal mechanisms of AZM resistance in recent Nanjing isolates were A2143G mutations (high-level resistance) and C2599T mutations (low- to mid-level resistance) in the 23S rRNA alleles. Characterization of NG-MAST STs and phylogenetic analysis indicated the genetic diversity of N. gonorrhoeae in Nanjing; however, ST1866 was the dominant genotype associated with HL-AZM-R isolates.

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.

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.

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

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.

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.

Phenotypic and genetic characterisation of bacterial sexually transmitted infections in Bissau, Guinea-Bissau, West Africa: a prospective cohort study

BackgroundKnowledge regarding characteristics and transmission of Neisseria gonorrhoeae, Chlamydia trachomatis and Mycoplasma genitalium and antibiotic resistance in N gonorrhoeae in Guinea-Bissau, West Africa, is entirely lacking.ObjectivesTo characterise N gonorrhoeae, C...

Genotypes and antimicrobial-resistant phenotypes of Neisseria gonorrhoeae in Portugal (2004–2009)

ObjectivesTo determine the antibiotic phenotype and MAST-genotype distribution of Neisseria gonorrhoeae isolates in Portugal between 2004 and 2009, and to evaluate specific associations between MAST-genotypes and sexual orientation, age and...

Various penA mutations together with mtrR, porB and ponA mutations in Neisseria gonorrhoeae isolates with reduced susceptibility to cefixime or ceftriaxone

To examine mutations within the penA, mtrR, porB, ponA and pilQ genes of Neisseria gonorrhoeae to determine their contribution to cephalosporin resistance. A total of 46 N. gonorrhoeae isolates with reduced susceptibility to cefixime or ceftriaxone (MICs > or = 0.12 mg/L) and two susceptible isolates were selected. The full sequence of penA and partial sequences previously reported as hot mutation sites of the other genes were analysed. Genotyping by N. gonorrhoeae multiantigen sequence typing (NG-MAST) was also performed. A mosaic penicillin-binding protein 2 (PBP 2) was found in a single isolate that exhibited the highest cefixime MIC (0.5 mg/L). The majority of the isolates with reduced susceptibility to cephalosporins contained non-mosaic PBP 2 sequences, of which PBP 2 pattern XIII was most common (28/46). All isolates with reduced susceptibility to cephalosporins also had mtrR and porB mutations. Two susceptible isolates had the PBP 2 pattern XIV and an incomplete MtrR protein, which was a new mutation. Isolates with identical PBP 2 patterns comprised multiple NG-MAST sequence types. Reduced susceptibility of N. gonorrhoeae to ceftriaxone and cefixime was associated with diverse penA mutations, particularly PBP 2 pattern XIII containing an Ala-501-->Val substitution, together with mtrR and porB mutations. The existence of only one strain having the mosaic penA sequence indicated that ceftriaxone and cefixime resistance in Korea is mostly not associated with a mosaic penA sequence. Highly heterogeneous NG-MAST sequence types excluded the clonal expansion of a particular subtype.

Neisseria gonorrhoeae isolates with reduced susceptibility to cefixime and ceftriaxone: association with genetic polymorphisms in penA, mtrR, porB1b, and ponA.

The recent emergence and transmission of Neisseria gonorrhoeae isolates with reduced susceptibility to expanded-spectrum cephalosporins such as cefixime and ceftriaxone have been reported. The aim of this study was to determine the correlation of different polymorphisms in the penA, mtrR, porB1b (penB), and ponA genes of N. gonorrhoeae with reduced susceptibility to cefixime and ceftriaxone. Eighteen gonococcal isolates with reduced cefixime and ceftriaxone susceptibility (Cef(i)) and two susceptible isolates were characterized using serovar determination, antibiograms, N. gonorrhoeae multiantigen sequence typing (NG-MAST), and sequencing of penA, mtrR, porB1b, and ponA alleles. For the Cef(i) isolates (n = 18), the MICs of cefixime and ceftriaxone ranged between 0.032 to 0.38 mug/ml and 0.064 to 0.125 mug/ml, respectively. These isolates were assigned five different serovars and six divergent NG-MAST sequence types. Eleven isolates (61%) with higher MICs of cefixime and ceftriaxone contained a nearly identical penA mosaic allele and previously described polymorphisms in mtrR (a single nucleotide [A] deletion in the promoter), penB (mutations in porB1b encoding loop 3 of PorB1b), and ponA (ponA1 polymorphism). The remaining seven Cef(i) isolates (39%), which had somewhat lower MICs of cefixime and ceftriaxone, contained an aspartic acid insertion (Asp-345a) in PBP 2 in conjunction with alterations of 4 to 10 amino acid residues in the C-terminal region of the transpeptidase domain of penA. In conclusion, an unambiguous association between penA mosaic alleles, in conjunction with genetic polymorphisms in mtrR, porB1b, and ponA, and greater reduced susceptibility to cefixime and ceftriaxone was identified.