Mosaic penA Allele Research Articles

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.

Antimicrobial resistance and molecular characterisation of Neisseria gonorrhoeae isolates in Fukuoka, Japan, 1996-2016.

Antimicrobial resistance and molecular characteristics of Neisseria gonorrhoeae isolates obtained from 1996-2005 (n=200) and 2008-2016 (n=200) in Fukuoka, Japan, were examined. MICs were determined by agar dilution. Sequence types (STs) were examined using N. gonorrhoeae multiantigen sequence typing (NG-MAST). Sequencing of major extended-spectrum cephalosporin (ESC) resistance determinants (penA, mtrR and ponA) was performed. Increases in the proportion of gonococci with decreased susceptibility or resistance to cefixime (from 18.0% in 1996-2005 to 46.0% in 2008-2016) and ceftriaxone (from 2.5% to 4.0%) were observed. Gonococcal isolates also showed increased resistance to ciprofloxacin and azithromycin. The four most prevalent NG-MAST STs with a multidrug-resistant phenotype were ST2958 (n=18), ST1407 (n=14), ST6798 (n=12) and ST4015 (n=10). The number of isolates belonging to these four STs rose between the first and second period. Among the 54 isolates belonging to the four major STs, 42 (77.8%) contained a penA mosaic allele and 12 (22.2%) contained a penA non-mosaic allele. The sequence pattern types in the 42 isolates with a penA mosaic allele included type X (64.3%), type XXXIV (33.3%) and a novel pattern type (2.4%). In contrast, all 12 isolates with the penA non-mosaic allele included the sequence pattern type V. Neisseria gonorrhoeae isolates with decreased susceptibility or resistance to ESC have increased over the years. Four major STs with a multidrug-resistant phenotype were identified. These isolates contained a penA mosaic allele or a non-mosaic allele.

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.

Genomic surveillance of Neisseria gonorrhoeae to investigate the distribution and evolution of antimicrobial-resistance determinants and lineages.

The first extensively drug resistant (XDR) Neisseria gonorrhoeae strain with high resistance to the extended-spectrum cephalosporin ceftriaxone was identified in 2009 in Japan, but no other strain with this antimicrobial-resistance profile has been reported since. However, surveillance to date has been based on phenotypic methods and sequence typing, not genome sequencing. Therefore, little is known about the local population structure at the genomic level, and how resistance determinants and lineages are distributed and evolve. We analysed the whole-genome sequence data and the antimicrobial-susceptibility testing results of 204 strains sampled in a region where the first XDR ceftriaxone-resistant N. gonorrhoeae was isolated, complemented with 67 additional genomes from other time frames and locations within Japan. Strains resistant to ceftriaxone were not found, but we discovered a sequence type (ST)7363 sub-lineage susceptible to ceftriaxone and cefixime in which the mosaic penA allele responsible for reduced susceptibility had reverted to a susceptible allele by recombination. Approximately 85 % of isolates showed resistance to fluoroquinolones (ciprofloxacin) explained by linked amino acid substitutions at positions 91 and 95 of GyrA with 99 % sensitivity and 100 % specificity. Approximately 10 % showed resistance to macrolides (azithromycin), for which genetic determinants are less clear. Furthermore, we revealed different evolutionary paths of the two major lineages: single acquisition of penA X in the ST7363-associated lineage, followed by multiple independent acquisitions of the penA X and XXXIV in the ST1901-associated lineage. Our study provides a detailed picture of the distribution of resistance determinants and disentangles the evolution of the two major lineages spreading worldwide.

Molecular screening for Neisseria gonorrhoeae antimicrobial resistance markers in Nigerian men who have sex with men and transgender women.

Antimicrobial-resistant Neisseria gonorrhoeae (NG) is a global public health issue that threatens effectiveness of current treatments of NG. Increased use of nucleic acid amplification tests (NAATs) in lieu of cultures makes obtaining clinical isolates for susceptibility testing difficult and samples collected in commercial transport buffer for NAATs do not preserve viable organism, while molecular methods of assessing antibiotic susceptibility do not require viable organism. We evaluated 243 NG-positive samples in Aptima transport media including urine, oral, and rectal swabs from Nigerian men who have sex with men for markers to penicillinase-producing NG, ciprofloxacin ( GyrA and ParC mutations), and extended spectrum cephalosporins (ESCs, PenA mosaic [allele X], PonA, mtrR, PorB mutations) by real-time PCR. NG DNA was recovered in 75% (183/243) of samples. Of these, 93% (171/183) were positive for at least one resistance marker. We observed a prevalence of dual resistance markers to penicillin and ciprofloxacin at 46.2% (79/171). Six percent of samples (10/171) tested positive for the PenA mosaic (allele X) ESC marker. These data indicate that antibiotic-resistant NG is common in Nigeria. Laboratory and clinical capacity building in Nigeria should include development of methods to culture NG and determine antimicrobial susceptibility.

Antimicrobial resistance and molecular epidemiology using whole-genome sequencing of Neisseria gonorrhoeae in Ireland, 2014-2016: focus on extended-spectrum cephalosporins and azithromycin.

High-level resistance and treatment failures with ceftriaxone and azithromycin, the first-line agents for gonorrhoea treatment are reported and antimicrobial-resistant Neisseria gonorrhoeae is an urgent public health threat. Our aims were to determine antimicrobial resistance rates, resistance determinants and phylogeny of N. gonorrhoeae in Ireland, 2014-2016. Overall, 609 isolates from four University Hospitals were tested for susceptibility to extended-spectrum cephalosporins (ESCs) and azithromycin by the MIC Test Strips. Forty-three isolates were whole-genome sequenced based on elevated MICs. The resistance rate to ceftriaxone, cefixime, cefotaxime and azithromycin was 0, 1, 2.1 and 19%, respectively. Seven high-level azithromycin-resistant (HLAzi-R) isolates were identified, all susceptible to ceftriaxone. Mosaic penA alleles XXXIV, X and non-mosaic XIII, and G120K plus A121N/D/G (PorB1b), H105Y (MtrR) and A deletion (mtrR promoter) mutations, were associated with elevated ESC MICs. A2059G and C2611T mutations in 23S rRNA were associated with HLAzi-R and azithromycin MICs of 4-32mg/L, respectively. The 43 whole-genome sequenced isolates belonged to 31 NG-MAST STs. All HLAzi-R isolates belonged to MLST ST1580 and some clonal clustering was observed; however, the isolates differed significantly from the published HLAzi-R isolates from the ongoing UK outbreak. There is good correlation between previously described genetic antimicrobial resistance determinants and phenotypic susceptibility categories for ESCs and azithromycin in N. gonorrhoeae. This work highlights the advantages and potential of whole-genome sequencing to be applied at scale in the surveillance of antibiotic resistant strains of N. gonorrhoeae, both locally and internationally.

First description of a cefixime- and ciprofloxacin-resistant Neisseria gonorrhoeae isolate with mutations in key antimicrobial susceptibility-determining genes from the country of Georgia.

Antimicrobial resistance in Neisseria gonorrhoeae is a global health problem. Enhanced international collaborative surveillance and disease control are needed to reduce the global burden of this important pathogen. Currently the antimicrobial resistance properties and molecular mechanisms of multidrug-resistant N. gonorrhoeae in the Republic of Georgia represent a significant knowledge gap. Here we report the isolation of a strain of N. gonorrhoeae exhibiting resistance to cefixime and ciprofloxacin with reduced susceptibility to penicillin and tetracycline from a patient being treated at a Georgian medical centre. Notably, this isolate was found to contain a mosaic penA allele and to harbour mutations in genes conferring susceptibility to the β-lactam, cephalosporin, fluoroquinolone, macrolide and penicillin classes of antibiotic. To our knowledge, this is the first report to describe the key mutations conferring the antimicrobial resistance properties of an isolate of N. gonorrhoeae from Georgia.

Cooperative Recognition of Internationally Disseminated Ceftriaxone-Resistant Neisseria gonorrhoeae Strain.

Ceftriaxone remains a first-line treatment for patients infected by Neisseria gonorrhoeae in most settings. We investigated the possible spread of a ceftriaxone-resistant FC428 N. gonorrhoeae clone in Japan after recent isolation of similar strains in Denmark (GK124) and Canada (47707). We report 2 instances of the FC428 clone in Australia in heterosexual men traveling from Asia. Our bioinformatic analyses included core single-nucleotide variation phylogeny and in silico molecular typing; phylogenetic analysis showed close genetic relatedness among all 5 isolates. Results showed multilocus sequence type 1903; N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) 233; and harboring of mosaic penA allele encoding alterations A311V and T483S (penA-60.001), associated with ceftriaxone resistance. Our results provide further evidence of international transmission of ceftriaxone-resistant N. gonorrhoeae. We recommend increasing awareness of international spread of this drug-resistant strain, strengthening surveillance to include identifying treatment failures and contacts, and strengthening international sharing of data.

Neisseria cinerea with High Ceftriaxone MIC Is a Source of Ceftriaxone and Cefixime Resistance-Mediating penA Sequences in Neisseria gonorrhoeae.

Mosaic penA alleles have caused most of the cephalosporin resistance in Neisseria gonorrhoeae, but their evolution is mostly unknown. The penA gene from Neisseria cinerea strain AM1601 (ceftriaxone MIC, 1.0 μg/ml) caused ceftriaxone resistance (MIC, 1 μg/ml) in a ceftriaxone-susceptible gonococcal strain. The 3'-terminal half of AM1601 penA was almost identical to that of the ceftriaxone-resistant gonococcal GU140106 and FC428 strains. N. cinerea can serve as a reservoir of ceftriaxone resistance-mediating penA sequences that can be transferred to gonococci.

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.

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.

Alanine 501 Mutations in Penicillin-Binding Protein 2 from Neisseria gonorrhoeae: Structure, Mechanism, and Effects on Cephalosporin Resistance and Biological Fitness.

Resistance of Neisseria gonorrhoeae to expanded-spectrum cephalosporins such as ceftriaxone and cefixime has increased markedly in the past decade. The primary cephalosporin resistance determinant is a mutated penA gene, which encodes the essential peptidoglycan transpeptidase, penicillin-binding protein 2 (PBP2). Decreased susceptibility and resistance can be conferred by mosaic penA alleles containing upward of 60 amino acid changes relative to wild-type PBP2, or by nonmosaic alleles with relatively few mutations, the most important of which occurs at Ala501 located near the active site of PBP2. Recently, fully cefixime- and ceftriaxone-resistant clinical isolates that harbored a mosaic penA allele with an A501P mutation were identified. To examine the potential of mutations at Ala501 to increase resistance to expanded-spectrum cephalosporins, we randomized codon 501 in a mosaic penA allele and transformed N. gonorrhoeae to increased cefixime resistance. Interestingly, only five substitutions of Ala501 (A501V, A501T, A501P, A501R, and A501S) that increased resistance and preserved essential transpeptidase function were isolated. To understand their structural implications, these mutations were introduced into the nonmosaic PBP2-6140CT, which contains four C-terminal mutations present in PBP2 from the penicillin-resistant strain FA6140. The crystal structure of PBP2-6140CT-A501T was determined and revealed ordering of a loop near the active site and a new hydrogen bond involving Thr501 that connects the loop and the SxxK conserved active site motif. The structure suggests that increased rigidity in the active site region is a mechanism for cephalosporin resistance mediated by Ala501 mutations in PBP2.

Genetic Resistance Determinants for Cefixime and Molecular Analysis of Gonococci Isolated in Italy.

A strictly defined subset of gonococci (n = 65) isolated in Italy from 2011 to 2014 was characterized by antimicrobial susceptibility for cefixime (CFM) and ceftriaxone (CRO) and by sequencing of resistance determinant genes (penA, mtrR, porB1b, ponA) for extended-spectrum cephalosporins and Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST). The penA mosaic alleles XXXIV and XXXV were found in all resistant (R) and decreased susceptibility (DS) gonococci to CFM, except for one. They were associated with an adenine deletion in the mtrR promoter plus amino acid substitutions, H105Y or G45D, in the coding region and ponA L421P. The penA mosaic allele XXXIV, and one variant, was found exclusively among genogroup (G) 1407 and its closely related sequence types (STs), as in CFM-DS as well as in CFM-R isolates. Single or combined mutation patterns in penA, mtrR, porB1b, and ponA genes were associated with different CFM susceptibility patterns and NG-MAST STs. Genotyping and antimicrobial resistance (AMR) determinant analyses can be valuable to enhance the gonococcal AMR surveillance.

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.

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.

Within host evolution – what does it say about the initial infecting population?

Background: In February, 2012, a 32 year old African American, heterosexual man in Oklahoma presented with gonorrhea exhibiting reduced susceptibility to ceftriaxone, with a minimum inhibitory concentration (MIC) of 0.5 g/ml. No history of travel, drug use, or sex work was reported, and the individual denied having prior gonococcal infections. Methods & Materials: Whole genome sequencing of this isolate was performed using next-generation sequencing techniques (Roche 454 and Pacific Biosciences SMRT), and a suite of known gonococcal resistance determinants were examined to identify mutationspotentially associatedwith reduced susceptibility to ceftriaxone. Results: The penicillin-binding protein 2 gene (penA) has been associated with both reduced susceptibility and treatment failure to the cephalosporins due to the presence of a mosaic penA allele; however, this particular isolate possessed a non-mosaic penA allele not normally associated with decreased susceptibility to cephalosporins. Conclusion: These results suggest that while a mosaic penA allele can be predictive for reduced susceptibility to cephalosporins in N. gonorrhoeae, it is not absolutely necessary. In this case, it appears that other mutations are responsible for the reduced susceptibility to ceftriaxone.

Genomic epidemiology of Neisseria gonorrhoeae with reduced susceptibility to cefixime in the USA: a retrospective observational study.

SummaryBackgroundThe emergence of Neisseria gonorrhoeae with decreased susceptibility to extended spectrum cephalosporins raises the prospect of untreatable gonorrhoea. In the absence of new treatments, efforts to slow the increasing incidence of resistant gonococcus require insight into the factors that contribute to its emergence and spread. We assessed the relatedness between isolates in the USA and reconstructed likely spread of lineages through different sexual networks.MethodsWe sequenced the genomes of 236 isolates of N gonorrhoeae collected by the Centers for Disease Control and Prevention's Gonococcal Isolate Surveillance Project (GISP) from sentinel public sexually transmitted disease clinics in the USA, including 118 (97%) of the isolates from 2009–10 in GISP with reduced susceptibility to cefixime (cefRS) and 118 cefixime-susceptible isolates from GISP matched as closely as possible by location, collection date, and sexual orientation. We assessed the association between antimicrobial resistance genotype and phenotype and correlated phylogenetic clustering with location and sexual orientation.FindingsMosaic penA XXXIV had a high positive predictive value for cefRS. We found that two of the 118 cefRS isolates lacked a mosaic penA allele, and rechecking showed that these two were susceptible to cefixime. Of the 116 remaining cefRS isolates, 114 (98%) fell into two distinct lineages that have independently acquired mosaic penA allele XXXIV. A major lineage of cefRS strains spread eastward, predominantly through a sexual network of men who have sex with men. Eight of nine inferred transitions between sexual networks were introductions from men who have sex with men into the heterosexual population.InterpretationGenomic methods might aid efforts to slow the spread of antibiotic-resistant N gonorrhoeae through augmentation of gonococcal outbreak surveillance and identification of populations that could benefit from increased screening for aymptomatic infections.FundingAmerican Sexually Transmitted Disease Association, Wellcome Trust, National Institute of General Medical Sciences, and National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Antimicrobial susceptibility/resistance and genetic characteristics of Neisseria gonorrhoeae isolates from Poland, 2010-2012.

BackgroundIn Poland, gonorrhoea has been a mandatorily reported infection since 1948, however, the reported incidences are likely underestimated. No antimicrobial resistance (AMR) data for Neisseria gonorrhoeae has been internationally reported in nearly four decades, and data concerning genetic characteristics of N. gonorrhoeae are totally lacking. The aims of this study were to investigate the AMR to previously and currently recommended gonorrhoea treatment options, the main genetic resistance determinant (penA) for extended-spectrum cephalosporins (ESCs), and genotypic distribution of N. gonorrhoeae isolates in Poland in 2010-2012.MethodsN. gonorrhoeae isolates cultured in 2010 (n = 28), 2011 (n = 92) and 2012 (n = 108) in Warsaw and Bialystok, Poland, were examined using antimicrobial susceptibility testing (Etest), pyrosequencing of penA and N. gonorrhoeae multi-antigen sequence typing (NG-MAST).ResultsThe proportions of N. gonorrhoeae isolates showing resistance were as follows: ciprofloxacin 61%, tetracycline 43%, penicillin G 22%, and azithromycin 8.8%. No isolates resistant to ceftriaxone, cefixime or spectinomycin were found. However, the proportion of isolates with an ESC MIC = 0.125 mg/L, i.e. at the resistance breakpoint, increased significantly from none in 2010 to 9.3% and 19% in 2012 for ceftriaxone and cefixime, respectively. Furthermore, 3.1% of the isolates showed multidrug resistance, i.e., resistance to ciprofloxacin, penicillin G, azithromycin, and decreased susceptibility to cefixime (MIC = 0.125 mg/L). Seventy-six isolates (33%) possessed a penA mosaic allele and 14 isolates (6.1%) contained an A501V/T alteration in penicillin-binding protein 2. NG-MAST ST1407 (n = 58, 25% of isolates) was the most prevalent ST, which significantly increased from 2010 (n = 0) to 2012 (n = 46; 43%).ConclusionsIn Poland, the diversified gonococcal population displayed a high resistance to most antimicrobials internationally previously recommended for gonorrhoea treatment and decreasing susceptibility to the currently recommended ESCs. The decreasing susceptibility to ESCs was mostly due to the introduction of the internationally spread multidrug-resistant NG-MAST ST1407 in 2011. It is essential to promptly revise the gonorrhoea treatment guidelines, improve the gonorrhoea laboratory diagnostics, and implement quality assured surveillance of gonococcal AMR (ideally also treatment failures) in Poland.

Neisseria gonorrhoeae and extended-spectrum cephalosporins in California: surveillance and molecular detection of mosaic penA.

BackgroundThe spread of Neisseria gonorrhoeae strains with mosaic penA alleles and reduced susceptibility to extended-spectrum cephalosporins is a major public health problem. While much work has been performed internationally, little is known about the genetics or molecular epidemiology of N. gonorrhoeae isolates with reduced susceptibility to extended-spectrum cephalosporins in the United States. The majority of N. gonorrhoeae infections are diagnosed without a live culture. Molecular tools capable of detecting markers of extended-spectrum cephalosporin resistance are needed.MethodsUrethral N. gonorrhoeae isolates were collected from 684 men at public health clinics in California in 2011. Minimum inhibitory concentrations (MICs) to ceftriaxone, cefixime, cefpodoxime and azithromycin were determined by Etest and categorized according to the U.S. Centers for Disease Control 2010 alert value breakpoints. 684 isolates were screened for mosaic penA alleles using real-time PCR (RTPCR) and 59 reactive isolates were subjected to DNA sequencing of their penA alleles and Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST). To increase the specificity of the screening RTPCR in detecting isolates with alert value extended-spectrum cephalosporin MICs, the primers were modified to selectively amplify the mosaic XXXIV penA allele.ResultsThree mosaic penA alleles were detected including two previously described alleles (XXXIV, XXXVIII) and one novel allele (LA-A). Of the 29 isolates with an alert value extended-spectrum cephalosporin MIC, all possessed the mosaic XXXIV penA allele and 18 were sequence type 1407, an internationally successful strain associated with multi-drug resistance. The modified RTPCR detected the mosaic XXXIV penA allele in urethral isolates and urine specimens and displayed no amplification of the other penA alleles detected in this study.ConclusionN. gonorrhoeae isolates with mosaic penA alleles and reduced susceptibility to extended-spectrum cephalosporins are currently circulating in California. Isolates with the same NG-MAST ST, penA allele and extended-spectrum cephalosporin MICs have caused treatment failures elsewhere. The RTPCR assay presented here may be useful for the detection of N. gonorrheoae isolates and clinical specimens with reduced extended-spectrum cephalosporin MICs in settings where antimicrobial susceptibility testing is unavailable. In an era of increasing antimicrobial resistance and decreasing culture capacity, molecular assays capable of detecting extended-spectrum cephalosporin of resistance are essential to public health.