penA Allele Research Articles

Recognition of the β-lactam carboxylate triggers acylation of Neisseria gonorrhoeae penicillin-binding protein 2

Resistance of Neisseria gonorrhoeae to extended-spectrum cephalosporins (ESCs) has become a major threat to human health. The primary mechanism by which N. gonorrhoeae becomes resistant to ESCs is by acquiring a mosaic penA allele, encoding penicillin-binding protein 2 (PBP2) variants containing up to 62 mutations compared with WT, of which a subset contribute to resistance. To interpret molecular mechanisms underpinning cephalosporin resistance, it is necessary to know how PBP2 is acylated by ESCs. Here, we report the crystal structures of the transpeptidase domain of WT PBP2 in complex with cefixime and ceftriaxone, along with structures of PBP2 in the apo form and with a phosphate ion bound in the active site at resolutions of 1-7-1.9 Å. These structures reveal that acylation of PBP2 by ESCs is accompanied by rotation of the Thr-498 side chain in the KTG motif to contact the cephalosporin carboxylate, twisting of the β3 strand to form the oxyanion hole, and rolling of the β3-β4 loop toward the active site. Recognition of the cephalosporin carboxylate appears to be the key trigger for formation of an acylation-competent state of PBP2. The structures also begin to explain the impact of mutations implicated in ESC resistance. In particular, a G545S mutation may hinder twisting of β3 because its side chain hydroxyl forms a hydrogen bond with Thr-498. Overall, our data suggest that acylation is initiated by conformational changes elicited or trapped by binding of ESCs and that these movements are restricted by mutations associated with resistance against ESCs.

Emergence and Spread of Cephalosporin-Resistant Neisseria gonorrhoeae with Mosaic penA Alleles, South Korea, 2012-2017.

In South Korea, surveillance of antimicrobial drug resistance in Neisseria gonorrhoeae is extremely limited. We describe the emergence and subsequent national spread of N. gonorrhoeae strains with mosaic penA alleles associated with decreased susceptibility and resistance to extended-spectrum cephalosporins. From 2012 through 2017, the proportion of mosaic penA alleles in gonococcal-positive nucleic acid amplification test (NAAT) specimens across South Korea increased from 1.1% to 23.9%. Gonococcal strains with mosaic penA alleles emerged in the international hubs of Seoul in Gyeonggi Province and Busan in South Gyeongsang Province and subsequently spread across South Korea. Most common was mosaic penA-10.001 (n = 572 isolates; 94.7%), which is associated with cefixime resistance. We also identified mosaic penA-34.001 and penA-60.001, both of which are associated with multidrug-resistant gonococcal strains and spread of cefixime and ceftriaxone resistance. Implementation of molecular resistance prediction from N. gonorrhoeae–positive nucleic acid amplification test specimens is imperative in South Korea and internationally.

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.

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.

The invasive MenC cc103 lineage with penicillin reduced susceptibility persisting in Brazil

Penicillin is the antibiotic of choice for the treatment of meningococcal infections, and mutations in penA gene are involved with reduced susceptibility (penI) emergence to this antibiotic. This study aimed to characterize the penA allelic diversity, their association with penI phenotype and distribution among prevalent meningococci serogroups in Brazil. The entire penA from 49 invasive strains of distinct serogroups circulating in Brazil for more than two decades were obtained by PCR and sequencing. Additionally, the penA from 22 publicly available complete Neisseria meningitidis genomes from Brazil were included in the study. The allelic diversity was determined and a genetic tree was built using the penA sequence alignment. The penicillin MIC was obtained by the E-Test method. In general, the identified penA alleles correlated with the observed penI phenotype. The canonical penA1 was the most prevalent allele, however, several altered penA were also identified in strains presenting increased penicillin MICs. It was identified a new penA amino acid position (residue 480) that possibly influence the penicillin MIC in some strains. Interestingly, the altered penA14 was found in penI invasive MenC cc103 strains spread in Brazil and persisting since 2011, indicating that the biological cost imposed by penI phenotype can be ameliorated by particular features present in this lineage, which represents an additional public health threat.

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.

Emergence of meningococci with reduced susceptibility to third-generation cephalosporins.

Reduced susceptibility to penicillin G in Neisseria meningitidis is mainly due to alterations in PBP2 encoded by the penA gene. However, this phenotype was not associated with reduced susceptibility to third-generation cephalosporins (C3Gs). We aimed to study the emergence of meningococci with reduced susceptibility to C3Gs (MIC >0.06 mg/L) in France since 2012. Invasive meningococcal isolates were typed by MLST and penA sequencing and their antibiotic susceptibility was tested. Isolates with reduced susceptibility to C3Gs represented 2% of all invasive isolates from 2012-15, but were absent before. They harboured a new penA allele, penA327, that was also detected in isolates from urethritis cases and in gonococci. Surveillance of these isolates should be enhanced as they may jeopardize the use of C3Gs in the management of invasive meningococcal disease.

Emerging Extended-Spectrum Cephalosporin Resistance in Neisseria gonorrhoeae

Over the last several decades, Neisseria gonorrhoeae has sequentially developed resistance to the agents recommended by the CDC and the Public Health Service for the treatment of gonorrhea. During the 1970s and through the 1990s, penicillin, tetracyclines, and the fluoroquinolones were recommended and subsequently removed as the therapy of choice for gonorrhea, leaving intramuscular ceftriaxone and the oral cephalosporin cefixime as the sole agents recommended for treatment of gonorrhea in 2010. In the late 1990s, treatment failures following oral cefixime therapy were recognized in Japan and the Far East, and subsequently, cefixime treatment failures were reported from the United Kingdom, Europe, and the United States. Gonococcal isolates from these cases showed diminished susceptibility to both cefixime and ceftriaxone, and molecular studies have shown that N. gonorrhoeae isolates with elevated cephalosporin MICs have genetic alterations primarily in the chromosomal penA gene, which encodes penicillin-binding protein 2. The altered proteins bind cephalosporins less efficiently, and isolates phenotypically demonstrate diminished cephalosporin susceptibility and elevated cefixime and ceftriaxone MICs. The penA alleles in these strains have a “mosaic” composition, as they contain nucleic acid sequences that were acquired from commensal Neisseria species. The strains also have genetic alterations in several other chromosomal loci that contribute to incremental cephalosporin resistance. With the utility of extended-spectrum cephalosporins for gonorrhea treatment being threatened, older agents (e.g., gentamicin and ertapenem) and newly developed antimicrobials (e.g., solithromycin) are now being evaluated as possible treatment options. The CDC has issued guidelines for the public health response to the emergence of cephalosporin-resistant N. gonorrhoeae. This plan includes improvements in gonococcal surveillance, identification by clinicians of “probable” and “suspected” treatment failures, the re-establishment of culture-based testing, and the timely performance of antimicrobial susceptibility testing on isolates obtained from treatment failures. Potential problems central to implementation of the response plan are also addressed.

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.

Role of penA polymorphisms for penicillin susceptibility in Neisseria lactamica and Neisseria meningitidis

In meningococci, reduced penicillin susceptibility is associated with five specific mutations in the transpeptidase region of penicillin binding protein 2 (PBP2). We showed that the same set of mutations was present in 64 of 123 Neisseria lactamica strains obtained from a carriage study (MIC range: 0.125–2.0mg/L). The PBP2 encoding penA alleles in these strains were genetically similar to those found in intermediate resistant meningococci suggesting frequent interspecies genetic exchange. Fifty-six N. lactamica isolates with mostly lower penicillin MICs (range: 0.064–0.38mg/L) exhibited only three of the five mutations. The corresponding penA alleles were unique to N. lactamica and formed a distinct genetic clade. PenA alleles with no mutations on the other hand were unique to meningococci. Under penicillin selective pressure, genetic transformation of N. lactamica penA alleles in meningococci was only possible for alleles encoding five mutations, but not for those encoding three mutations; the transfer resulted in MICs comparable to those of meningococci harboring penA alleles that encoded PBP2 with five mutations, but considerably lower than those of the corresponding N. lactamica donor strains. Due to a transformation barrier the complete N. lactamica penA could not be transformed into N. meningitidis. In summary, penicillin MICs in N. lactamica were associated with the number of mutations in the transpeptidase region of PBP2. Evidence for interspecific genetic transfer was only observed for penA alleles associated with higher MICs, suggesting that alleles encoding only three mutations in the transpeptidase region are biologically not effective in N. meningitidis. Factors other than PBP2 seem to be responsible for the high levels of penicillin resistance in N. lactamica. A reduction of penicillin susceptibility in N. meningitidis by horizontal gene transfer from N. lactamica is unlikely to happen.

Antimicrobial susceptibility of Neisseria gonorrhoeae isolates from symptomatic men attending the Nanjing sexually transmitted diseases clinic (2011-2012): genetic characteristics of isolates with reduced sensitivity to ceftriaxone.

BackgroundEvolving gonococcal antimicrobial resistance (AMR) poses a serious threat to public health. The aim of this study was to: update antimicrobial susceptibility data of Neisseria gonorrhoeae recently isolated in Nanjing, China and identify specific deteminants of antimicrobial resistance and gentoypes of isolates with decreased sensitivity to ceftriaxone.Methods334 N. gonorrhoeae isolates were collected consecutively from symptomatic men attending the Nanjing STD Clinic between April 2011 and December 2012. The minimum inhibitory concentrations (MICs) for penicillin, tetracycline, ciprofloxacin, spectinomycin and ceftriaxone were determined by agar plate dilution for each isolate. Penicillinase-producing N. gonorrhoeae (PPNG) and tetracycline-resistant N. gonorrhoeae (TRNG) were examined and typed for β-lactamase and tetM encoding plasmids respectively. Isolates that displayed elevated MICs to ceftriaxone (MIC ≥0.125 mg/L) were also tested for mutations in penA, mtrR, porB1b, ponA and pilQ genes and characterized by Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST).Results98.8% (330/334) of N. gonorrhoeae isolates were resistant to ciprofloxacin; 97.9% (327/334) to tetracycline and 67.7% (226/334) to penicillin. All isolates were susceptible to ceftriaxone (MIC ≤0.25 mg/L) and spectinomycin (MIC ≤32 mg/L). Plasmid mediated resistance was exhibited by 175/334 (52%) of isolates: 120/334 (36%) of isolates were PPNG and 104/334 (31%) were TRNG. 90.0% (108/120) of PPNG isolates carried the Asia type β-lactamase encoding plasmid and 96% (100/104) of TRNG isolates carried the Dutch type tetM containing plasmid. Elevated MICs for ceftriaxone were present in 15 (4.5%) isolates; multiple mutations were found in penA, mtrR, porB1b and ponA genes. The 15 isolates were distributed into diverse NG-MAST sequence types; four different non-mosaic penA alleles were identified, including one new type.ConclusionsN. gonorrhoeae isolates in Nanjing generally retained similar antimicrobial resistance patterns to isolates obtained five years ago. Fluctuations in resistance plasmid profiles imply that genetic exchange among gonococcal strains is ongoing and is frequent. Ceftriaxone and spectinomycin remain treatments of choice of gonorrhea in Nanjing, however, decreased susceptibility to ceftriaxone and rising MICs for spectinomycin of N. gonorrhoeae isolates underscore the importance of maintaining surveillance for AMR (both phenotypic and genotypic).Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-014-0622-0) contains supplementary material, which is available to authorized users.

Surveillance of invasive Neisseria meningitidis with a serogroup Y update, Sweden 2010 to 2012.

An increase of invasive meningococcal disease caused by Neisseria meningitidis serogroup Y has been noted in Sweden since 2005, and to a lower extent throughout Europe. The present study describes the epidemiology of invasive N. meningitidis isolates in Sweden in the period between 2010 and 2012, with a focus on serogroup Y. We also aimed to find an optimal molecular typing scheme for both surveillance and outbreak investigations. All invasive N. meningitidis isolates in Sweden during the study period (n=208) were genetically characterised. Serogroup Y predominated with 22/57, 31/61 and 44/90 of all invasive isolates (incidence 0.23, 0.33 and 0.46 per 100,000 population) in 2010, 2011 and 2012 respectively. In each of these years, 15/22, 22/31 and 19/44 of serogroup Y isolates were genetically clonal (Y: P1.5–2,10–1,36–2: F4–1: ST-23(cc23), ‘porB allele 3–36, fHbp allele 25 and penA allele 22). Our findings further support those of others that currently recommended FetA typing could be replaced by FHbp. Moreover, in line with a previous study that we conducted, the current results indicate that highly variable multilocus variable-number tandem repeat analysis (HV-MLVA) can be used as a first-hand rapid method for small outbreak investigations.

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.