Increasing Azithromycin Resistance in Neisseria gonorrhoeae Due to NG-MAST 12302 Clonal Spread in Canada, 2015 to 2018.

Azithromycin-resistant (AZIR) gonorrhea has been steadily increasing in Canada over the past decade, which is cause for alarm, as azithromycin (AZI) has been part of the combination therapy recommended by the Canadian Guidelines on Sexually Transmitted Infections (CGSTI) since 2012. Neisseria gonorrhoeae with AZI MICs ≥1 mg/L collected between 2015 and 2018 as part of the Gonococcal Antimicrobial Surveillance Program-Canada underwent antimicrobial susceptibility testing, molecular typing, and whole-genome sequencing. Regional, demographic, and clinical isolation site comparisons were made to aid in our understanding of AZI susceptibility trending. We identified 3,447 N. gonorrhoeae with AZI MICs of ≥1 mg/L in Canada, which increased from 6.3% in 2015 to 26.5% of isolates in 2018. Central Canada had the highest proportion, rising from 9.2% in 2015 to 31.2% in 2018. We identified 273 different N. gonorrhoeae multiantigen sequence types (NG-MAST) among these isolates, with ST-12302 the most prevalent (50.9%). Whole-genome sequencing identified the Neisseria lactamica-like mosaic mtr locus as the mechanism of AZIR in isolates of ST-12302 and isolates genetically similar (differing by ≤5 bp), designated the ST-12302 genogroup, accounting for 65.2% of study isolates which were originally identified in central Canada but spread to other regions by 2018. Genomic analysis indicated that AZIR in Canadian N. gonorrhoeae expanded rapidly due to clonal spread of the ST-12302 genogroup. The rapid expansion of this AZIR clonal group in all regions of Canada is of concern. CGSTI are currently under review to address the increase in AZIR in Canada.

16S ribosomal assay for early diagnosis of gonococcal meningitis with negative CSF culture: A case report.

Gonococcal meningitis is an exceedingly rare infectious disease, and if not diagnosed and treated in time, it can be severe. We present a case of gonococcal meningitis occurring in a 31-year old healthy woman. She was admitted with fever and persistent headache without urogenital symptoms. Blood cultures were positive and identified as N.gonorrhoeae, but CSF and cervical secretions cultures were both negative. Further testing confirmed the presence of N.gonorrhoeae by 16S ribosomal gene amplification and sequencing in all samples. These results suggest that the case may be a disseminated infection caused by untreated gonorrhea. Our case also shows that nucleic acid detection plays an important role in the rapid and precise diagnosis of gonococcal meningitis and in finding the origin of the pathogen.

Molecular Mechanisms of Drug Resistance and Epidemiology of Multidrug-Resistant Variants of Neisseria gonorrhoeae.

The paper presents various issues related to the increasing drug resistance of Neisseria gonorrhoeae and the occurrence and spread of multidrug-resistant clones. One of the most important is the incidence and evolution of resistance mechanisms of N. gonorrhoeae to beta-lactam antibiotics. Chromosomal resistance to penicillins and oxyimino-cephalosporins and plasmid resistance to penicillins are discussed. Chromosomal resistance is associated with the presence of mutations in the PBP2 protein, containing mosaic variants and nonmosaic amino acid substitutions in the transpeptidase domain, and their correlation with mutations in the mtrR gene and its promoter regions (the MtrCDE membrane pump repressor) and in several other genes, which together determine reduced sensitivity or resistance to ceftriaxone and cefixime. Plasmid resistance to penicillins results from the production of beta-lactamases. There are different types of beta-lactamases as well as penicillinase plasmids. In addition to resistance to beta-lactam antibiotics, the paper covers the mechanisms and occurrence of resistance to macrolides (azithromycin), fluoroquinolones and some other antibiotics. Moreover, the most important epidemiological types of multidrug-resistant N. gonorrhoeae, prevalent in specific years and regions, are discussed. Epidemiological types are defined as sequence types, clonal complexes and genogroups obtained by various typing systems such as NG-STAR, NG-MAST and MLST. New perspectives on the treatment of N. gonorrhoeae infections are also presented, including new drugs active against multidrug-resistant strains.

The Mosaic mtr Locus as Major Genetic Determinant of Azithromycin Resistance of Neisseria gonorrhoeae-Germany, 2018.

Within the German Gonococcal Resistance Network's (GORENET) Neisseria gonorrhoeae (NG) sample collection, azithromycin-resistant NG isolates increased from 4.3% in 2016 to 9.2% in 2018. We aim to understand this observed increase using whole genome sequencing of NG isolates combined with epidemiological and clinical data. Reduced susceptibility to azithromycin in 2018 was predominately clonal (NG multiantigen sequence typing G12302) and could mainly be attributed to the recently described mosaic-like mtr locus. Our data suggest that, together with horizontal gene transfer of resistance determinants and well-established point mutations, international spread of resistant lineages plays a major role regarding azithromycin resistance in Germany.

Antimicrobial resistance and molecular epidemiological typing of Neisseria gonorrhoeae isolates from Kyrgyzstan in Central Asia, 2012 and 2017.

BACKGROUND: Gonorrhoea and antimicrobial resistance (AMR) in Neisseria gonorrhoeae are significant public health concerns globally. Nearly no gonococcal AMR data are available from Central Asia, and no data from Kyrgyzstan has been published. We examined, for the first time, AMR and molecular epidemiology of N. gonorrhoeae isolates cultured in Kyrgyzstan in 2012 and 2017, in order to inform refinements of the Kyrgyz national gonorrhoea management guidelines. METHODS: N. gonorrhoeae isolates cultured in 2012 (n = 84) and 2017 (n = 72) in Kyrgyzstan were examined. MICs of nine antimicrobials were determined using Etest and, where available, clinical breakpoints from the EUCAST were applied. N. gonorrhoeae multiantigen sequence typing (NG-MAST) was also performed. RESULTS: The overall resistance levels were high to ciprofloxacin (88.5%), tetracycline (56.9%), benzylpenicillin (39.1%), and kanamycin (4.7%). Resistance to cefixime (0.6%, n = 1 isolate), azithromycin (0.6%, n = 1), and gentamicin (0.6%, n = 1) was rare. No resistance to ceftriaxone or spectinomycin was found. However, the proportion of isolates with decreased susceptibility (MIC = 0.125 mg/L) to ceftriaxone and cefixime was 12.8 and 11.5%, respectively. Gonococcal isolates were assigned 69 sequence types, of which 52 (75.4%) were new. CONCLUSIONS: The gonococcal population in Kyrgyzstan in 2012 and 2017 showed a high genetic diversity. Ceftriaxone, 500-1000 mg, in combination with azithromycin 2 g or doxycycline, particularly when chlamydial infection has not been excluded, should be recommended as empiric first-line treatment. Spectinomycin 2 g could be an alternative treatment, and given with azithromycin 2 g if pharyngeal gonorrhoea has not been excluded. Fluoroquinolones, aminoglycosides, benzylpenicillin, or tetracyclines should not be used for empiric treatment of gonorrhoea in Kyrgyzstan. Timely updating and high compliance to national gonorrhoea treatment guidelines based on quality-assured AMR data is imperative. Expanded and improved gonococcal AMR surveillance in Kyrgyzstan is crucial.

Multiresistant Neisseria gonorrhoeae: a new threat in second decade of the XXI century.

Neisseria gonorrhoeae is an etiologic agent of gonorrhoea, one of the most common sexually transmitted diseases caused by bacteria. For many years, infections caused by N. gonorrhoeae were considered to be relatively easy to treat; however, resistance has emerged successively to all therapeutic agents used in treatment of the disease, e.g., penicillin, ciprofloxacin or azithromycin. Currently, the global problem is the emergence and a threat of spread of N. gonorrhoeae strains resistant to extended-spectrum cephalosporins (ESC), such as injectable ceftriaxone and oral-used cefixime. Especially, dangerous are multi-resistant strains resistant simultaneously to ESC and azithromycin. Three strains with high-level resistance to azithromycin and resistant to ESC were first time isolated in 2018. Moreover, in 2018, the first ESBL was described in N. gonorrhoeae and that makes the threat of appearing the ESBL mechanism of resistance in N. gonorrhoeae more real, even though the strain was sensitive to ceftriaxone. Molecular typing revealed that variants resistant to ESC occurred also among strains belonging to epidemic clonal complex CC1 (genogroup G1407) distinguished in NG-MAST typing system. The G1407 genogroup, in particular the ST1407 sequence type, is currently dominant in most European countries. The presence of different mechanisms of drug resistance significantly affects clinical practice and force changes in treatment regimens and introduction of new drugs.

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.

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.

Epidemiology, molecular characterisation and antimicrobial susceptibility of Neisseria gonorrhoeae isolates in Madrid, Spain, in 2016.

With the aim to elucidate gonococcal antimicrobial resistance (AMR)-risk factors, we undertook a retrospective analysis of the molecular epidemiology and AMR of 104 Neisseria gonorrhoeae isolates from clinical samples (urethra, rectum, pharynx and cervix) of 94 individuals attending a sexually transmitted infection clinic in Madrid (Spain) from July to October 2016, and explored potential links with socio-demographic, behavioural and clinical factors of patients. Antimicrobial susceptibility was determined by E-tests, and isolates were characterised by N. gonorrhoeae multi-antigen sequence typing. Penicillin resistance was recorded for 15.4% of isolates, and most were susceptible to tetracycline, cefixime and azithromycin; a high incidence of ciprofloxacin resistance (~40%) was found. Isolates were grouped into 51 different sequence types (STs) and 10 genogroups (G), with G2400, ST5441, ST2318, ST12547 and G2992 being the most prevalent. A significant association (P = 0.015) was evident between HIV-positive MSM individuals and having a ciprofloxacin-resistant strain. Likewise, a strong association (P = 0.047) was found between patient age of MSM and carriage of isolates expressing decreased susceptibility to azithromycin. A decrease in the incidence of AMR gonococcal strains and a change in the strain populations previously reported from other parts of Spain were observed. Of note, the prevalent multi-drug resistant genogroup G1407 was represented by only three strains in our study, while the pan-susceptible clones such as ST5441, and ST2318, associated with extragenital body sites were the most prevalent.

Cephalosporin-Resistant Neisseria gonorrhoeae Clone, China.

Cephalosporin-resistant Neisseria gonorrhoeae is a major public health concern. N. gonorrhoeae of multiantigen sequence type G1407 and multilocus sequence type 1901 is an internationally spreading cephalosporin-resistant clone. We detected 4 cases of infection with this clone in China and analyzed resistance determinants by using N. gonorrhoeae sequence typing for antimicrobial resistance.

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 4-32 mg/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.

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.

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

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

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

A total of 128 Neisseria gonorrhoeae isolates were consecutively obtained in Changsha, China, between April 2015 and June 2016. The minimum inhibitory concentrations (MICs) of ceftriaxone were measured by agar dilution method for each isolate. Ceftriaxone resistance determinants involving penA, mtrR, porB and ponA were amplified by PCR, and the PCR products were sequenced. N. gonorrhoeae multi-antigen sequence typing (NG-MAST) was conducted for genotyping. The results exhibited 11% (14/128) of isolates were reduced susceptibility or resistant to ceftriaxone. One isolate with a susceptibility MIC of 0.03 mg/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.

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

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

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

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

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

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

Emergence of decreased susceptibility and resistance to extended-spectrum cephalosporins in Neisseria gonorrhoeae in Korea.

OBJECTIVES: Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a major concern globally; however, no comprehensive AMR data for gonococcal isolates cultured after 2006 in Korea have been published internationally. We determined the susceptibility of N. gonorrhoeae isolates cultured in 2011-13, the mechanism of extended-spectrum cephalosporin (ESC) resistance and the molecular epidemiology of gonococcal strains in Korea. METHODS: In 2011-13, 210 gonococcal isolates were collected in Korea and their AMR profiles were examined by the agar dilution method. The penA, mtrR, penB, ponA and pilQ genes were sequenced in 25 isolates that were resistant to ESCs and 70 randomly selected isolates stratified by year. For molecular epidemiology, N. gonorrhoeae multiantigen sequence typing and MLST were performed. RESULTS: None of the N. gonorrhoeae isolates was susceptible to penicillin G and most were resistant to tetracycline (50%) and ciprofloxacin (97%). The rates of resistance to ceftriaxone, azithromycin, cefpodoxime and cefixime were 3%, 5%, 8% and 9%, respectively. However, all isolates were susceptible to spectinomycin. Twenty-one (84%) of the 25 ESC-resistant isolates contained the non-mosaic PBP2 XIII allele; however, the remaining 4 (16%) possessed the mosaic PBP2 X allele, which has been previously associated with ESC resistance including treatment failures. CONCLUSIONS: In Korea, susceptibility to spectinomycin remains high. However, the recent emergence of ESC-resistant N. gonorrhoeae strains, including strains possessing the PBP2 mosaic X and non-mosaic XIII alleles, is a major concern and enhanced AMR surveillance is necessary to prevent transmission of these strains.

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

OBJECTIVES: 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. METHODS: 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. RESULTS: All isolates were resistant to ciprofloxacin, 42.4% produced ß-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. CONCLUSIONS: 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.

Treatment failure with 2 g of azithromycin (extended-release formulation) in gonorrhoea in Japan caused by the international multidrug-resistant ST1407 strain of Neisseria gonorrhoeae.

OBJECTIVES: Antimicrobial resistance in Neisseria gonorrhoeae is a major public health concern globally. We report the first verified treatment failure of gonorrhoea with 2 g of azithromycin (extended-release formulation) in Japan and characteristics of the corresponding N. gonorrhoeae isolates. METHODS: Pre- and post-treatment isolates (n = 4) were investigated by Etest for antimicrobial susceptibility. The isolates were examined for molecular epidemiology by multilocus sequence typing (MLST), N. gonorrhoeae multi-antigen sequence typing (NG-MAST) and multiple-locus variable-number tandem repeat analysis (MLVA), and for the presence of azithromycin resistance determinants (23S rRNA gene mutations, erm genes and mtrR mutations). RESULTS: All isolates were resistant to azithromycin (MIC 4 mg/L) and ciprofloxacin, but remained susceptible to cefixime, ceftriaxone and spectinomycin. All isolates were assigned to MLST ST1901 and NG-MAST ST1407 and three of four isolates possessed MLVA profile 8-3-21-16-1. All isolates contained the previously described C2599T mutation (N. gonorrhoeae numbering) in all four 23S rRNA alleles and the previously described single-nucleotide (A) deletion in the mtrR promoter region. CONCLUSIONS: This verified treatment failure occurred in a patient infected with an MLST ST1901/NG-MAST ST1407 strain of N. gonorrhoeae. While this international strain commonly shows resistance or decreased susceptibility to multiple antimicrobials, including extended-spectrum cephalosporins, the strain reported here remained fully susceptible to the latter antimicrobials. Hence, two subtypes of azithromycin-resistant gonococcal MLST ST1901/NG-MAST ST1407 appear to have evolved and to be circulating in Japan. Azithromycin should not be recommended as a single antimicrobial for first-line empirical treatment of gonorrhoea.