Gonorrhoeae Isolates Research Articles

Emergence and Characterization of a Ceftriaxone-Resistant Neisseria gonorrhoeae FC428 Clone Evolving Moderate-Level Resistance to Azithromycin in Shenzhen, China.

We here described a ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone (YL201) with moderate-level resistance to azithromycin in Shenzhen, South China in 2020. The NG-STAR type of YL201 is ST2238, containing a mosaic penA-60.001 allele, which is a typical characteristic of FC428 clone. YL201 harbours four copies of the 23S rRNA C2611T mutation, conferring moderate-level resistance to azithromycin. The MLST type is ST1600, identical with two N. gonorrhoeae FC428 clones identified in Hangzhou. Genome-wide phylogeny analysis demonstrates that YL201 is clustered with other FC428 clones from Hangzhou (South-east China) and Chengdu (South-west China). Isolates within this cluster have relatively higher MIC for ceftriaxone and display closely related MLST STs (ST1600 and ST7363) but are different from the ST of typical FC428 clone (ST1903). As ST1600 and ST7363 are common STs in Shenzhen, the further spread of FC428 clones may increase the severity of gonococcal resistance. In summary, identifying a multidrug-resistant (MDR) N. gonorrhoeae isolate in Shenzhen showed FC428 clones have undergone further transmission in China and presented more extensive and concerning antimicrobial resistance (AMR) characteristics during the spread.

Antimicrobial Susceptibility of Ertapenem in Neisseria gonorrhoeae Isolates Collected Within the China Gonococcal Resistance Surveillance Programme (China-GRSP) 2018

PurposeThis study aimed to determine the minimum inhibitory concentrations (MICs) of ertapenem on Neisseria gonorrhoeae collected from eight Chinese provinces in 2018.MethodsThe MICs of ertapenem on 503 Neisseria gonorrhoeae isolates (415 isolates selected randomly and 88 isolates selected with preference) were measured using the agar dilution method. For comparison, the MICs of ceftriaxone and azithromycin were detected.ResultsAmong 415 randomly selected isolates, the MIC range for ertapenem was from ≤0.008 mg/L to 0.5 mg/L. The corresponding MIC50 and MIC90 were 0.06 and 0.125 mg/L, respectively. Twelve of 415 isolates (2.9%) exhibited MIC values ≥0.25 mg/L, and only one isolate (0.2%) had a MIC of 0.5 mg/L. By comparing all 503 tested isolates, a correlation of r = 0.487 (P <0.001) between ertapenem and ceftriaxone MIC was observed, and the correlation between MICs of ertapenem and azithromycin was low (r = −0.12, P = 0.007). In 24 ceftriaxone-decreased susceptibility isolates, four isolates (16.7%) showed a MIC ≥0.25 mg/L for ertapenem. In 85 azithromycin resistant isolates, three isolates (3.5%) showed a MIC ≥0.25 mg/L for ertapenem.ConclusionThe in vitro results suggest that ertapenem has satisfactory susceptibility in isolates collected from eight provinces in China; hence, it might be a promising treatment option for resistant gonococcal infections.

Antimicrobial susceptibility of Neisseria gonorrhoeae isolates and syndromic treatment of men with urethral discharge in Kingston, Jamaica, 2018–19

ObjectivesTo quantitatively determine the antimicrobial susceptibility of clinical Neisseria gonorrhoeae isolates from men with urethral discharge in Jamaica and to describe the syndromic treatment therapies administered.MethodsUrethral eSwabs (Copan) were collected from 175 men presenting with urethral discharge to the Comprehensive Health Centre STI Clinic, Kingston, Jamaica. Clinical information was collected and MICs of eight antimicrobials were determined for N. gonorrhoeae isolates (n = 96) using Etest and interpreted using CLSI criteria.ResultsThe median age of the subjects was 28 years (range: 18–73 years) with a median of 2 sexual partners (range: 1–25) per male in the previous 3 months. All examined N. gonorrhoeae isolates were susceptible to ceftriaxone (96/96), azithromycin (91/91), cefixime (91/91) and spectinomycin (91/91). For ciprofloxacin and gentamicin, respectively, 98.9% (91/92) and 91.3% (84/92) of the isolates were susceptible and 1.1% (1/92) and 8.7% (8/92) showed intermediate susceptibility/resistance. For tetracycline and benzylpenicillin, respectively, 38.0% (35/92) and 22.0% (20/91) of the isolates were susceptible, 52.2% (48/92) and 74.7% (68/91) showed intermediate susceptibility/resistance and 9.8% (9/92) and 3.3% (3/91) were resistant. Syndromic treatment was administered as follows: 93.1% received 250 mg of ceftriaxone intramuscularly plus 100 mg of doxycycline orally q12h for 1–2 weeks and 6.9% received 500 mg of ciprofloxacin orally plus 100 mg of doxycycline orally q12h for 1 week.ConclusionsCeftriaxone (250 mg) remains appropriate for gonorrhoea treatment in the examined population of men in Kingston, Jamaica. Surveillance of N. gonorrhoeae AMR should be expanded in Jamaica and other Caribbean countries to guide evidence-based treatment guidelines.

Antimicrobial Susceptibility of Urogenital and Extragenital Neisseria gonorrhoeae Isolates Among Men Who Have Sex With Men: Strengthening the US Response to Resistant Gonorrhea and Enhanced Gonococcal Isolate Surveillance Project, 2018 to 2019.

We investigated differences in gonococcal antimicrobial susceptibility by anatomic site among cisgender men who have sex with men (MSM) using specimens collected through the Centers for Disease Control and Prevention's enhanced Gonococcal Isolate Surveillance Project and Strengthening the US Response to Resistant Gonorrhea. During the period January 1, 2018-December 31, 2019, 12 enhanced Gonococcal Isolate Surveillance Project and 8 Strengthening the US Response to Resistant Gonorrhea sites collected urogenital, pharyngeal, and rectal isolates from cisgender MSM in sexually transmitted disease clinics. Gonococcal isolates were sent to regional laboratories for antimicrobial susceptibility testing by agar dilution. To account for correlated observations, linear mixed-effects models were used to calculate geometric mean minimum inhibitory concentrations (MICs), and mixed-effects logistic regression models were used to calculate the proportion of isolates with elevated or resistant MICs; comparisons were made across anatomic sites. Participating clinics collected 3974 urethral, 1553 rectal, and 1049 pharyngeal isolates from 5456 unique cisgender MSM. There were no significant differences in the geometric mean MICs for azithromycin, ciprofloxacin, penicillin, and tetracycline by anatomic site. For cefixime and ceftriaxone, geometric mean MICs for pharyngeal isolates were higher compared with anogenital isolates (P < 0.05). The proportion of isolates with elevated ceftriaxone MICs (≥0.125 μg/mL) at the pharynx (0.67%) was higher than at rectal (0.13%) and urethral (0.18%) sites (P < 0.05). Based on data collected from multijurisdictional sentinel surveillance projects, antimicrobial susceptibility patterns of Neisseria gonorrhoeae isolates may differ among MSM at extragenital sites, particularly at the pharynx. Continued investigation into gonococcal susceptibility patterns by anatomic site may be an important strategy to monitor and detect the emergence of antimicrobial resistant gonorrhea over time.

Prediction of ceftriaxone MIC inNeisseria gonorrhoeae usingDNA microarray technology and regression analysis

Decreased susceptibility of Neisseria gonorrhoeae to extended-spectrum cephalosporins is a major concern. Elucidation of the phenotypic and genetic characteristics of such isolates is a priority task. We developed a method for predicting the N. gonorrhoeae ceftriaxone susceptibility level (MICcro) by identifying genetic determinants of resistance using low-density hydrogel microarrays and a regression equation. A training dataset, containing 5631 isolates from the Pathogenwatch database and 181 isolates obtained in the Russian Federation during 2018-19, was used to build a regression model. The regression equation was tested on 14 WHO reference strains. Ceftriaxone resistance determinants for the 448 evaluated clinical isolates collected in Russia were identified using microarray analysis, and MICcro values were calculated using the regression equation and compared with those measured by the serial dilution method. The regression equation for calculating MICcro values included 20 chromosomal resistance determinants. The greatest contributions to the increase in MICcro were shown to be PBP2: Ala-501→Pro, Ala-311→Val, Gly-545→Ser substitutions, Asp(345-346) insertion; and PorB: Gly-120→Arg substitution. The substitutions PBP2: Ala-501→Thr/Val, PorB: Gly-120→Asn/Asp/Lys and PBP1: Leu-421→Pro had weaker effects. For 94.4% of the isolates in the evaluation set, the predicted MICcro was within one doubling dilution of the experimentally determined MICcro. No ceftriaxone-resistant isolates were identified in the analysed samples from Russia, and no interpretative errors were detected in the MICcro calculations. The developed strategy for predicting ceftriaxone MIC can be used for the continuous surveillance of known and emerging resistant N. gonorrhoeae isolates.

The Accuracy of Molecular Detection Targeting the Mutation C2611T for Detecting Moderate-Level Azithromycin Resistance in Neisseria gonorrhoeae: A Systematic Review and Meta-Analysis.

Background: Neisseria gonorrhoeae (N. gonorrhoeae) is now recognized as a commonly reported sexually transmitted pathogen, and the increasing drug resistance of N. gonorrhoeae has become a serious public health problem. The accuracy of molecular detection for detecting moderate-level azithromycin resistance is not well-established. We summarized the data from studies of the N. gonorrhoeae 23S rRNA mutation at position 2611 with azithromycin resistance to determine the relationship between the mutation and resistance. Methods and Findings: In this systematic review and meta-analysis, two researchers independently searched six databases for studies with data for the azithromycin minimum inhibitory concentrations (MICs) and the 23S rRNA mutation C2611T of each N. gonorrhoeae isolate. Since the breakpoint of moderate-level resistance to azithromycin (ML-AzmR) was not determined, we divided the moderate level into two groups according to the range of MICs (moderate resistance limited to 2–128 mg/L or 4–128 mg/L) for data extraction. A random-effects model was used to calculate the pooled sensitivity rate, the specificity rate, the pooled positive likelihood ratio (PLR), the negative likelihood ratio (NLR), and the diagnostic odds ratio (DOR). Meta-regression analyses by detection method, isolates sampling (a random sample or not), location, and sample size were performed to explore the possible causes of heterogeneity. The potential publication bias of the included studies was conducted by the Deeks’ test. We included 20 studies in our study: 20 studies have data of N. gonorrhoeae with MICs between 2 and 128 mg/L with mutation or without mutation at position 2611(4759 samples), and 14 studies have data of N. gonorrhoeae with MICs between 4 and 128 mg/L (3367 samples). In the group with the moderate level of 2–128 mg/L, the pooled sensitivity rate of the molecular assays was determined to be 71.9% (95% CI, 67.6–74%), the pooled specificity rate was 98.7% (95% CI, 98.2–99.0%), and the DOR ranged from 55.0 to 351.3 (mean, 139.1). In the 4–128 mg/L group, the pooled sensitivity rate was 91.9% (95% CI, 88.9–94.2%), the pooled specificity rate was 95.9% (95% CI, 95.1–96.6%), and the DOR ranged from 41.9 to 364.1 (mean, 123.6). Conclusion: Through this meta-analysis, we found that the C2611T mutation of 23S rRNA is valuable for the molecular diagnostic of moderate-level azithromycin resistance (ML-AzmR) in N. gonorrhoeae, especially when the moderate level is set at 4–128 mg/L. This rapid molecular detection method can be used for the rapid identification of ML-AzmR isolates in the clinic.

Large Increase in Azithromycin-Resistant Neisseria gonorrhoeae in Northern Spain

The aim of this study was to characterize the evolution of gonorrhea in the general population by correlating epidemiological, genotypic, and antimicrobial resistance data of Neisseria gonorrhoeae isolates collected in northern Spain from 2014 to 2018. One hundred ninety-four strains underwent antimicrobial susceptibility testing and were genetically analyzed by N. gonorrhoeae multiantigen sequence typing. Increasing cases of gonococcal infections have been observed after 2015. Most occurred in male with urethritis. Sequence type (ST)-9972 and ST-1576, the predominant genotypes identified, have not been previously described as epidemic clones. Of great concern was the significant increase in azithromycin-resistant N. gonorrhoeae. More than 30% of these isolates were obtained from men who have sex with men (MSM). ST-12302 was the most prevalent clone among the azithromycin-resistant strains, and was also resistant to penicillin, ciprofloxacin, and tetracycline. This multidrug-resistant clone was exclusively isolated from MSM during 2018. The incidence rates of gonorrhea and azithromycin-resistant N. gonorrhoeae have significantly increased due to the emergence of new clones. ST-12302 has recently been recognized as an epidemic clone; therefore, its surveillance could be the key in controlling further dissemination of azithromycin resistance. These data highlight the need to perform local studies to update treatment guidelines and reinforce preventive measures against gonorrhea.

Genomic Analysis of the Predominant Strains and Antimicrobial Resistance Determinants Within 1479 Neisseria gonorrhoeae Isolates From the US Gonococcal Isolate Surveillance Project in 2018.

The prevalence of Neisseria gonorrhoeae (GC) isolates with elevated minimum inhibitory concentrations to various antibiotics continues to rise in the United States and globally. Genomic analysis provides a powerful tool for surveillance of circulating strains, antimicrobial resistance determinants, and understanding of transmission through a population. Neisseria gonorrhoeae isolates collected from the US Gonococcal Isolate Surveillance Project in 2018 (n = 1479) were sequenced and characterized. Whole-genome sequencing was used to identify sequence types, antimicrobial resistance profiles, and phylogenetic relationships across demographic and geographic populations. Genetic characterization identified that (1) 80% of the GC isolates were represented in 33 multilocus sequence types, (2) isolates clustered in 23 major phylogenetic clusters with select phenotypic and demographic prevalence, and (3) common antimicrobial resistance determinants associated with low-level or high-level decreased susceptibility or resistance to relevant antibiotics. Characterization of this 2018 Gonococcal Isolate Surveillance Project genomic data set, which is the largest US whole-genome sequence data set to date, sets the basis for future prospective studies, and establishes a genomic baseline of GC populations for local and national monitoring.

Antimicrobial Resistance Profiling and Phylogenetic Analysis of Neisseria gonorrhoeae Clinical Isolates From Kenya in a Resource-Limited Setting.

BackgroundAfrica has one of the highest incidences of gonorrhea. Neisseria gonorrhoeae is gaining resistance to most of the available antibiotics, compromising treatment across the world. Whole-genome sequencing (WGS) is an efficient way of predicting AMR determinants and their spread in the population. Recent advances in next-generation sequencing technologies like Oxford Nanopore Technology (ONT) have helped in the generation of longer reads of DNA in a shorter duration with lower cost. Increasing accuracy of base-calling algorithms, high throughput, error-correction strategies, and ease of using the mobile sequencer MinION in remote areas lead to its adoption for routine microbial genome sequencing. To investigate whether MinION-only sequencing is sufficient for WGS and downstream analysis in resource-limited settings, we sequenced the genomes of 14 suspected N. gonorrhoeae isolates from Nairobi, Kenya.MethodsUsing WGS, the isolates were confirmed to be cases of N. gonorrhoeae (n = 9), and there were three co-occurrences of N. gonorrhoeae with Moraxella osloensis and N. meningitidis (n = 2). N. meningitidis has been implicated in sexually transmitted infections in recent years. The near-complete N. gonorrhoeae genomes (n = 10) were analyzed further for mutations/factors causing AMR using an in-house database of mutations curated from the literature.ResultsWe observe that ciprofloxacin resistance is associated with multiple mutations in both gyrA and parC. Mutations conferring tetracycline (rpsJ) and sulfonamide (folP) resistance and plasmids encoding beta-lactamase were seen in all the strains, and tet(M)-containing plasmids were identified in nine strains. Phylogenetic analysis clustered the 10 isolates into clades containing previously sequenced genomes from Kenya and countries across the world. Based on homology modeling of AMR targets, we see that the mutations in GyrA and ParC disrupt the hydrogen bonding with quinolone drugs and mutations in FolP may affect interaction with the antibiotic.ConclusionHere, we demonstrate the utility of mobile DNA sequencing technology in producing a consensus genome for sequence typing and detection of genetic determinants of AMR. The workflow followed in the study, including AMR mutation dataset creation and the genome identification, assembly, and analysis, can be used for any clinical isolate. Further studies are required to determine the utility of real-time sequencing in outbreak investigations, diagnosis, and management of infections, especially in resource-limited settings.

Visual and Rapid Diagnosis of Neisseria gonorrhoeae Using Loop-Mediated Isothermal Amplification Combined With a Polymer Nanoparticle-Based Biosensor in Clinical Application.

Neisseria gonorrhoeae is a host-adapted human pathogen that causes sexually transmitted gonorrhea and remains to be a serious global public health challenge, especially in low- and middle-income regions. It is vital to devise a reliable, simple, cost-saving, and easy-to-use assay for detecting the N. gonorrhoeae agent. In the current study, we firstly report a novel approach, loop-mediated isothermal amplification linked with a polymer nanoparticle–based biosensor (LAMP-PNB), that was used for identifying N. gonorrhoeae in clinical samples. The results showed that the LAMP primers based on the orf1 gene were valid for development of the N. gonorrhoeae-LAMP-PNB assay. The detection system with optimal conditions could be performed at a fixed temperature of 64°C for 40 min. The whole process, including genomic DNA preparation (approximately 10 min), LAMP reaction (40 min), and PNB reporting (approximately 2 min), could be accomplished within 60 min. The limit of detection (LoD) of the N. gonorrhoeae-LAMP-PNB assay was 50 copies per test. The specificity of the current assay was 100%, and no cross-reactions to non–N. gonorrhoeae isolates were observed. These results confirmed that the N. gonorrhoeae-LAMP-PNB technique is a reliable, specific, sensitive, rapid, low-cost, and easy-to-use method for detecting gonococci isolates. More importantly, this assay has great potential to develop a point-of-care (POC) testing method in clinical practice, especially in resource-constrained regions.

Antimicrobial Resistance Mechanisms, Multilocus Sequence Typing, and NG-STAR Sequence Types of Diverse Neisseria gonorrhoeae Isolates in KwaZulu-Natal, South Africa.

Antimicrobial resistance (AMR) is a major challenge to managing infectious diseases. Africa has the highest incidence of gonorrhoea, but there is a lack of comprehensive data from sparse surveillance programs. This study investigated the molecular epidemiology and AMR profiles of Neisseria gonorrhoeae isolates in KwaZulu-Natal province (KZN), South Africa. Repository isolates from patients attending public health care clinics for sexually transmitted infection (STI) care were used for phenotypic and genotypic analysis. An Etest was performed to determine antimicrobial susceptibility. Whole-genome sequencing (WGS) was used to determine epidemiology and to predict susceptibility by detecting resistance-associated genes and mutations. Among the 61 isolates, multiple sequence types were identified. Six isolates were novel, as determined by multilocus sequence typing. N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) determined 48 sequence types, of which 35 isolates had novel antimicrobial profiles. Two novel penA alleles and eight novel mtrR alleles were identified. Point mutations were detected in gyrA, parC, mtrR, penA, ponA, and porB1. This study revealed a high prevalence of AMR (penicillin 67%, tetracycline 89%, and ciprofloxacin 52%). However, spectinomycin, cefixime, ceftriaxone, and azithromycin remained 100% effective. This study is one of the first to comprehensively describe the epidemiology and AMR of N. gonorrhoeae in KZN, South Africa and Africa, using WGS. KZN has a wide strain diversity and most of these sequence types have been detected in multiple countries; however, more than half of our isolates have novel antimicrobial profiles. Continued surveillance is crucial to monitor the emergence of resistance to cefixime, ceftriaxone, and azithromycin.

Analysis of antimicrobial susceptibility testing methods and variables and in vitro activity of gepotidacin against urogenital Neisseria gonorrhoeae in men

Gepotidacin is a triazaacenaphthylene antibiotic with activity against Neisseria gonorrhoeae including strains resistant to current agents. We tested 145 N. gonorrhoeae isolates by agar dilution according to Gonococcal Isolate Surveillance Program and Clinical and Laboratory Standards Institute methodologies. Gepotidacin demonstrated a minimum inhibitory concentration (MIC)50 of 0.25 μg/mL and a MIC90 of 0.5 μg/mL (highest gepotidacin MIC was 1 μg/mL) against the 145 N. gonorrhoeae isolates tested. We also assessed the impact of test variables on antimicrobial susceptibility test results for gepotidacin, ciprofloxacin, and ceftriaxone against 10 N. gonorrhoeae isolates. Media type had the biggest effect but wasn't specific to gepotidacin. Gepotidacin MICs were also affected by inoculum, pH, and 10% CO2. These in vitro data indicate that further study of gepotidacin is warranted for potential use in treating gonorrhea infections and highlight the importance of controlling for media type, inoculum, CO2, and pH when performing MIC testing with gepotidacin.

Phylogenomic analysis reveals persistence of gonococcal strains with reduced-susceptibility to extended-spectrum cephalosporins and mosaic penA-34

The recent emergence of strains of Neisseria gonorrhoeae associated with treatment failures to ceftriaxone, the foundation of current treatment options, has raised concerns over a future of untreatable gonorrhea. Current global data on gonococcal strains suggest that several lineages, predominately characterized by mosaic penA alleles, are associated with elevated minimum inhibitory concentrations (MICs) to extended spectrum cephalosporins (ESCs). Here we report on whole genome sequences of 813 N. gonorrhoeae isolates collected through the Gonococcal Isolate Surveillance Project in the United States. Phylogenomic analysis revealed that one persisting lineage (Clade A, multi-locus sequence type [MLST] ST1901) with mosaic penA-34 alleles, contained the majority of isolates with elevated MICs to ESCs. We provide evidence that an ancestor to the globally circulating MLST ST1901 clones potentially emerged around the early to mid-20th century (1944, credibility intervals [CI]: 1935–1953), predating the introduction of cephalosporins, but coinciding with the use of penicillin. Such results indicate that drugs with novel mechanisms of action are needed as these strains continue to persist and disseminate globally.

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

BackgroundGonorrhoea 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.MethodsN. 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.ResultsThe 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.ConclusionsThe 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.

Antimicrobial resistance in Neisseria gonorrhoeae isolates and gonorrhoea treatment in the Republic of Belarus, Eastern Europe, 2009\u20132019

BackgroundLimited antimicrobial resistance (AMR) data for Neisseria gonorrhoeae are available in Eastern Europe. We investigated AMR in N. gonorrhoeae isolates in the Republic of Belarus from 2009 to 2019, antimicrobial treatment recommended nationally, and treatment given to patients with gonorrhoea.MethodsN. gonorrhoeae isolates (n = 522) cultured in three regions of Belarus in 2009–2019 were examined. Determination of minimum inhibitory concentrations (MICs) of eight antimicrobials was performed using Etest. Resistance breakpoints from the European Committee on Antimicrobial Susceptibility Testing were applied where available. A Nitrocefin test identified β-lactamase production. Gonorrhoea treatment for 1652 patients was also analysed. Statistical significance was determined by the Z-test, Fisher’s exact test, or Mann-Whitney U test with p-values of < 0.05 indicating significance.ResultsIn total, 27.8% of the N. gonorrhoeae isolates were resistant to tetracycline, 24.7% to ciprofloxacin, 7.0% to benzylpenicillin, 2.7% to cefixime, and 0.8% to azithromycin. No isolates were resistant to ceftriaxone, spectinomycin, or gentamicin. However, 14 (2.7%) isolates had a ceftriaxone MIC of 0.125 mg/L, exactly at the resistance breakpoint (MIC > 0.125 mg/L). Only one (0.2%) isolate, from 2013, produced β-lactamase. From 2009 to 2019, the levels of resistance to ciprofloxacin and tetracycline were relatively high and stable. Resistance to cefixime was not identified before 2013 but peaked at 22.2% in 2017. Only sporadic isolates with resistance to azithromycin were found in 2009 (n = 1), 2012 (n = 1), and 2018–2019 (n = 2). Overall, 862 (52.2%) patients received first-line treatment according to national guidelines (ceftriaxone 1 g). However, 154 (9.3%) patients received a nationally recommended alternative treatment (cefixime 400 mg or ofloxacin 400 mg), and 636 (38.5%) were given non-recommended treatment.ConclusionsThe gonococcal resistance to ciprofloxacin and tetracycline was high, however, the resistance to azithromycin was low and no resistance to ceftriaxone was identified. Ceftriaxone 1 g can continuously be recommended as empiric first-line gonorrhoea therapy in Belarus. Fluoroquinolones should not be prescribed for treatment if susceptibility has not been confirmed by testing. Timely updating and high compliance with national evidence-based gonorrhoea treatment guidelines based on quality-assured AMR data are imperative. The need for continued, improved and enhanced surveillance of gonococcal AMR in Belarus is evident.

Retrospective Analysis of Drug Sensitivity of Neisseria gonorrhoeae in Teaching Hospitals of South China.

The aim of this study was retrospective analysis of drug sensitivity of Neisseria gonorrhoeae in two teaching hospitals of South China. A total of 304 Neisseria gonorrhoeae isolates obtained from patients in South China from 2016 to 2020 were evaluated. The MICs of penicillin, cefuroxime, ceftriaxone (CRO), cefepime, ciprofloxacin, ceftazidime and azithromycin (AZM) against the isolates were determined by the agar dilution method. Then, Neisseria gonorrhoeae isolates were categorized into sensitive, moderately sensitive and resistant according to MICs. Also, β-lactamases were detected by enzyme linked immunosorbent assay (ELISA). Ureaplasma urealyticum and Mycoplasma hominis were determined by culture in liquid medium, and Chlamydia was detected by rapid antigen test. The result showed there was 50.99%, 20.72%, 9.87%, 14.47%, 86.84%, 7.57%, 6.91%, 11.18% resistance to penicillin, cefuroxime, ceftriaxone, cefepime, ciprofloxacin, ceftazidime and azithromycin, respectively. Also, β-lactamase positivity was 53.29% and Chlamydia antigen positivity was 20.07%. Ureaplasma urealyticum and Mycoplasma hominis positivity was 11.84% and 6.25%, respectively. From 2016 to 2020, the resistant rate of ceftriaxone and azithromycin gradually increased. In conclusion, Southern China is among the area reporting gonococci with high-level resistance to AZM and CRO, so N. gonorrhoeae culture and drug sensitivity test will be vital for monitoring trends in antimicrobial resistance.

Molecular characteristics and antimicrobial susceptibility of penicillinase-producing Neisseria gonorrhoeae isolates in Fukuoka, Japan, 1996–2018

The objective of this study was to investigate the molecular characteristics and antimicrobial susceptibility of penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates collected in Fukuoka, Japan, from 1996-2018. Antimicrobial susceptibility to seven antibiotics was determined by the agar dilution method. Molecular characteristics were determined by Sanger sequencing of the blaTEM allele, plasmid typing and N. gonorrhoeae multiantigen sequence typing (NG-MAST). Furthermore, full sequences of the penA gene, encoding penicillin-binding protein 2 (PBP2), of PPNG isolates with reduced susceptibility to cefixime were analysed. Among 50 PPNG isolates, 17 and 33 were collected during 1996-2006 and 2007-2018, respectively. In 1996-2006, blaTEM-1 in African plasmid was most frequent (64.7%), followed by blaTEM-1 in Asian plasmid (29.4%) and blaTEM-135 in Toronto/Rio plasmid (5.9%). In 2007-2018, blaTEM-135 in Toronto/Rio plasmid was predominant (54.5%), followed by blaTEM-1 in African plasmid (36.4%) and blaTEM-135 in Asian plasmid (6.1%). Among isolates with the blaTEM-135-carrying Toronto/Rio plasmid in 2007-2018, a novel genogroup G15576 was predominant (66.7%). Isolates with the TEM-135 β-lactamase were more resistant to ciprofloxacin but were more susceptible to ceftriaxone and tetracycline than isolates with TEM-1. Seven PPNG isolates less susceptible to cefixime possessed the plasmidic blaTEM-1 allele and had mosaic or non-mosaic alterations within PBP2. The proportion of PPNG with the blaTEM135-carrying Toronto/Rio plasmid increased during the last 12 years. The increase in PPNG carrying the blaTEM-135 allele is of particular concern as it is considered a possible direct precursor of an extended-spectrum β-lactamase (ESBL).

Molecular characterization of decreased susceptibility to ceftriaxone and genotyping of Neisseria gonorrheae isolates in New Delhi, India

Data on genetic characteristics of Neisseria gonorrhoeae isolates exhibiting decreased susceptibility to extended-spectrum cephalosporins in India is deficient. In this study, we have sequenced penA, porB, mtrR and ponA and blaTEM genes in 70 clinical isolates of NG with varying ceftriaxone MICs. Amongst these, 22 (31.4%) were PPNG. Additionally, N. gonorrheae Multiantigen Sequence Typing was performed. Fisher exact and χ2 were used to evaluate significance of mutations with MICs. A total of six non-mosaic penA (Penicillin binding protein 2 [PBP2]) amino acid patterns were seen (II, IV, IX, XII, XIX, XXII) of which, pattern IX was significantly associated with decreased susceptibility to ceftriaxone. Other significant associations were noted in porB & mtrR genes. There were no mutations in blaTEM gene. ST6069 was significantly associated with decreased susceptibility to ceftriaxone. To conclude, development of decreased susceptibility to ceftriaxone in gonococci involves cumulation of different mutations in the four chromosomal genes investigated.

Genomic surveillance of Neisseria gonorrhoeae in the Philippines, 2013-2014.

Antimicrobial-resistant Neisseria gonorrhoeae is a major threat to public health and is of particular concern in the Western Pacific Region, where the incidence of gonorrhoea is high. The Antimicrobial Resistance Surveillance Program (ARSP) has been capturing information on resistant gonorrhoea since 1996, but genomic epidemiology studies on this pathogen are lacking in the Philippines.We sequenced the whole genomes of 21 N. gonorrhoeae isolates collected in 2013–2014 by ARSP. The multilocus sequence type, multiantigen sequence type, presence of determinants of antimicrobial resistance and relatedness among the isolates were all derived from the sequence data. The concordance between phenotypic and genotypic resistance was also determined.Ten of 21 isolates were resistant to penicillin, ciprofloxacin and tetracycline, due mainly to the presence of the blaTEM gene, the S91F mutation in the gyrA gene and the tetM gene, respectively. None of the isolates was resistant to ceftriaxone or cefixime. The concordance between phenotypic and genotypic resistance was 92.38% overall for five antibiotics in four classes. Despite the small number of isolates studied, they were genetically diverse, as shown by the sequence types, the N. gonorrhoeae multiantigen sequence typing types and the tree. Comparison with global genomesplaced the Philippine genomes within global lineage A and led to the identification of an international transmission route.This first genomic survey of N. gonorrhoeae isolates collected by ARSP will be used to contextualize prospective surveillance. It highlights the importance of genomic surveillance in the Western Pacific and other endemic regions for understanding the spread of drug-resistant gonorrhoea worldwide.

Emergence and evolution of antimicrobial resistance genes and mutations in Neisseria gonorrhoeae

BackgroundAntimicrobial resistance in Neisseria gonorrhoeae is a global health concern. Strains from two internationally circulating sequence types, ST-7363 and ST-1901, have acquired resistance to third-generation cephalosporins, mainly due to mosaic penA alleles. These two STs were first detected in Japan; however, the timeline, mechanism, and process of emergence and spread of these mosaic penA alleles to other countries remain unknown.MethodsWe studied the evolution of penA alleles by obtaining the complete genomes from three Japanese ST-1901 clinical isolates harboring mosaic penA allele 34 (penA-34) dating from 2005 and generating a phylogenetic representation of 1075 strains sampled from 35 countries. We also sequenced the genomes of 103 Japanese ST-7363 N. gonorrhoeae isolates from 1996 to 2005 and reconstructed a phylogeny including 88 previously sequenced genomes.ResultsBased on an estimate of the time-of-emergence of ST-1901 (harboring mosaic penA-34) and ST-7363 (harboring mosaic penA-10), and > 300 additional genome sequences of Japanese strains representing multiple STs isolated in 1996–2015, we suggest that penA-34 in ST-1901 was generated from penA-10 via recombination with another Neisseria species, followed by recombination with a gonococcal strain harboring wildtype penA-1. Following the acquisition of penA-10 in ST-7363, a dominant sub-lineage rapidly acquired fluoroquinolone resistance mutations at GyrA 95 and ParC 87-88, by independent mutations rather than horizontal gene transfer. Data in the literature suggest that the emergence of these resistance determinants may reflect selection from the standard treatment regimens in Japan at that time.ConclusionsOur findings highlight how antibiotic use and recombination across and within Neisseria species intersect in driving the emergence and spread of drug-resistant gonorrhea.