Gonorrhoeae Isolates Research Articles

Tetracycline-resistant Neisseria gonorrhoeae strains isolated in a Grade III hospital in eastern China: molecular characteristics and genetic diversity

Objective To analyze the genotypes of Neisseria gonorrhoeae (N.gonorrhoeae) epidemic strains in Wenzhou, eastern China, and to study the mechanism of tetracycline resistance in these strains. Methods A total of 77 N. gonorrhoeae strains were isolated from patients with gonorrhea. Antimicrobial susceptibility of these strains to penicillin, tetracycline, ciprofloxacin, spectinomycin, ceftriaxone and azithromycin was analyzed using E-test. PCR and DNA sequencing were used to detect the genes associated with tetracycline resistance, such as Tet-M, mtrR promoter region and mtrR coding region. N. gonorrhoeae multi-antigen sequence typing (NG-MAST) and multilocus sequence typing (MLST) were used to determine the molecular characteristics of all clinical isolates and tetracycline-resistant isolates, respectively. Results Among the 77 N. gonorrhoeae isolates, 74 (96.10%), 27 (35.06%) , 70 (90.91%) and 15 (19.48%) were resistant to penicillin, tetracycline, ciprofloxacin and azithromycin, respectively. All tested isolates were susceptible to spectinomycin and ceftriaxone. Nineteen isolates were resistant to tetracycline and all of them carried Tet-M gene. Among them, 17 had one deletion mutation of base A in mtrR promoter region and three had G45D mutation in mtrR coding region. NG-MAST classified the 19 tetracycline-resistant isolates into 11 different sequence types (ST). ST14781, ST1766 and ST1866 each accounted for 15.79% (three strains). Two ST (10.52%, 2/19) found in the present study had not been reported previously in the NG-MAST database. MLST showed the 19 tetracycline-resistant isolates belonged to 12 different STs, in which ST10899 accounted for 26.32% (five strains) and ST1600 accounted for 15.79% (three strains). Conclusions Mutations in mtrR promoter region and carrying Tet-M gene were associated with tetracycline resistance in N. gonorrhoeae. Clinical strains isolated in Wenzhou showed considerable molecular diversity. Measures should be implemented to monitor the spread of NG-MAST ST1766 and MLST ST1600 N. gonorrhoeae clones with high resistance to tetracycline in Wenzhou. Key words: Neisseria gonorrhoeae; Resistance rate; Tetracycline; NG-MAST; MLST

In vitro activity of the novel oral antimicrobial SMT-571, with a new mechanism of action, against MDR and XDR Neisseria gonorrhoeae: future treatment option for gonorrhoea?

BackgroundLack of effective treatment of gonorrhoea due to increasing antimicrobial resistance in Neisseria gonorrhoeae is a serious threat to the management and control of the infection. Novel antimicrobials are required to prevent the infection becoming untreatable.ObjectivesHerein, we investigated the in vitro activity of a novel small-molecule antimicrobial with a new mechanism of action, SMT-571, against a large collection of clinical N. gonorrhoeae isolates (n = 228) and international gonococcal reference strains (n = 34), including numerous MDR and XDR gonococcal isolates.MethodsMICs of SMT-571 were determined by agar dilution and MICs of ceftriaxone, cefixime, azithromycin, ciprofloxacin, ampicillin, spectinomycin and tetracycline were determined by Etest.ResultsSMT-571 showed potent in vitro activity against all the tested N. gonorrhoeae isolates (n = 262). The MICs ranged from 0.064 to 0.125 mg/L and the MIC50, MIC90 and modal MIC were all 0.125 mg/L. No cross-resistance or correlation between the MICs of SMT-571 and comparator agents was seen.ConclusionsSMT-571 demonstrated potent in vitro activity against all tested gonococcal isolates and no cross-resistance to previously and currently used antimicrobials was seen. With its promising supplementary in vitro and in vivo preclinical data, including high levels of oral bioavailability, SMT-571 could be an effective option for the oral treatment of gonorrhoea. Randomized controlled clinical trials for gonorrhoea that examine the treatment efficacy, pharmacokinetics/pharmacodynamics, toxicity and safety of SMT-571, and include urogenital and extragenital (rectal and pharyngeal) samples, are crucial.

Evaluation of Susceptibilities to Carbapenems and Faropenem Against Cephalosporin-Resistant Neisseria gonorrhoeae Clinical Isolates with penA Mosaic Alleles.

Neisseria gonorrhoeae is a principal pathogen for sexually transmitted infections, especially for male urethritis. Currently, the prevalence of multidrug resistance is increasing. Carbapenems are broad-spectrum antimicrobials that are widely used in the clinical setting, especially for multidrug-resistant Gram-negative bacteria. However, susceptibility to carbapenems has not been well evaluated for cephalosporin-resistant N. gonorrhoeae isolates. In this study, we determined the susceptibility to a series of carbapenems (meropenem, imipenem, doripenem, and biapenem) and faropenem against cephalosporin-resistant (resistant to cefixime, but susceptible to ceftriaxone) and cephalosporin-susceptible N. gonorrhoeae clinical isolates. The gene mutations associated with β-lactam resistance were evaluated. All cephalosporin-resistant N. gonorrhoeae isolates possessed mosaic mutation alleles in penA (NG-STAR penA-10.001, 27.001, or 108.001). They exhibited a low minimum inhibitory concentration (MIC) (≤0.125 mg/L) for meropenem and markedly high MICs (0.5-2 mg/L) for other carbapenems and faropenem. The strongest association was observed between the mosaic alleles in penA and decreased susceptibility to carbapenems and faropenem compared with mutations in mtrR, porB, and ponA. These results suggest that meropenem may serve as an alternative therapeutic agent for cephalosporin-resistant N. gonorrhoeae with a mosaic allele in penA, whereas other carbapenems and faropenem may be ineffective.

Evidence for Clonally Associated Increasing Rates of Azithromycin Resistant Neisseria gonorrhoeae in Rio de Janeiro, Brazil.

Azithromycin is one of the drugs used in the combined therapy for syndromic treatment of gonorrhoea in many countries, including Brazil. Our research group, which receives isolates from clinical laboratories since 2006, has detected, after 2016, a tendency of rising rates of azithromycin resistance, with isolates showing higher minimal inhibitory concentrations (MICs) than those previously reported in this country. In this study, we report the susceptibility to azithromycin of 93 N. gonorrhoeae isolates obtained between 2014 and 2017. Strains with MIC ≥2 μg/mL were characterized according to azithromycin resistance mechanisms and strain typing. Results indicate that azithromycin resistance has emerged in all these years in unrelated MLST-STs, but after 2016 a clonal complex connected with ST1901 has been more frequently detected, grouping isolates with MIC varying from 2 to 64 μg/mL, with DelA mutations at the mtrR promoter region associated or not with mutations at rrl alleles. High rates of azithromycin resistance may compromise the use of this drug in the combined therapy with ceftriaxone. Inclusion of Rio de Janeiro in the Brazilian gonococcal surveillance program is important to evaluate if this data indicates an epidemiological phenomenon in the country.

Comparison of Neisseria gonorrhoeae minimum inhibitory concentrations obtained using agar dilution versus microbroth dilution methods

With increasing antibiotic resistance observed amongst clinical isolates of Neisseria gonorrhoeae, the second most prevalent sexually transmitted bacterial disease in the United States, there is still a need for antimicrobial susceptibility testing (AST). The current method recommended by the Clinical and Laboratory Standards Institute is agar dilution.In this study, we show that a commercially available version of Fastidious Broth is capable of supporting N. gonorrhoeae in the evaluation of minimum inhibitory concentrations of 4 antibiotics (ceftriaxone, azithromycin, ciprofloxacin, and tetracycline), when comparing the agar dilution (AD) versus microbroth dilution (MBD) method and the susceptibilities obtained for 32 N. gonorrhoeae isolates. Herein, 3 out of the 4 antibiotics tested showed 94% or greater essential agreement (EA) and 91% or greater categorical agreement (CA) respectively, when comparing the MBD versus AD methods.

Using the genetic characteristics of Neisseria gonorrhoeae strains with decreased susceptibility to cefixime to develop a molecular assay to predict cefixime susceptibility.

Background:In the last two decades, gonococcal strains with decreased cefixime susceptibility and cases of clinical treatment failure have been reported worldwide. Gonococcal strains with a cefixime minimum inhibitory concentration (MIC) ≥0.12 μg mL−1 are significantly more likely to fail cefixime treatment than strains with an MIC <0.12 μg mL−1. Various researchers have described the molecular characteristics of gonococcal strains with reduced cefixime susceptibility, and many have proposed critical molecular alterations that contribute to this decreased susceptibility.Methods:A systematic review of all published articles in PubMed through 1 November 2018 was conducted that report findings on the molecular characteristics and potential mechanisms of resistance for gonococcal strains with decreased cefixime susceptibility. The findings were summarised and suggestions were made for the development of a molecular-based cefixime susceptibility assay.Results:The penicillin-binding protein 2 (PBP2) encoded by the penA gene is the primary target of cefixime antimicrobial activity. Decreased cefixime susceptibility is conferred by altered penA genes with mosaic substitute sequences from other Neisseria (N.) species (identifiable by alterations at amino acid position 375–377) or by non-mosaic penA genes with at least one of the critical amino acid substitutions at positions 501, 542 and 551. Based on this review of 415 international cefixime decreased susceptible N. gonorrhoeae isolates, the estimated sensitivity for an assay detecting the aforementioned amino acid alterations would be 99.5% (413/415).Conclusions:Targeting mosaic penA and critical amino acid substitutions in non-mosaic penA are necessary and may be sufficient to produce a robust, universal molecular assay to predict cefixime susceptibility.

Antibiotic resistance of Neisseria species in Iran: A systematic review and meta-analysis

Objective: To estimate the prevalence of antibiotic resistance of Neisseria species in Iran. Methods: A systematic and electronic search using relevant keywords in major national and international databases was performed until 6th July, 2018 in order to find studies reporting the prevalence of antibiotic resistance of Neisseria species in Iran. Results: A total of nine studies were found to be eligible based on predefined inclusion and exclusion criteria. Our analysis indicated that the prevalence of Neisseria gonorrhoeae resistance to different antibiotics was as follows: 66.9% to penicillin, 59.1% to ciprofloxacin, 11.1% to ceftriaxone, 21.6% to spectinomycin, 13.8% to cefixime, 82.4% to co-trimoxazole, 52.7% to tetracycline, 29.9% to gentamicin, 87.5% to ampicillin, 11.1% to azithromycin, 2.2% to chloramphenicol, 50.1% to cefepime and 50.0% to vancomycin. Antimicrobial resistance rates of Neisseria meningitidis was as follows: 30.0% to penicillin, 33.3% to amoxicillin, 33.3% to cephalexin, 55.6% to ampicillin and 0.0% to ciprofloxacin, ceftriaxone, cefotaxime, amikacin, co-trimoxazole, gentamicin, kanamycin, chloramphenicol and ceftizoxime. Conclusion: Neisseria gonorrhoeae and Neisseria meningitidis isolates of Iran show resistance to different types of antibiotics. Therefore, care should be exercised for the use of penicillin, ciprofloxacin, co-trimoxazole, tetracycline, gentamicin, ampicillin, cefepime and vancomycin for gonococcal infections, and also with respect to the use of penicillin, amoxicillin, ampicillin and cephalexin for meningococcal infections in Iran.

Temporal evolution of antimicrobial resistance among Neisseria gonorrhoeae clinical isolates in the most populated South American Metropolitan Region.

A total of 124 Neisseria gonorrhoeae isolates recovered during a 12-year period (2003-2015) from outpatients assisted at Centro de Referência e Treinamento DST/AIDS-CRT of São Paulo city, Brazil, were analysed. The following resistance rates were observed: penicillin-59.6%, ciprofloxacin-15.3%, and azithromycin-6.7%. Although reduced susceptibility to these drugs was observed since 2003, no ceftriaxone-resistant isolates were detected. Ciprofloxacin- and azithromycin non-susceptible isolates were grouped in 11 clusters. Mutations were detected in GyrA and ParC of isolates 124 and 260, and a C2611T substitution on 23S rRNA alleles was also observed in isolate 260. Both isolates belonged to ST1901/ST6210 (MSLT/NG-MAST schemes).

Stably high azithromycin resistance and decreasing ceftriaxone susceptibility in Neisseria gonorrhoeae in 25 European countries, 2016

BackgroundThe European Gonococcal Antimicrobial Surveillance Programme (Euro-GASP) performs annual sentinel surveillance of Neisseria gonorrhoeae susceptibility to therapeutically relevant antimicrobials across the European Union/European Economic Area (EU/EEA). We present the Euro-GASP results from 2016 (25 countries), linked to patient epidemiological data, and compared with data from previous years.MethodsAgar dilution and minimum inhibitory concentration (MIC) gradient strip methodologies were used to determine the antimicrobial susceptibility (using EUCAST breakpoints) of 2660 N. gonorrhoeae isolates from 25 countries across the EU/EEA. Significance of differences compared with Euro-GASP results in previous years was analysed using Z-tests.ResultsNo isolates with resistance to ceftriaxone (MIC > 0.125 mg/L) were detected in 2016 (one in 2015). However, the proportion of isolates with decreased susceptibility to ceftriaxone (MICs from 0.03 mg/L to 0.125 mg/L) increased significantly (p = 0.01) from 2015 to 2016. There were 14 (0.5%) isolates with ceftriaxone MICs 0.125 mg/L (on the resistance breakpoint), of which one isolate was resistant to azithromycin and four showed intermediate susceptibility to azithromycin. Cefixime resistance was detected in 2.1% of isolates in 2016 compared with 1.7% in 2015 (p = 0.26) and azithromycin resistance in 7.5% in 2016 compared with 7.1% in 2015 (p = 0.74). Seven (0.3%) isolates from five countries displayed high-level azithromycin resistance (MIC≥256 mg/L) in 2016 compared with five (0.2%) isolates in 2015. Resistance rate to ciprofloxacin was 46.5% compared with 49.4% in 2015 (p = 0.06). No isolates were resistant to spectinomycin and the MICs of gentamicin remained stable compared with previous years.ConclusionsOverall AMR rates in gonococci in EU/EEA remained stable from 2015 to 2016. However, the ceftriaxone MIC distribution shifted away from the most susceptible (≤0.016 mg/L) and the proportion of isolates with decreased susceptibility to ceftriaxone increased significantly. This development is of concern as current European gonorrhoea management guideline recommends ceftriaxone 500 mg plus azithromycin 2 g as first-line therapy. With azithromycin resistance at 7.5%, the increasing ceftriaxone MICs might soon threaten the effectiveness of this therapeutic regimen and requires close monitoring.

Evaluation of the accuracy of molecular assays targeting the mutation A2059G for detecting high-level azithromycin resistance in Neisseria gonorrhoeae: a systematic review and meta-analysis.

BackgroundNeisseria gonorrhoeae resistance to azithromycin has become a significant public health concern globally, and high-level azithromycin-resistant (HL-AzmR) isolates have emerged frequently. However, high-level azithromycin resistance is considered to be caused by mutated alleles of 23S rRNA gene at position 2059, and identification of HL-AzmR isolates mainly relies on agar dilution method or E-test method. This study aimed to assess the accuracy of the molecular assays targeting the mutation A2059G for identifying HL-AzmR isolates and thereby determine the association between the mutation and high-level azithromycin resistance.MethodsTwo researchers independently searched six databases to identify studies published from the launch of each database to October 15, 2017. The fixed effects model was used to estimate the pooled sensitivity rate, specificity rate, positive predictive value (PPV), and negative predictive value (NPV). Summary receiver operating characteristic curves were generated, and the area under the curve (AUC) was determined to estimate the overall performance of the assays. The Deeks’ test was conducted to evaluate potential publication bias.ResultsTen relevant studies were included in the meta-analysis to assess the synthetic accuracy of the molecular assays. The molecular assays had the synthetic sensitivity rate of 97.8% and the synthetic specificity rate of 99.1%. And the aggregated PPV and NPV were 96.4% and 99.5%, respectively. AUC was 0.99, suggesting a close relation existing between the mutation A2059G and high-level azithromycin resistance. This indicated that the molecular assays targeting the mutation A2059G have relatively high overall accuracy for identifying HL-AzmR N. gonor-rhoeae isolates. Publication bias was statistically significant.ConclusionThe mutation A2059G is the critical factor causing high-level azithromycin resistance. Hence, molecular methods are recommended to be put into clinical practice by commercialization, which will assist clinicians to prescribe more precisely.

1489. Trends in Antimicrobial Resistance in N. gonorrhoeae Isolated in Korea During 2015–2017

BackgroundThe trends of antimicrobial resistance (AMR) and the molecular epidemiology of gonococcal strains were analyzed in Korean during 2015 to 2017.MethodsThe susceptibility tests of 187 N. gonorrhoeae isolates were investigated. The minimum inhibitory concentration (MIC) of N. gonorrhoeae isolates were determined by the agar dilution method. Penicillinase-producing N. gonorrhoeae (PPNG) and plasmid-mediated tetracycline-resistant N. gonorrhoeae (TRNG) were evaluated. Isolates were tested for mutations in penA and also genotyped using N. gonorrhoeae multi-antigen sequence typing (NG-MAST) to determine the molecular epidemiological characteristics of the isolates.ResultsThe rates of resistance to ceftriaxone (CRO) increased from 6.5% in 2015 to 13.4% in 2017. Azithromycin resistance was documented only in 1.1%, but 53.5% of isolates showed intermediate susceptibility. All isolates were susceptible to spectinomycin. However, none of isolates was susceptible to ciprofloxacin and penicillin (PEN) and 4.3% of isolates were susceptible to tetracycline. The PPNG and TRNG increased from 25.7% and 39.4% in 2015 to 47.8% and 81.5% in 2017, respectively. 74.7% of the extended-spectrum cephalosporins (ESCs)-resistant isolates contained the mosaic PBP2 X allele, which has been previously associated with ESC resistance including treatment failures. penA mosaic alleles X were found in 83.8% of all isolates that harbored decreased susceptibility to cefixime. Among the 16 N. gonorrhoeae isolates with decreased susceptibility to CRO, penA mosaic X allele was identified in the 11 isolates. The 189 N. gonorrhoeae isolates were assigned to 112 different NG-MASTs; seven sequence types (STs) were novel. The most common ST was 10,668 (16.1%), followed by ST15024 (7.1%). Two ST1407 strains have been isolated in 2015. Although ST1407 was known to display decreased susceptibility to ESCs or full resistance, they were susceptible for CRO (MIC = 0.06 μg/mL) and cefixime (MIC = 0.12 μg/mL).ConclusionThe recent emergence of ESC-resistant N. gonorrhoeae strains which was often associated with mutations in the penA gene is a major concern and enhanced AMR surveillance is necessary to prevent transmission of these strains.Disclosures All authors: No reported disclosures.

Multidrug-resistant Neisseria gonorrhoeae isolate, belonging to the internationally spreading Japanese FC428 clone, with ceftriaxone resistance and intermediate resistance to azithromycin, Ireland, August 2018.

We describe a multidrug-resistant Neisseria gonorrhoeae urethritis case with ceftriaxone resistance and azithromycin intermediate resistance in a heterosexual man in Ireland, August 2018. Whole-genome sequencing showed that the isolate IR72 belongs to the internationally spreading multidrug-resistant ceftriaxone-resistant FC428 clade, initially described in Japan in 2015. IR72 was assigned MSLT ST1903, NG-MAST ST17842 and NG-STAR type 1133, including the ceftriaxone resistance-mediating penA-60.001. Global awareness of spreading ceftriaxone-resistant gonococcal strains that threaten recommended dual therapies is essential.

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

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

Antimicrobial resistance and molecular characteristics of Neisseria gonorrhoeae isolates from men who have sex with men

ObjectivesTo analyze the susceptibility of Neisseria gonorrhoeae isolates to penicillin (Pen), cefixime (Cfm), ceftriaxone (Cro), tetracycline (Tet), ciprofloxacin (Cip), azithromycin (Azm), and spectinomycin (Spt), and to verify the presence of mutations in resistance genes. MethodsAntibiotic susceptibility testing was performed by Etest method on 30 N. gonorrhoeae isolates collected from the MSM (men who have sex with men) population. PCR and DNA sequencing were performed to identify mutations within the penA, mtrR, gyrA, and parC genes in intermediately resistant and fully resistant isolates. ResultsN. gonorrhoeae isolates showed intermediate or full resistance to Pen (73%), Cfm (3%), Tet (60%), Cip (37%), and Azm (13%). One isolate with resistance to Cfm presented a penicillin-binding protein 2 (PBP2) mosaic XXXIV. All isolates with intermediate or full resistance to Pen (except at PBP2 mosaic) presented a D345a in PBP2. All Cip-resistant isolates had an S91F in the gyrA gene together with mutations in the parC gene. All intermediate or fully resistant isolates to substrates of the MtrCDE efflux pump had an A39T or G45D mutation in the mtrR gene or an adenine deletion within the mtrR promoter. One isolate presented a Neisseria meningitidis-like mtrR promoter sequence. ConclusionsThe results of this study are consistent with the findings of other studies and reinforce the importance of the expedient development of new therapeutic options.

Complete ciprofloxacin resistance in gonococcal isolates in an urban Ugandan clinic: findings from a cross-sectional study.

Antimicrobial resistance (AMR) to gonorrhoea is a threat to global health security. There have been concerns expressed that countries with high rates of disease have poor surveillance. The objectives of the study were to determine the AMR patterns of Neisseria gonorrhoeae clinical isolates to antimicrobial agents in patients with HIV or high risk of HIV acquisition, to compare the concordance of disk diffusion and agar dilution as methods for determining AMR to N. gonorrhoeae, and to describe methodological challenges to carrying out AMR testing. The study was conducted at an HIV outpatient service for at-risk populations and an outreach clinic for commercial sex workers in Kampala. Patients were offered a sexually transmitted infection screen using a polymerase chain reaction (PCR)-based assay. Samples positive for gonorrhoea were cultured. Antimicrobial susceptibility testing was performed using disk diffusion and isolates were sent to a reference laboratory for agar dilution direct susceptibility testing. Five hundred and seventy-five patients were screened. There were 33 (5.7%) patients with gonorrhoea detected by PCR. Of the 16 viable N. gonorrhoeae isolates, 100% were resistant to ciprofloxacin and tetracycline by disk diffusion and 31% exhibited reduced susceptibility to ceftriaxone and cefixime. By agar dilution, 100% of isolates were resistant to ciprofloxacin and all isolates were susceptible to ceftriaxone and cefixime. There was concordance between disk diffusion and agar dilution for ciprofloxacin and tetracycline resistance and a significant discordance for third-generation cephalosporins. More than half the women with gonorrhoea were asymptomatic and represent a potential reservoir for ongoing transmission. AMR testing of N. gonorrhoeae isolates is needed to ensure optimal treatment and prevention of antibiotic resistance progression.

Identification and expression analysis of ceftriaxone resistance-related genes in Neisseria gonorrhoeae integrating RNA-Seq data and qRT-PCR validation.

The aim of the study is to identify ceftriaxone resistance-related genes in Neisseria gonorrhoeae. Differences in gene expression were compared between ceftriaxone-susceptible N. gonorrhoeae isolates [minimum inhibitory concentration (MIC)=0.002-0.004mg/L] and isolates with decreased ceftriaxone susceptibility (MIC=0.125-0.5mg/L) using RNA-Seq (RNA sequencing). Total RNA of 10 clinical isolates was used to make libraries and generated an average of 24.07Mb reads per sample; these were assembled into 1871 mRNA genes. Moreover, 21 differentially expressed genes (DEGs) were found between the N. gonorrhoeae isolates with susceptibility and decreased susceptibility to ceftriaxone with a fold change of ≥2 (P<0.05), among which 11 were upregulated and 10 were downregulated. Furthermore, all DEGs were verified by quantitative reverse transcription PCR (qRT-PCR), which detected 25 clinical isolates with decreased ceftriaxone susceptibility and 21 ceftriaxone-susceptible isolates. In addition, seven DEGs revealed relative expression levels by 2-ΔΔCt and showed a statistical significance (P≤0.05). Analysis of Gene Ontology (GO) terms and KEGG pathway for functional enrichment showed that six DEGs were related to the cellular component and one DEG was related to the biosynthesis of antibiotics, and these results might be related to ceftriaxone resistance. Examining ceftriaxone resistance-related genes in N. gonorrhoeae is necessary owing to the high morbidity and antimicrobial resistance of N. gonorrhoeae, especially its eventual resistance to third-generation extended-spectrum cephalosporins (cefixime and ceftriaxone). Moreover, this report provides a new direction for the study and control of ceftriaxone-resistant N. gonorrhoeae.

Azithromycin Susceptibility Among Neisseria gonorrhoeae Isolates and Seasonal Macrolide Use.

Rising azithromycin nonsusceptibility among Neisseria gonorrhoeae isolates threatens current treatment recommendations, but the cause of this rise is not well understood. We performed an ecological study of seasonal patterns in macrolide use and azithromycin resistance in N. gonorrhoeae, finding that population-wide macrolide use is associated with increased azithromycin nonsusceptibility. These results, indicative of bystander selection, have implications for antibiotic prescribing guidelines.

Evaluation of a Hydrogel-Based Diagnostic Approach for the Point-of-Care Based Detection of Neisseria gonorrhoeae.

Eleven primer pairs were developed for the identification of Neisseria gonorrhoeae. The sensitivity and specificity of these primers were evaluated by Real Time (RT)-PCR melt curve analyses with DNA from 145 N. gonorrhoeae isolates and 40 other Neisseria or non-Neisseria species. Three primer pairs were further evaluated in a hydrogel-based RT-PCR detection platform, using DNA extracted from 50 N. gonorrhoeae cultures. We observed 100% sensitivity and specificity in the hydrogel assay, confirming its potential as a point-of-care test (POCT) for N. gonorrhoeae diagnosis.

Association of Neisseria gonorrhoeae genogroups and specific PBP2/MtrR/PorB mutation patterns with susceptibility to penicillin in a susceptible gonococcal population.

To ascertain whether the antimicrobial susceptibility of Neisseria gonorrhoeae isolates with differing susceptibilities to penicillin is associated with genogroups (GGs) and combined mutation patterns in PBP2 (penA), the multiple transfer resistance repressor (MtrR; mtrR) and porin B (PorB; porB). The susceptibility of 146 clinical N. gonorrhoeae isolates to penicillin was determined using the agar dilution method and the interpretation criteria of CLSI. The DNA sequences of penA, mtrR and porB in isolates were compared with WT sequences and mutation patterns were determined. Isolates were typed by N. gonorrhoeae multi-antigen sequence typing (NG-MAST) and STs were grouped into specific GGs. The isolates tested carried 9 mutation patterns in PBP2 and 12 mutation patterns in each of MtrR and PorB. Of the 146 isolates, 121 (82.9%) were grouped into 13 different GGs. Isolates with penicillin MICs of 0.03-0.06 mg/L were significantly associated with GG25 (P < 0.05) and PBP2/MtrR/PorB mutation pattern I/WT/WT (P < 0.01). Isolates with a penicillin MIC of 1.0 mg/L were associated (P < 0.05) with: (i) GG3655 and mutation pattern XXII/A-;G45D/G120K;A121N; (ii) GG921 and mutation pattern IX/G45D/G120D;A121N; and (iii) GG1109 and mutation pattern IX/G45D/WT. Sixty percent (9/15) of penicillin-resistant isolates (MIC ≥2 mg/L) were GG3654 (P < 0.0001) and carried mutation pattern IX/G45D/G120K;A121D or IX/G45D/G120D;A121D (P < 0.05). Specific mutation patterns in PBP2/MtrR/PorB were associated with specific GGs and penicillin susceptibility. This approach of typing strains and resistance patterns is ideal for predicting antimicrobial resistance and should be used in instances in which gonococcal culture is not available but DNA can be obtained from clinical specimens.

Determining the in vitro susceptibility of Neisseria gonorrhoeae isolates from 8 cities in Guangdong Province through an improved microdilution method

A microdilution method for the antibiotic susceptibility testing of Neisseria gonorrhoeae was established and improved, and the antibiotic resistance of N. gonorrhoeae samples isolated from 8 cities of Guangdong in 2016 was determined. The improved microdilution method was compared with the agar dilution method recommend by the World Health Organization (WHO) Western Pacific Region by testing the susceptibility of 100 clinical N. gonorrhoeae isolates. The essential agreement (EA), categorical agreement (CA), very major error (VME), major error (ME), and minor error (MIE) levels of the two methods were analyzed; the acceptable performance rates were measured as follows: ≥90% for EA or CA, ≤3% for VME or ME, and ≤7% for MIE. The EA, CA, VME, ME, and MIE of each method for 7 antibiotics, penicillin, tetracycline, ciprofloxacin, spectinomycin, ceftriaxone, cefixime, and azithromycin, were 96%–100%, 94%–100%, 0%–3%, 0%–2%, and 0%–6%, respectively. The Wilcoxon signed-rank test results indicated 94%–100% agreement between the 2 methods after excluding off-scale values (P > 0.05). The susceptibility of 634 N. gonorrhoeae strains to the 7 antibiotics above were tested through the microdilution method. The resistant rates of the isolates against ciprofloxacin, tetracycline, penicillin, and azithromycin were 99.8%, 88.3%, 53.8%, and 11%, and the percentages of the isolates with decreased susceptibility to ceftriaxone (minimum inhibitory concentration [MIC] ≥0.125 μg/mL) and cefixime (MIC ≥0.25 μg/mL) were 2.1% and 12%, respectively, in Guangdong. Among 8 cities, Shenzhen had the highest rates of resistance against penicillin (77.8%) and decreased susceptibility against ceftriaxone (5.6%). Zhuhai had the highest rates of decreased susceptibility against cefixime (30.1%), and Jiangmen had the highest azithromycin-resistant isolates (16.8%). The findings from this study indicated that the improved microdilution method is an alternative for testing the antimicrobial susceptibility of N. gonorrhoeae. The resistance rates of N. gonorrhoeae against penicillin, tetracycline, and ciprofloxacin were high. While ceftriaxone, cefixime, and spectinomycin remained effective against N. gonorrhoeae, their effectiveness seemed to be decreasing over time. Azithromycin therapy requires timely susceptibility test results.