Preliminary Strategies For Antimicrobial Resistance In Neisseria Gonorrhoeae To First-line Therapeutic Drugs

Section 1:Molecular research for the ceftriaxone-resistant Neisseria gonorrhoeae Part 1:Genotyping of the ceftriaxone-resistant clinical gonococcal isolate Objective:Ceftriaxone is the last option for empiric treatment of Neisseria gonorrhoeae.This study aimed to genotyping the ceftriaxone-resistant clone BJ16148,isolated from annual China Gonococcal Antimicrobial Surveillance Programme.Methods:Minimum Inhibitory Concentrations(MICs)to azithromin,spectinomycin,tetracycline,cipfloxacin,and cefixime were obtained using Etest methods.Multiantigen Sequence Typing(NG-MAST),Multilocus Sequence Typing(MLST)and Sequence Typing for Antimicrobial Resistance(NG-STAR)were used for its identification.Results:MICs of BJ16148 to ceftriaxone,azithromycin,spectinomycin,tetracycline,cipfloxacin,and cefixime were 0.5,0.25,16,4,>32,and 1mg/L.And its NG-MAST,MLST,NG-STAR,and penA genetyping related to ceftriaxone-resistance were 3435,1903,233,and 60.Conclusions:The ceftriaxone-resistant gonococcal clone in this study is homogenous to the resistant one found in Japan,2015.Part 2:Establishment of a molecular assay for ceftriaxone-resistant clones Objective:Nowadays resistant clones to ceftriaxone has been transmitted globally.This study aimed to establish a molecular dual-detection assay for the rapid discovery of gono-coccal isolates and globally disseminted ceftriaxone-resistant clones.Methods:The prin-ciple of real-time fluorescence PCR method was used to develop the detection assay for two specific gonococcal sequences of porA and penA gene segments.We assessed the fea-sibility and detection limits.Results:The dual-detection assay can precisely amplify the corresponding fragments with the detection limits of about 160 copies/reaction Conclu-sions:The rapid dual-detection assay established in this study can detect both Neisseria gonorrhoeae isolates and globally transmitted ceftriaxone-resistant clones with good de-tection efficacy.Part 3:Comparison of molecular detection assays for ceftriaxone-resistant clones Objective:To compare the accuracy and clinical applicability of four molecular detection assays for ceftriaxone-resistant gonococcal clones.Methods:The real-time fluorescence PCR methods of simple probe and locked nucleic acid(LNA)probe,and high resolution melting(HRM)method were established to detect ceftriaxone-resistant clones according to descriptions in different literatures.Then 18 positive samples of ceftriaxone-resistant gonococcal clones recently collected in China and 51 negative samples with different penA genotypes were used to evaluate their accuracy.Results:Four methods were sensitive in detecting positive samples.Of three probe methods,the LNA probe assay produced the most false-positive results,followed by the simple probe assay,and no false-positive re-sults were found in the dual-detection probe assay.Regarding the HRM assay,melting temperatures of positive samples ranged from 78.25 to 78.95?,which were much lower than the minimum melting temperature of negative samples,79.35?.The melting tem-perature of 79? can be a distinguishing threshold for ceftriaxone-resistant clones.Con-clusions:All four assays were able to detect the globally disseminated ceftriaxone-re-sistant gonococcal clones accurately.The HRM assay is recommended for high throughput screening tests,and the dual-detection method is more apt for rapid clinical application.Section 2:Antimicrobial susceptibility surveillance to gentamicinPart 1:Preliminary evaluation on gonococcal susceptibility to gentamicin in Dalian Objective:Under the severe antimicrobial resistance circumstances,this study preliminary evaluate the susceptibility profiles to gentamicin of clinical strains in Dalian.Methods:We selected 120 gonococcal isolates from 2016 to 2018,and detected their MICs to gen-tamicin.The Kruskal-Wallis H test was used to analyse their annual MICs.Results:Within 120 gonococcal isolates,no resistant strains were detected.103(85.8%)isolates were sus-ceptible to gentamicin(MIC?4mg/L),and 17 strains were intermediate susceptible(8mg/L).No significant difference was found in their annual MIC values.Conclusions:Neisseria gonorrhoeae isolates in Dalian were mostly susceptible to gentamicin,with no annual variation.A larger sample size is essential to appraise its clinical potency.Part 2:Cross-sectional evaluation on gonococcal susceptibility to gentamicin in ChinaObjective:This study assessed the gentamicin susceptibility of gonococcal isolates col-leted in China,and their relationships with ceftriaxone,azithromycin,and spectinomycin.Methods:In this study,470 clinical isolates collected from seven provinces in China,were tested by the agar dilution method to obtain their MICs to four antibiotics.And we ana-lyzed their associations between susceptibility profiles.Results:The MICs of gentamicin ranged from 1 to 8mg/L,and no resistant clones were found.19(4.0%)strains were re-sistant to ceftriaxone,and 73(1 5.5%)isolates were resistant to azithromycin.No correla-tion was found between the gentamicin MICs and the susceptibility or resistance profiles of different antibiotics.Conclusions:In vitro tests found that most gonococcal strains were susceptible to gentamicin.And clinical trials can be conducted to evaluate its therapeutic effects.In addition,the decreased susceptibility of Neisseria gonorrhoeae to ceftriaxone and/or azithromycin,the first-line therapeutic agents,is gradually emerging in China,and enhanced antimicrobial resistance surveillance is urgently needed.