| Research BackgroundAntimicrobial resistance in Neisseria gonorrhoeae has become a global public health problem.In response to the increasingly severe resistance situation,it is necessary to strengthen continuous monitoring of gonococcal antibiotic resistance,and to address the two major needs of rapid detection of resistant strains and alternative treatment for gonorrhea.Developing rapid detection methods for antibiotic resistance is beneficial for timely detection of resistant strains and can complement the long detection time of traditional methods in clinical medication guidance.In addition,screening of currently approved antibiotics and newly developed antibiotics can ensure alternative treatment options in case of treatment failure with first-line drugs.This study is divided into three parts,with the aim of providing strategies for promoting antibiotic susceptibility monitoring,rapid detection of resistant strains,and alternative treatment for gonorrhea.Part 1:Supplements assessment and consistency comparison of traditional antimicrobial susceptibility testingSection 1:The impact of alternative growth supplements on antimicrobial susceptibility testing results in the agar dilution method[Objective]To evaluate the impact of alternative growth supplements on antimicrobial susceptibility testing results in the agar dilution method.[Methods]The agreement of the antimicrobial susceptibility testing results of eight antibiotics(penicillin,gentamicin,tetracycline,spectinomycin,azithromycin,ciprofloxacin,ceftriaxone and zoliflodacin)with the addition of sterile defibrinated sheep blood or bovine hemoglobin as a growth supplement was assessed using the group added Vitox supplement as a control group.[Results]The essential agreement(EA)and category agreement(CA)of the sheep blood group for eight antibiotics were above 90.00%,and the minor error rate was less than 7.00%.The consistency of the MIC results of zoliflodacin,penicillin and ceftriaxone in the hemoglobin group was lower than 90%.[Conclusion]Sterile defibrinated sheep blood has good consistency with Vitox supplement as an alternative growth supplement for gonococcal antibiotic susceptibility surveillance,with lower cost and easier availability than Vitox supplement.Section 2:Comparison of the consistency of disk diffusion method and agar dilution method[Objective]To evaluate the accuracy of disk diffusion method on gonococcal antibiotic susceptibility testing.[Method]The antibiotic susceptibility of ceftriaxone,cefixime,and spectinomycin was determined using agar dilution method and disk diffusion method,respectively.The consistency between the two methods was calculated.[Results]The CA of spectinomycin,ceftriaxone and cefixime by disk diffusion method were 87.64%,83.45%and 80.31%,respectively,which were lower than the acceptable agreement rates.The results of the paired chi-square test indicated that there was a significant difference between the MIC results detected by the two methods.[Conclusion]The disk diffusion method is less accurate and may not be an appropriate choice for screening gonococcal antibiotic resistance strains in clinical practice.Part 2:Study of rapid molecular detection method for gonococcal azithromycin resistanceSection 1:Meta-analysis of the accuracy of 23S rRNA C2611T mutation in diagnosing azithromycin resistance[Objective]To evaluate the accuracy of the mutation C2611T in diagnosing the azithromycin resistance in gonococci.[Methods]Relevant studies were searched in multiple databases.Sensitivity and specificity of mutation C2611T in diagnosing the azithromycin resistance were metaanalyzed.[Results]Based on the meta-analysis of 20 included studies,the sensitivity of C2611T mutation for detecting gonococcal azithromycin minimal inhibitory concentration(MIC)in 4-128 mg/L was 91.9%and the specificity was 95.9%.[Conclusion]23S rRNA C2611T mutation has high accuracy for detecting gonococcal azithromycin MIC in 4-128 mg/L.Section 2:Establishment of MAMA-PCR method for rapid detection of gonococcal azithromycin resistance[Objective]To establish a rapid molecular detection method based on mismatch amplification mutation assay by SYBR Green qPCR(MAMA-PCR)according to 23S rRNA A2059G and C2611T mutations.[Method]Mismatched primers were designed based on the A2059G and C2611T mutations.The double-stranded DNA was detected by SYBR Green I fluorescent dye,and the △Ct values of the two genes were compared with the Ct values of the internal reference gene to distinguish the mutant strain and wild-type strain.[Results]The △Ct value of A2059G mutant gene was selected as 3,and that of C2611T mutant gene was selected as 3.If the △Ct value was less than or equal to this value,the strain was identified as mutant.The detection limit was 100 copies/μl in this method.[Conclusion]The MAMA-PCR method is a rapid molecular detection method that can detect 23S rRNA A2059G and C2611T mutations.Section 3:Application of MAMA-PCR for detection of azithromycin resistance in gonococcal clinical isolates[Objective]To evaluate the sensitivity and specificity of MAMA-PCR method for the diagnostic of azithromycin resistance in clinical isolates.[Methods]367 gonococcal clinical isolates were selected from multiple regions in China.The MAMA-PCR method was used to detect 23S rRNA mutations and diagnose azithromycin resistance.The MIC results was compared with that of the agar dilution method to assess the accuracy of this method.[Results]5 C2611T mutant strains and 4 A2059G mutant strains were found among the 367 gonococcal clinical isolates.The sensitivity of MAMA-PCR method was 100%and the specificity was 99.72%in predicting azithromycin MIC of 4-128 mg/L by detecting C2611T mutation in 367 clinical isolates;the sensitivity and specificity of detecting A2059G mutation in predicting azithromycin MIC≥256 mg/L were both 100%.[Conclusion]MAMA-PCR has high sensitivity and specificity in detecting gonococcal clinical isolates with azithromycin MIC≥2mg/L.Part 3:Evaluation of the in vitro effects of alternative antibiotics for gonorrheaSection 1:In vitro study of the interaction of gentamicin with ceftriaxone and azithromycin against gonococci[Objective]To analyze the susceptibility to gentamicin of gonococci isolates and the interaction between gentamicin,azithromycin and ceftriaxone.[Methods]The susceptibility testing of gentamicin,ceftriaxone and azithromycin was performed on 380 gonococcal clinical isolates from multiple regions in China using the agar dilution method,and the interaction between three antibiotics was detected by the agar dilution checkerboard method.[Results]36.60%of isolates showed complete susceptibility to gentamicin and 61.30%showed intermediate susceptibility.The overall results of the three antibiotic combinations revealed indifferent effects.Among azithromycin-resistant bacteria,the MIC geometric mean for azithromycin alone and in combination were 2.532 mg/L and 0.397 mg/L,respectively(84.3%reduction in combination compared to monotherapy).[Conclusion]Most of the gonococci isolates demonstrated susceptibility to gentamicin.Gentamicin significantly reduces MIC when used in combination with other drugs,and may be an alternative for the future treatment of gonorrhea in China.Section 2:In vitro study of the susceptibility to new tetracycline antibiotics on gonococci[Objective]To analyze the susceptibility of gonococci to three third-generation tetracycline antibiotics and the correlation between MICs.[Methods]The MICs of ceftriaxone,azithromycin,tetracycline,minocycline,tigecycline,eravacycline and omadacycline of 412 gonococci isolates were measured by the agar dilution method.The correlations between MICs were determined by the value of the correlation coefficient R.[Results]The MIC90 of 412 gonococci isolates was 0.25mg/L to ceftriaxone,1 mg/L to azithromycin,64mg/L to tetracycline,64mg/L to minocycline,0.5mg/L to tigecycline,0.5mg/L to eravacycline,4mg/L to omadacycline.A weak correlation between MIC results of omadacycline,eravacycline and tetracycline was observed.The MIC results of tigecycline and azithromycin showed a weak correlation.The MIC results of minocycline and tetracycline showed a strong correlation.[Conclusion]Comparing the in vitro effects and feasibility of three third-generation tetracyclines,this study suggested that tigecycline had better in vitro activity and might be alternative antibiotic for the treatment of gonorrhea. |
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