Research Of The Gonococcal PenA Allele On Ceftriaxone Resistance And Biological Fitness

Chapter ⅠConstruction of penA Gene Mutant strain of Neisseria gonorrhoeae resistant ceftriaxoneObjective:To construct exchange mosaic penA10.001 and penA60.001 ceftriaxone resistant gonococcal mutant strainMethods:The recombinant plasmid pUC57-penA10.001-kanarpsL-down and pUC57-penA 60.001-kanarpsL-down of E.coli were constructed by secondary homologous recombination reverse screening and complete exchange of two clinical isolates SZ20(penA 60.001)and SRRSH78(penA 10.001).Results:The mosaic penA gene of highly resistant cephalosporin SZ20(penA 60.001)was successfully replaced into SZ20(penA10.001)mutant by secondary homologous recombination reverse screening method,and the mosaic penA gene of less sensitive ceftriaxone(penA10.001)was replaced into SRRSH78(penA 60.001)mutant.Conclusion:Recombinant plasmids containing double antibiotic screening markers were used to perform secondary homologous recombination reverse screening without introducing any other genes which could affect the mosaic genes of gonococcal.Chapter ⅡA study on the high resistance of mosaic penA gene mutation to ceftriaxone of gonococcus and the cost of biological fitness in vitroObjective:To study the effect of penA 60.001 to ceftriaxone resistance and its impact on biological fitness.Methods:Use agar dilution method to determine MIC of the transformed gonococcal wild strain SZ20(penA 60.001)/mutation strain SZ20(penA 10.001)and wild strain SRRSH78(penA 10.001)/mutation strain SRRSH78(penA 60.001)in cefixime and ceftriaxone.The growth of gonococcal wild SZ20(penA 60.007)/mutant SZ20(penA 10.001)and wild SRRSH78(penA 10.001)/mutant SRRSH78(penA 60.001)at different time was determined by ordinary liquid culture.The growth of gonococcal wild SZ20(penA 60.001)/mutation SZ20(penA 10.001)and wild SRRSH78(penA 10.001)/mutation SRRSH78(penA 60.001)were determined by adding pressure stress conditions to liquid culture environment.Spots assay determination of gonococcal wild strain SZ20(penA 60.001)/mutant strain SZ20(penA 10.001)and wild strain SRRSH78(penA 10.001)/mutant strain SRRSH78(penA 60.001)under pressure stress.The competetion growth ability of gonococcal wild strain SZ20(penA 60.001)/mutant SZ20(penA 10.001)and wild strain SRRSH78(penA 10.001)/mutant SRRSH78(penA 60.001)was compared with that of normal liquid culture and pressure stress in vitro.Results:Subsequent antimicrobial susceptibility analyses showed that mutants containing penA 60.001 displayed eight-to sixteen-fold higher ceftriaxone and cefixime minimal inhibitory concentrations(MICs)compared with otherwise isogenic mutants containing penA 10.001.Further analysis of biological fitness showed that in vitro liquid growth of single strains and in competition was identical between the isogenic penA allele swap mutants.However,in the presence of high concentrations of palmitic acid or lithocholic acid,the penA 60.001-containing mutants grew better then the isogenic penA 10.001-containing mutants when grown as single strains.In contrast,the penA 10.001 mutants outcompeted the penA 60.001 mutants when grown in competition at slightly lower palmitic acid or lithocholic acid concentrations.Conclusion:penA 60.001 is essential for ceftriaxone resistance of the FC428 clone,while its impact on biological fitness is dependent on the specific growth conditions,At higher palmitic acid or lithocholic acid concentrations in vitro pressure conditions,the penA60.001 mutants outcompeted the penA10.001 mutants,In contrast when grown in competition at slightly lower concentrations,the penA 10.001 mutants outcompeted the penA60.001 mutants.Chapter ⅢExperiment on Competition between Wild and Mutant Strain of Neisseria gonorrhoeae with Mosaic penA GeneObjective:The animal infection model of Neisseria gonorrhoeae was successfully established and the competition in vivo was compared between wild SZ20(penA60.001)/mutant strain SZ20(penA10.001)and wild SRRSH78(penA1 0.001)/mutant strain SRRSH78(penA 60.001).Methods:A mouse model BACL gonococcal vaginal infection was established by subcutaneous injection of estrogen in female mice with clean grade.the established animal model was used to carry out equal volume infection in gonococcal wild SZ20(penA60.001)/mutant strain SZ20(penA 10.001)and wild SRRSH78(penA10.001)/mutant strain SRRSH78(penA60.001)respectively.In total,6 mice were selected in each group.the vaginal samples of mice were compared daily after successful infection to observe the competitive survival in vivo.Results:The average infection time was 4.5 days in the SZ20(penA60.001)/mutant strain SZ20(penA10.001)and 4.5 days in the wild strain SRRSH78(penA10.001)/mutant(penA60.001).The colonization and survival of wild plant SRRSH78(penA10.001)was better than that of mutant SRRSH78(penA 60.001).Conclusion:In conclusion the penA 60.001 mutants were outcompeted by their penA 10.001 counterparts for in vivo colonization and survival in a mouse vaginal tract infection model.