| Chapter one: Surveillance of antimicrobial susceptibilities in Neisseriagonorrhoeae in Nanjing, 2003-2006Objective To monitor antibiotic resistance in Neisseria gonorrhoeae in Nanjing cityand to report the result obtained between 2003 and 2006 Methods The production ofβ-lactamase was determined by paper acidometric testing. Minimum inhibitoryconcentrations(MIC) for penicillin, ceftriaxone, tetracycline, ciprofloxacin andspectinomycin were determined by an agar plate dilution method. Results 791 N.gonorrhoeae isolates were examined. Rate of PPNG maintained high, from 42.23％to57.36％. During the period between 2003 and 2006, prevalence of TRNG increasedfrom 19.47％(37/190) in 2003 to 32.82％(65/198) in 2006. Among non-PPNG,chromosomally mediated resistance to penicillin ranged from 57.84％to 87.27％.Chromosomal resistance to ciprofloxacin was between 97.89％and 99.51％. None ofgonococcal isolates tested were resistant to ceftriaxone, and rate of isolates lesssusceptible to ceftriaxone ranged from 30.58％to 57.89％. One or twospectinomycin-resistant N. gonorrhoeae strains were detected each year. ConclusionsAmong N. gonorrhoeae strains isolated in Nanjing, PPNG maintained at higher level,while TRNG has increased rapidly. Chromosomally mediated resistance to bothpenicillin and ciprofloxacin was very high. Spectinomycin-resistant N. gonorrhoeaestrain was sporadic. Ceftriaxone and spectinomycin are considered as efficient drugsfor treatment of gonorrhea.Chapter two: Molecular epidemiology of tetM genes in high-leveltetracycline-resistant Neisseria gonorrhoeae (TRNG) in Nanjing, 1999-2006Objective To investigate the epidemiology of the tetM gent in Neisseria gonorrhoeaestrains with high level resistance to tetracycline (TRNG). Methods The detection ofTRNG was performed by an agar dilution method, The production ofβ-lactamase wasdetermined by paper acidometric testing. A single tube PCR was used to subtype tetM gene in TRNC..Results The rate of TRNG increased year by year from 1999 to 2006,and a total of 260(21.52％) TRNG strains were detected out of 1208 Neisseriagohorrhoeae strains in Nanjing during these period. Of these TRNG 68.08％(177/260) wereβ-lactamase producing Neisseria gonorrhoeae (PPNG/TRNG). Theresult of tetM subtyping showed that 258 TRNG were Dutch variant while two strainswere American variant. Conclusions In Nanjing, TRNG with Dutch type tetMdeterminant is predominant, whereas American variant is the sporadic.Chapter three: Study on penicillin resistance in Neisseria gonorrhoeae andgenotypes of plasmid in penicillinase-producing Neisseria gonorrhoeae inNanjing, 1999-2006Objective To study penicillin resistance in Neisseria gonorrhoeae isolates andgenotypes of plasmid in penicillinase-producing Neisseria gonorrhoeae in Nanjing.Methods Minimum inhibitory concentrations(MIC) for penicillin were determined byan agar plate dilution method. The production ofβ-lactamase was determined bypaper acidometric testing. Subtyping of resistant plasmid in PPNG was performed bya single tube PCR. Results 1208 Neisseria gonorrhoeae isolates were examined, atotal of 1015(83.21％) Neisseria gonorrhoeae strains were resistant to penicillin. Rateof chromosomally mediated resistance to Neisseria gonorrhoeae was 45.53％(550/1208). Prevalence of PPNG was 38.49％(465/1208), in which 177(38.06％)strains were also TRNG. The proportion of PPNG increased year by year, from20.58％in 1999 to 57.36％in 2004, and decreased slightly in 2005 and 2006. Theresult of genotyping showed that all (409) PPNG tested contained Asian type plasmid.Conclusions Among Neisseria gonorrhoeae strains isolated in Nanjing, theprevalence of CMRNG maintains at higher level. The ratio of PPNG increased rapidly.Asian type plasmid is prevalent in PPNG strains. Other types of plasmid was notfound.Chapter four: Study on mechnisma of spectinomycin-resistant Neisseriagonorrhoeae Objiective To detect the mutions of 16S rRNA gene in Neisseria gonorrhoeaeresistant to spectinomycin. Methods 10 Neisseria gonorrhoeae strains include 6spectinomycin-resistant (MIC≥128μg/mL) isolates and 4 susceptible strains wereinvestigated. 16S rRNA gene in those strains were amplified by polymerase chainreaction and followed by DNA sequencing. Results Mutations of 16S rRNA genewere founded in 6 Neisseria gonorrhoeae strains resistant to spectinonmycin. Ofthese mutations, C to T substitution at 1192 was detected in 2 strains(MIC＞256μg/mL). Double mutations were found in two strains (MIC 256μg/mL),with C1344T and T1345A in one strain, and T990G and T991C in the another.Triple mutations were detected in one strain (MIC 128μg/mL) with changes atT990G,G1343C and C1344T. A mutation of A991G were founded in one strain (MIC128μg/mL). No mutation was detected in 16S rRNA gene in 4 Neisseria gonorrhoeaestrains sensitive to spectinomycin. Conclusions Transversions at various sites in 16SrRNA gene in Neisseria gonorrhoeae are related to different levels of spectinomycinresistance. Among these mutations C1192T mutation may lead to a higher levelresistance to spectinomycin in Neisseria gonorrhoeae, the other single mutation ormultisite mutations are associated to varying degrees of resistance less than transitionat C1192T.Chapter five: Study on Amino acid substitutions or insertions patterns of PBP2in Neisseria gonorrhoeae decreased susceptibility to ceftriaxoneObjiective To investigate the change of amino acid patterns in penicillin-blindingprotein 2(PBP2) in Neisseria gonorrhoeae isolates with decreased susceptibility toceftriaxone. Methods 13 gonococcal isolates including 11 strains with decreasedsusceptibility to ceftriaxone and 2 isolates sensitive to ceftriaxone were investigated.The penA gene encoding the penicillin-blinding protein 2 was amplified by PCR andthen sequenced and analyzed by BLASTN and BLASTX. Results There are fivedifferent PBP2 amino acid substitutions or insertions patterns in 13 gonococcalisolates, among these patterns no PBP2 mosaic structure was found. ConclusionsPBP2 mosaic structure may not be related to the decreased susceptibility to ceftriaxone in Neisseria gonorrhoeae. The result indicated that additional factors arecontributing to the emergence of decreased ceftriaxone susceptibility.
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