| Chapter one:Analysis on antimicrobial resistance of Neisseria gonorrhoeae isolated in Nanjing, ChinaObjective:The aim of this study was to investigate the antimicrobial susceptibilities and resistant trend of clinical Neisseria gonorrhoeae (NG) isolates in Nanjing from 2013 to 2014.Methods:NG isolates (n=384) from symptomatic male patients were collected consecutively at the Nanjing STD Clinic between 2013 and 2014. Minimum inhibitory concentrations (MICs) to penicillin, tetracycline, ciprofloxacin, spectinomycin, cefixime, ceftriaxone and azithromycin were determined by the agar dilution method. Penicillinase-producing NG (PPNG) isolates were analyzed by the paper acidometric method. Plasmid subtypes of PPNG and tetracycline-resistant NG (TRNG) isolates were determined using multiplex polymerase chain reaction (PCR). In addition, blaTEM genes were detected in PPNG.Results:A total of 100%(384/384) NG isolates displayed resistance to ciprofloxacin,85.9% (330/384) to tetracyclines,72.1%(277/384) to penicillin and 32.3%(124/384) to azithromycin.10.4%(40/384) of isolates were high-level azithromycin resistance and 9.9% (38/384) of isolates displayed reduced susceptibility to ceftriaxone. No resistance strain was found for ceftriaxone, cefixime and spectinomycin. Among all isolates, PPNG amount to 46.9%(180/384) and TRNG account for 34.6%(133/384). Genotyping of plasmids among PPNG showed that the majority (73.3%,132/180) of the isolates, while the African type plasmid significantly increased from 10.0%(12/120) in 2011～2012 to 26.7%(48/180) in 2013 ～ 2014 (χ2=12.500,P<0.001). For TRNG,91.7%(122/133) of isolates carried the Dutch type plasmid. The percentage of PPNG carrying the blaTEM-135 gene was 42.2% (76/180).Conclusions:Ceftriaxone and spectinomycin are still recommended as the first-line treatment of gonorrhea in Nanjing. However, reduced susceptibility to ceftriaxone and upward trend in MICs of spectinomycin in NG isolates stress that strengthening surveillance of antibiotic resistance is needed. The emergence of high-level azithromycin-resistant NG strains with a considerable proportion implies that ceftriaxone plus azithromycin dual therapy might not be ideal in Nanjing. Compared with the report obtained two years ago, the proportion of plasmid mediated resistant isolates and type of plasmids among TRNG have no significant change. A rise in the percentage of African type PPNG indicates genetic exchange across intercontinental among NG isolates may exist. The prevalence of PPNG carrying the blaTEM-135 gene is a high proportion in Nanjing.Chapter two:Study on the mechanism and molecular epidemiology of azithromycin resistant Neisseria gonorrhoeaeObjective:The purpose of this study was to investigate the resistance-related gene mutations, genotypes and evolutionary relationships in azithromycin resistant NG strains.Methods:A total of 124 NG isolates with azithromycin resistance were selected and divided into two groups, namely high-level azithromycin resistance (AZM-HR, MIC> 256mg/L, n=40) group and moderate azithromycin resistance (AZM-MR, MIC 1-64mg/L, n=84) group. All isolates were screened for mutations in domain V of each 23S rRNA allele, mtrR, rplD and rplV. Genotyping by NG multiantigen sequence typing (NG-MAST) and phylogenetic construction were also performed.Results:A2143G mutation in all four alleles of 23S rRNA gene was detected in all AZM-HR isolates, but not in AZM-MR isolates. The C2599T mutation in 4 alleles were present in 59.5%(50/84) of AZM-MR isolates, but not in AZM-HR isolates. The percentage of isolates carrying the G45D alteration in mtrR-coding region was significantly higher in AZM-HR (70.0%,28/40) isolates than that in AZM-MR (13.1%,11/84) isolates (χ2=40.698, P<0.001). Significantly more AZM-MR isolates (71.4%,60/84) exhibited the H105Y alteration in mtrR-coding region as compared with the AZM-HR (22.5%,9/40) isolates (χ2=26.283, P< 0.001). The mutations in rplD or rplV were not detected in any of the isolates. Among 124 azithromycin-resistant isolates,71 sequence types (STs) were identified by NG-MAST, of which 28 STs were novel. ST1866 was identified as the most prevalent type (45.0%,18/40) in AZM-HR isolates. Clonal coevolution of AZM resistance and reduced susceptibility to ceftriaxone was not detected in Nanjing using phylogenetic analysis.Conclusions:Specific point mutations in domain V of 23S rRNA alleles play an important role in azithromycin resistant NG. The A2143G mutation is associated with AZM-HR, while the C2599T mutation is associated with AZM-MR. The G45D alteration in mtrR was statistically associated with AZM-HR, while the presence of H105Y alteration was statistically associated with AZM-MR. AZM-resistant strains in Nanjing have the diversity of genetic background. ST1866 is a dominant genotype associated with AZM-HR in Nanjing.Chapter three:Study on the mechanism and molecular epidemiology in Neisseria gonorrhoeae with reduced susceptibility to ceftriaxoneObjective:This study aimed to investigate resistance-related loci, genotypes and evolutionary relationships in NG strains with reduced susceptibility to ceftriaxone.Methods:38 NG isolates with reduced susceptibility to ceftriaxone (CRORed, MIC 0.125mg/L) and 20 ceftriaxone-susceptible isolates (CROS, MIC ≤0.002～0.015 mg/L) were selected. Mutation patterns in penicillin-binding protein 2 (PBP2; penA), multiple transfer resistance repressor (MtrR; mtrR), porin B (porB), ponA and pilQ were ascertained by DNA sequence. Genotyping by NG-MAST and phylogenetic construction were also performed.Results:The mosaic PBP2 was not observed.11 amino acid sequence patterns in non-mosaic PBP2 were identified, including one new type. The major patterns of non-mosaic PBP2 in CRORed isolates were ⅩⅧ (n=17) an ⅩⅢ (n=16). All CRORed isolates had an A501T/V mutation in PBP2, which was significant difference with CROS isolates (χ2=41.981, P< 0.001). The percentage of isolates carrying dual mutations in the promoter (A deletion) and DNA-coding region (H105Y) of mtrR was significantly higher in CRORed (63.2%,24/38) isolates than that in CROS isolates (χ2=5.770, P=0.016). Other mutations including A39T/G45D alteration in mtrR-coding region, G120/A121 substitutions in porBlb and ponA (L421P) were not significantly difference between CRORed and CROS isolates. No pilQ mutations were found. Highly heterogeneous NG-MAST STs and phylogenetic polymorphism excluded the clonal expansion in CRORed isolates.Conclusions:Specific non-mosaic penA mutation patterns, particularly PBP2 containing an A501 substitution are mostly account for the reduced susceptibility to ceftriaxone in Nanjing. Nucleotide (A) deletion in the mtrR promoter and an H105Y alteration in mtrR could be associated with decreased susceptibility to ceftriaxone. |
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