| Chapter one: Analysis on antimicrobial resistance of Neisseria gonorrhoeae isolated in Hefei,ChinaObjective: The aim of this study was to investigate the antimicrobial susceptibilities and resistant trend of clinical Neisseria gonorrhoeae(NG)isolates in Hefei from 2014 to 2015.Methods: All N.gonorrhoeae clinical isolates(n =126)investigated in this study were collected from male urethritis or female cervicitis of the patients with gonorrhoeae in the clinic of Anhui Provincial Hospital,China between 2014 and 2015.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).Results: Resistance to penicillin and tetracycline was 73.8%(93/126)and 81.7%(103/126),respectively;All isolates(100%)were resistant to ciprofloxacin.A total of28.6%(36/126)of isolates were resistant to azithromycin,among them 10.3%(13/126)isolates displayed high-level azithromycin resistance(MIC ≥256 mg/L).No isolates were found to be resistant to spectinomycin,ceftriaxone and cefixime.However,10.3%(13/126)and 3.2%(4/126)of the isolates displayed decreased susceptibility to ceftriaxone(MIC=0.125 mg/L)and cefixime(MIC=0.25 mg/L),respectively.Of all isolates,39.7%(50/126)were PPNG and 31.7%(40/126)were TRNG.80%(40/50)PPNG isolates carried the Asia type β-lactamase encoding plasmid and 20%(10/50)harbored African type plasmid.No Toronto plasmid was detected.97.5%(39/40)of TRNG isolates carried the Dutch type.Conclusions: Reduced susceptibility to ceftriaxone,grim situation of azithromycin and upward trend in MICs of spectinomycin in NG isolates stress that strengthening surveillance of antibiotic resistance is needed.Ceftriaxone and spectinomycin are still recommended as the first-line treatment of gonorrhea in Hefei.The plasmid mediated resistance ratio and TRNG plasmid type distribution in Hefei region were similar to that in the southeast of China,such as Nanjing.Chapter two: Sequencing of N.gonorrhoeae genetic determinants associated with resistance to azithromycin and decreased susceptibility to ESCs.Objective: To investigate the resistance-related gene mutations,genotypes and evolutionary relationships in azithromycin resistant NG strains and strains with decreased susceptibility to ESCs.Methods: 1.A total of 126 NG isolates were selected and divided into two groups,namely azithromycin resistance(MIC≥ 1mg/L)group and azithromycin sensitive(MIC<1 mg/L)group.Genotyping by NG multiantigen sequence typing(NG-MAST)and phylogenetic construction were also performed.2.Tests for 23 Sr RNA mutations was performed on 36 Azithromycin-resistant strainsand 5 clinical azithromycin sensitive strains,comparing the mutations difference of moderate resistance group(MIC 1~64mg/L)and high resistance group(MIC≥256mg/L).3.14 strains with decreased susceptibility to ESCs were detected for pen A genes.Results: A deletion in the promoter region of mtrR was detected in 107 strains and higher percentage was seen in the higher azithromycin MIC group(1mg/L or higher)(33/36;91.7%)compared with lower MIC group(0.5mg/L or less)(74/90;82.2%),but no significant difference statistically(p=0.181).The G45 D mutation in the mtrR gene was identified more often in azithromycin-resistant isolates(69.4%,25/36)than in azithromycin – susceptible isolates(10%,9/90)(p<0.01).The A40 D was seen in 3stains exhibited reduced cefixime or ceftriaxone susceptibility.The A39 T mutation was found in 16 isolates of azithromycin MICs between 0.125mg/L to 0.5 mg/L.The A2143G(N.gonorrhoeae numbering)mutation in all four alleles was identified in all the 13 isolates with high-level azithromycin resistance(MICs≥256 mg/L).The C2599 T mutation in all four alleles was found in four isolates(MIC8 to 32 mg/L).No mutation was found in five azithromycin-susceptible isolates.Six PBP2 amino acid sequence patterns were identified,including a mosaic allele(XXXV,n=3).Among 11 nonmosaic isolates,seven isolates(allele XIII,XVII,XXI)had an A501 V substitution in PBP2,3 isolate(allele XVIII)possessed an A501 T substitution,and one isolate have a G542 S mutation.Among 126 NG isolates,86 sequence types(STs)were identified by NG-MAST,of which 53 STs were novel.ST7469 was identified as the most prevalent type(N=6)isolates,the second was ST1866(n=5),which were all present in azithromycin resistant strains.Clonal coevolution of AZM resistance and reduced susceptibility to ceftriaxone was not detected in Hefei using phylogenetic analysis.Conclusions: G45 D mutations in mtrR alleles play an important role in azithromycin resistant NG.The A2143 G mutation is associated with AZM-HR,while the C2599 T mutation is associated with AZM-MR.The G45 D alteration in mtrR was statistically associated with AZM-HR.ST1866 is dominant genetype associated with AZM-HR in Hefei.Mosaic pen A mutation pattern may play an important role in the decreased susceptibility to ceftriaxone of this area.Specific non-mosaic pen A mutation patterns, particularly PBP2 containing an A501 substitution are mostly account for the reduced susceptibility to ceftriaxone in Hefei.AZM-resistant strains in Hefei have the diversity of genetic background. |
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