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Objective To analyze the genotypes of Neisseria gonorrhoeae ( N. gonorrhoeae) epi-demic strains in Wenzhou, eastern China, and to study the mechanism of tetracycline resistance in these strains. Methods A total of 77 N. gonorrhoeae strains were isolated from patients with gonorrhea. Antimi-crobial susceptibility of these strains to penicillin, tetracycline, ciprofloxacin, spectinomycin, ceftriaxone and azithromycin was analyzed using E-test. PCR and DNA sequencing were used to detect the genes associ-ated with tetracycline resistance, such as Tet-M, mtrR promoter region and mtrR coding region. N. gonor-rhoeae multi-antigen sequence typing ( NG-MAST) and multilocus sequence typing ( MLST) were used to determine the molecular characteristics of all clinical isolates and tetracycline-resistant isolates, respectively. Results Among the 77 N. gonorrhoeae isolates, 74 (96. 10％), 27 (35. 06％) ,70 (90. 91％) and 15 (19. 48％) were resistant to penicillin, tetracycline, ciprofloxacin and azithromycin, respectively. All tested isolates were susceptible to spectinomycin and ceftriaxone. Nineteen isolates were resistant to tetracycline and all of them carried Tet-M gene. Among them, 17 had one deletion mutation of base A in mtrR promoter region and three had G45D mutation in mtrR coding region. NG-MAST classified the 19 tetracycline-resistant isolates into 11 different sequence types (ST). ST14781, ST1766 and ST1866 each accounted for 15. 79％(three strains). Two ST (10. 52％, 2/19) found in the present study had not been reported previously in the NG-MAST database. MLST showed the 19 tetracycline-resistant isolates belonged to 12 different STs, in which ST10899 accounted for 26. 32％ (five strains) and ST1600 accounted for 15. 79％ (three strains). Conclusions Mutations in mtrR promoter region and carrying Tet-M gene were associated with tetracycline resistance in N. gonorrhoeae. Clinical strains isolated in Wenzhou showed considerable molecular diversity. Measures should be implemented to monitor the spread of NG-MAST ST1766 and MLST ST1600 N. gonor-rhoeae clones with high resistance to tetracycline in Wenzhou.
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BACKGROUND: We investigated the molecular epidemiological characteristics and antimicrobial susceptibility pattern of penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates to monitor the change in distribution of bla(TEM) in Korea. METHODS: We collected 804 PPNG isolates from diverse hospitals and clinics mainly located in Seoul, Korea, over a period of 11 years (2005–2015). Isolate susceptibility to seven antimicrobials was determined using the agar dilution test. The molecular epidemiological characteristics of the isolates were determined by Sanger sequencing of bla(TEM), N. gonorrhoeae multiantigen sequence typing (NG-MAST) and plasmid typing. RESULTS: Among 72 fully sequenced PPNG isolates, sixteen (22.2%) possessed TEM-135. All TEM-135 isolates had a common silent mutation (c.18C>T), which was previously unreported. We observed a pattern of continuous increase in the number of TEM-135 isolates since 2012. The median and 90% minimum inhibitory concentration of azithromycin were substantially lower in the TEM-135 group than in the non-PPNG and TEM-1 groups. All TEM-135 isolates showed different NG-MAST types and predominantly harbored Toronto/Rio (75%) plasmids. A comprehensive comparative analysis of PPNG with TEM-135 according to NG-MAST, plasmid type, and year of isolation revealed a wide distribution. CONCLUSIONS: The proportion of TEM-135 PPNG has continuously increased since 2012, in association with clonal spread. The difference at position 18 of the TEM-135 sequence can be interpreted as the existence of multiple clonal complexes. The possibility that TEM-135 was acquired via foreign plasmids requires careful follow-up and continuous monitoring of TEM-135 to ascertain whether it constitutes a step towards evolutionary change.
Subject(s)
Agar , Azithromycin , Drug Resistance, Microbial , Follow-Up Studies , Incidence , Korea , Microbial Sensitivity Tests , Neisseria gonorrhoeae , Neisseria , Plasmids , Seoul , Silent MutationABSTRACT
Objective To analyze the characteristics of genotyping and gene polymorphism of Neisseria gonorrhoeae(N.go) with azithromycin(AZM)-resistance(AZM-R) and decreased susceptibility to ceftriaxone(CROD).Methods The minimum inhibitory concentration(MIC) of AZM and CRO were determined.AZM-R isolates were detected for mutations in 23S rRNA,mtrR and penA genes.Genotypes were analyzed by using N.go multi-antigen sequence typing(NG-MAST).Results All total of 485 isolates of N.go were detected.77(15.9%) strains were AZM-R(MIC≥1 mg/L),including 33(6.8%) isolates of AZM low-level resistant(AZM-LLR,MIC=1 mg/L) strains and 44(9.1%) isolates of AZM middle-level resistant(AZM-MLR,MIC≥2 mg/L) strains.There were more CROD(MIC≥0.125 mg/L) strains in AZM-MLR isolates(43.2%),compared with those in AZM-LLR isolates(18.2%,P0.05).Similar results were found between combined AZM-LLR/CROD isolates and combined AZM-MLR/CROD isolates(P>0.05).No mutation of A2059G and AZM high-level resistant(AZM-HLR,MIC≥256 mg/L) isolate were found.Among 77 AZM-R isolates,67 sequence types(ST) were identified by NG-MAST,of which 30 types were novel.Most ST were represented by a single isolate.Conclusion AZM-R and CROD isolates,presented in this area,might be deserved continuous surveillance to identify the mechanism of concurrent resistance.
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Objective To investigate the prevalence, molecular mechanism and genetic characteristics of azithromycin-resistant Neisseria gonorrhoeae (N.gonorrhoeae) strains isolated in Shenzhen.Methods N.gonorrhoeae strains were collected in Shenzhen from 2011 to 2015.Agar dilution method and E-test were used to detect the minimum inhibitory concentrations (MIC) of these strains to azithromycin.All azithromycin-resistant (AZM-R) strains (MIC≥2 μg/ml) and some azithromycin-sensitive strains (MIC≤0.25 μg/ml) which were randomly selected as the control group were screened for mutations in 23S rRNA, mtrR and erm genes and genotyped by using N.gonorrhoeae multi-antigen sequence typing (NG-MAST).Results A total of 788 N.gonorrhoeae strains were collected, 148 (18.8%) of which were AZM-R strains (MIC≥1 μg/ml).Eighteen out of 21 high-level AZM-R (AZM-HLR) strains had A2143G mutations in the four copies of the 23S rRNA gene.Twelve out of 29 middle-level AZM-R (AZ-MLR) strains had missense mutations, among which C2611T mutations in the four copies of the 23S rRNA were detected in 10 strains.Incidence of G45D/Y105H mutation in AZM-HLR strains was higher than that in AZM-MLR (χ2=12.702, P=0.000) or AZ-S (χ2=4.462, P=0.035) strains according to the analysis of the promoter and coding region of mtrR gene.PCR analysis revealed that only one strain carried ermB gene (MIC=2 μg/ml).The 788 N.gonorrhoeae strains were typed into 81 sequence types (STs) by NG-MAST, most of which were represented by one strain only.STs of ST3356 and ST1866 that were identified in the AZ-R strains in the current study had been noted in a previous report of emerging AZM-R N.gonorrhoeae strains in Nanjing, Chongqing and Guangzhou.Neighbor-joining (NJ) phylogenetic tree showed that the resistant strains did not form a separate cluster.Conclusion Currently, it is not suitable to use azithromycin as a monotherapy for gonorrhea in Shenzhen.Mutations of A2059G and C2611T in 23S rRNA of N.gonorrhoeae were respectively responsible for high-level and middle-level resistance to azithromycin.Repeated emergence of ST1866 and ST3356 will help us monitor and analyze the epidemiological characteristics of N.gonorrhoeae strains resistant to azithromycin in Shenzhen.
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Objective To investigate genetic characteristics of Neisseria gonorrhoeae (N.gonorrhoeae) isolates from Guangzhou city in 2014,and to analyze the relationship of N.gonorrhoeae multi-antigen sequence typing (NGMAST) sequence types (STs) with ciprofloxacin resistance.Methods An agar dilution method was used to determine the minimal inhibitory concentration (MIC) of ciprofloxacin in 97 N.gonorrhoeae isolates from Guangzhou city.PCR was performed to amplify the gyrA,parC,porB and tbpB genes from these isolates,followed by gene sequencing and determination of NG-MAST STs.Results Of the 97 N.gonorrhoeae isolates,95 (97.9％) were resistant to ciprofloxacin,including 41 high-level (MIC ≥ 16 mg/L) and 54 low-level (1 mg/L ≤ MIC ＜ 16 mg/L) resistant strains.Mutations were detected at codons 91 and 95 encoding serine in the gyrA gene of all the 95 ciprofloxacin-resistant strains,and in the parC gene of 93 resistant strains.The frequency of the mutation at codon 87 in the parC gene was 85.4％ (35/41) in high-level resistant strains,significantly higher than that in low-level resistant strains (59.3％[32/54],x2 =7.64,P ＜ 0.05).MAST STs were successfully determined for all the 97 N.gonorrhoeae isolates except 1 isolate with incorrect PCR amplicons.Of the 96 genotyped isolates,50 were assigned to 35 known STs by using the NG-MAST website (www.ngmast.net),among which,10 STs each contained 2 to 4 isolates.The most common ST was ST5309.Phylogenetic tree analysis revealed that the 96 genotyped N.gonorrhoeae isolates could be classified into 2 groups,and the proportion of isolates with MIC ≥ 16 mg/L is 46.4％ (39/84) in group 1,but only 1/12 in group 2 (x2 =6.27,P=0.012).Conclusions High-level resistance of N.gonorrhoeae to ciprofloxacin may be mainly associated with the mutation at codon 87 in the parC gene.NG-MAST STs may be related to the degree of ciprofloxacin resistance.
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PURPOSE: The detection of high-level tetracycline-resistant strains of Neisseria gonorrhoeae (TRNG) can make important epidemiological contributions that are relevant to controlling infections from this pathogen. In this study, we aimed to determine the incidence of TRNG isolates over time and also to investigate the characteristics and genetic epidemiology of these TRNG isolates in Korea. MATERIALS AND METHODS: The antimicrobial susceptibilities of 601 isolates of N. gonorrhoeae from 2004 to 2011 were tested by standard Clinical and Laboratory Standards Institute methods. To determine the molecular epidemiological relatedness, N. gonorrhoeae multi-antigen sequence typing was performed. RESULTS: The incidence of TRNG increased from 2% in 2004 to 21% in 2011. The minimum inhibitory concentration distributions of ceftriaxone and susceptibility of ciprofloxacin in TRNG were different from non-TRNG and varied according to the year of isolation. Most of the TRNG isolates collected from 2004 to 2007 exhibited genetic relatedness, with sequence type (ST) 1798 being the most common. From 2008 to 2011, the STs of the isolates became more variable and introduction of genetically unrelated TRNG were noted. CONCLUSION: The increased incidence of TRNG strains until 2007 appears to be due, at least in part, to clonal spread. However, we propose that the emergence of various STs since 2008 could be associated with foreign import.
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Humans , Anti-Bacterial Agents/pharmacology , Ceftriaxone/pharmacology , Ciprofloxacin/pharmacology , DNA, Bacterial/analysis , Drug Resistance, Multiple, Bacterial/genetics , Gonorrhea/drug therapy , Incidence , Microbial Sensitivity Tests , Molecular Epidemiology , Neisseria gonorrhoeae/drug effects , Republic of Korea/epidemiology , Sequence Analysis, DNA , Tetracycline/pharmacology , Tetracyclines/pharmacologyABSTRACT
To identify the genomic species of Neisseria gonorrhoeae, evaluate the difference between two molecular epidemiological methods and examine the relationship between sex partners and genotypes of bacteria, 24 strains of Neisseria gonorrhoeae isolated from the outpatients with gonorrhea were identified by using the Opa genotyping and NG-MAST genotyping and the relationship between genotypes and phenotypes was studied. Twenty-four strains of Neisseria gonorrhoeae fell into 10 ST genotypes by NG-MAST genotyping, whereas these strains were classified into 12 OT Opa genotypes by Opa genotyping. A new epidemic strain of ST genotype (217-86% homologisation 178) in China was identified. It is concluded that genotypes of each pair of strains from a pair of patient/sex partner besides 45/46 are the same, indicating that contagious infection take place between patient and the sex partner. Opa genotyping was more effective than NG-MAST genotyping in identifying the genomic species of Neisseria gonorrhoeae. ST genotype could be further classified into different Opa-types.