中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2010年
5期
466-471
,共6页
结核分枝杆菌%耐药%聚合酶链反应%基因%分子特征
結覈分枝桿菌%耐藥%聚閤酶鏈反應%基因%分子特徵
결핵분지간균%내약%취합매련반응%기인%분자특정
Mycobacterium tuberculosis%Drug resistant%PCR%Gene%Molecular characterization
目的 研究深圳地区2007-2008年分离的结核分枝杆菌耐药株分子特征与表型特征的相关性.方法 参照WHO/IUATLD标准,使用L-J药敏培养基,1%比例法药敏试验筛选针对异烟肼、利福平、链霉素、氧氟沙星、卡那霉素5种药物耐药或敏感的临床分离株,通过PCR扩增经筛选菌株的rpoB、katG、rpsL、rrs1、gyrAB、rrs2基因的相关序列,运用DNAStar和BLASTN进行序列分析,应用二倍稀释法测定其表型最低抑菌浓度(MIC)值.结果 筛得实验菌株123株,其中耐药株73株,全敏感株50株.异烟肼耐药株katG基因突变率为84.6%,突变位点全部为S315T或S315N.利福平耐药株rpoB基因突变率为93.6%,突变位点主要集中在S531L(30/44,68.2%)和H526D(9/44,20.5%)或H526R(1/44,2.3%).链霉素耐药株以rpsl基因突变为主,突变位点为K43R(19/27,70.4%)和K88Q(6/27,22.2%);rrs1基因突变较少见,仅491C→T(2/27,7.4%)及513A→C(1/27,3.7%);2个基因突变率合计为65.9%.氧氟沙星耐药株突变率为100%,以gyrA基因突变为主,突变位点包括D94A(2/11,18.2%)、S91P(4/11,36.4%)和A90V(3/11,27.3%),3个突变位点总突变率为81.8%(9/11);S95T存在于所有氧氟沙星耐药株及部分全敏感株gyrA基因中;gyrB未发现突变.卡那霉索耐药株rrs2基因突变率为61.1%;突变位点为1400 A→C(9/11,81.8%)和1483 G→T(2/11,18.2%).其他突变位点在全敏感株中未发现.临床耐药株同一耐药组别所包含耐药类型不同,相关耐药基因的突变位点相同,其MIC值基本一致;相关耐药基因突变位点不同,其MIC值存在明显差异.结论 结核分枝杆菌耐药基因突变存在一定的地域特性,表型特征因耐药基因突变位点不同存在耐药程度差异.
目的 研究深圳地區2007-2008年分離的結覈分枝桿菌耐藥株分子特徵與錶型特徵的相關性.方法 參照WHO/IUATLD標準,使用L-J藥敏培養基,1%比例法藥敏試驗篩選針對異煙肼、利福平、鏈黴素、氧氟沙星、卡那黴素5種藥物耐藥或敏感的臨床分離株,通過PCR擴增經篩選菌株的rpoB、katG、rpsL、rrs1、gyrAB、rrs2基因的相關序列,運用DNAStar和BLASTN進行序列分析,應用二倍稀釋法測定其錶型最低抑菌濃度(MIC)值.結果 篩得實驗菌株123株,其中耐藥株73株,全敏感株50株.異煙肼耐藥株katG基因突變率為84.6%,突變位點全部為S315T或S315N.利福平耐藥株rpoB基因突變率為93.6%,突變位點主要集中在S531L(30/44,68.2%)和H526D(9/44,20.5%)或H526R(1/44,2.3%).鏈黴素耐藥株以rpsl基因突變為主,突變位點為K43R(19/27,70.4%)和K88Q(6/27,22.2%);rrs1基因突變較少見,僅491C→T(2/27,7.4%)及513A→C(1/27,3.7%);2箇基因突變率閤計為65.9%.氧氟沙星耐藥株突變率為100%,以gyrA基因突變為主,突變位點包括D94A(2/11,18.2%)、S91P(4/11,36.4%)和A90V(3/11,27.3%),3箇突變位點總突變率為81.8%(9/11);S95T存在于所有氧氟沙星耐藥株及部分全敏感株gyrA基因中;gyrB未髮現突變.卡那黴索耐藥株rrs2基因突變率為61.1%;突變位點為1400 A→C(9/11,81.8%)和1483 G→T(2/11,18.2%).其他突變位點在全敏感株中未髮現.臨床耐藥株同一耐藥組彆所包含耐藥類型不同,相關耐藥基因的突變位點相同,其MIC值基本一緻;相關耐藥基因突變位點不同,其MIC值存在明顯差異.結論 結覈分枝桿菌耐藥基因突變存在一定的地域特性,錶型特徵因耐藥基因突變位點不同存在耐藥程度差異.
목적 연구심수지구2007-2008년분리적결핵분지간균내약주분자특정여표형특정적상관성.방법 삼조WHO/IUATLD표준,사용L-J약민배양기,1%비례법약민시험사선침대이연정、리복평、련매소、양불사성、잡나매소5충약물내약혹민감적림상분리주,통과PCR확증경사선균주적rpoB、katG、rpsL、rrs1、gyrAB、rrs2기인적상관서렬,운용DNAStar화BLASTN진행서렬분석,응용이배희석법측정기표형최저억균농도(MIC)치.결과 사득실험균주123주,기중내약주73주,전민감주50주.이연정내약주katG기인돌변솔위84.6%,돌변위점전부위S315T혹S315N.리복평내약주rpoB기인돌변솔위93.6%,돌변위점주요집중재S531L(30/44,68.2%)화H526D(9/44,20.5%)혹H526R(1/44,2.3%).련매소내약주이rpsl기인돌변위주,돌변위점위K43R(19/27,70.4%)화K88Q(6/27,22.2%);rrs1기인돌변교소견,부491C→T(2/27,7.4%)급513A→C(1/27,3.7%);2개기인돌변솔합계위65.9%.양불사성내약주돌변솔위100%,이gyrA기인돌변위주,돌변위점포괄D94A(2/11,18.2%)、S91P(4/11,36.4%)화A90V(3/11,27.3%),3개돌변위점총돌변솔위81.8%(9/11);S95T존재우소유양불사성내약주급부분전민감주gyrA기인중;gyrB미발현돌변.잡나매색내약주rrs2기인돌변솔위61.1%;돌변위점위1400 A→C(9/11,81.8%)화1483 G→T(2/11,18.2%).기타돌변위점재전민감주중미발현.림상내약주동일내약조별소포함내약류형불동,상관내약기인적돌변위점상동,기MIC치기본일치;상관내약기인돌변위점불동,기MIC치존재명현차이.결론 결핵분지간균내약기인돌변존재일정적지역특성,표형특정인내약기인돌변위점불동존재내약정도차이.
Objective To characterize the relationship between such phenotypes and the patterns of genetic mutations in the corresponding resistance genes in drug-resistant Mycobacterium tuberculosis (MTB)isolates in Shenzhen of China during 2007-2008.Methods According to standard of WHO,International Union Against Tuberculosis and Lung Disease(IUATLD),136 strains of MTB were collected by performing drug sensitivity test(DST)to isoniazid,rifampicin,streptomycin,ofloxacin and kanamycin on Lowenstein-Jensen in 1%proportion method.Genetic mutations in the corresponding resistance genes (rpoB,katG,rpsL,rrs1,gyrAB,rrs2)in these MTB isolates were identified by PCR,followed by DNA sequencing of the purified PCR products.The minimal inhibitory concentration(MIC)values of the aforementioned anti-tuberculosis drugs were determined for these MTB clinical isolates by two-fold dilution method in vitro.Results A total of 123 isolates were collected,73 isolates were drug resistant.50 isolates were drug susceptible.Among the isolates that were resistant to isoniazid,rifampicin,streptomycin.ofloxacin and kanamycin,the proportion of isolates that harboured mutations in the respective genes was 84.6%,93.6%,65.9%,100%,61.1%.For katG gene,the mutation detected were S315T or S315N.For rpoB,the most frequently found changes were S531L(30/44,68.2%)and H526D(9/44,20.5%)or H526R(1/44,2.3%).The reported mutations that K43R and KS8Q were founded in the rpsL locus and 491C→T and 513A→C were founded in the rrs1 gene related with streptomycin-resistant strains.For gyrA,all gyrA mutations were clustered in codons 90,91,and 94 apart from the S95T that was natural polymorphism.accounted for 81.1% of the ofloxacin-resistant isolates,and condon 91 was the most frequently mutated.No mutation were found in gyrB.The most frequent substitution were 1400 A→G(9/11,81.8%)and 1483 G→T(2/11,18.2%)in a specific region of the rrs2 gene related with kanamycin-resistant strains.No mutations except S95T of gyrA detected in the drug-susceptible isolates.The MIC values of clinical drug-resistant strains that the same drug-resistant group contains a different resistance phenotype are basically the same with the relevant resistance genes in the same mutation.Associated resistance mutations in different sites varied significantly with their MIC values.Conclusion The mutation characterization of drug-resistant and drug-suscep-tible isolates of MTB have been shown to vary according to geographic region,phenotypic characteristics exist difference in resistance levels due to different muntants of drug-resistant gene.
