rpoB基因在结核分枝杆菌利福平／利福布丁交叉耐药株中突变情况的差异分析

The differences of rpoB mutations in rifampicin/rifabutin cross-resistant clinical isolates of Mycobacterium tuberculosis

目的 了解结核分枝杆菌利福平/利福布丁交叉耐药株与利福平耐药/利福布丁敏感株rpoB全基因突变特征的差异.方法 测定rpoB全基因序列,比较278株利福平/利福布丁交叉耐药（rifampicin/rifabutin cross-resistant,RIF/Rfb-R）株和40株利福平耐药/利福布丁敏感（rifampicinresistant/rifabutin-susceptible,RIF-R/Rfb-S）株,30株利福平/利福布丁敏感（rifampicin-susceptible/rifabutin-susceptible,RIF-S/Rfb-S）株及标准株H37 Rv之间的rpoB全基因序列突变差异.结果 利福平/利福布丁敏感株和H37Rv rpoB全基因未发现突变；利福平/利福布丁交叉耐药株的常见突变位点是531（70.5％）和526（ 20.9％）.223（ 80.2％）株单点突变株分别含有S531L、S531W、H526D、H526Y、H526R、Q513K、Q513P、Q510H、V176F、P287R、Y395C及H404Y单点突变,55（ 19.8％）株多重突变株分别以S531L、H526R、H526Y、H526D、D516G和Q513K位点与其他位点联合突变为主.利福平耐药/利福布丁敏感株的常见突变位点分别是516（65.0％）、526（ 17.5％）和533（ 10.0％）.21（52.5％）株单点突变株分别含有L533P、H526L、H526S、S522L、D516V、D516Y和D516F单点突变,19（47.5％）株多重突变株分别以D516V和L533P位点与其他位点联合突变为主.结论 本批标本中,利福霉素耐药株rpoB基因突变率为100％,利福平/利福布丁交叉耐药株与利福平耐药/利福布丁敏感株rpoB全基因单点突变株的突变位置或氨基酸替换类型、多重突变株的突变位置或组合类型以及常见突变位点或其氨基酸替换类型完全不同,rpoB全基因DNA序列分析对临床科学应用利福平和利福布丁有指导意义.

Objective To learn the rpoB mutation difference in rifampicin/rifabutin cross-resistant （RIF/Rfb-R） clinical isolates and in rifampicin-resistant/rifabutin-susceptible （RIF-R/Rbo-S） clinical isolates of Mycobacterium tuberculosis.Methods To sequence the full-length genome of rpoB gene,and analyze the rpoB full-length gene mutation differences in 278 RIF/Rfb-R clinical isolates,40 RIF-R/Rfb-S clinical isolates,30 rifampicin-susceptible/rifabutin-susceptible （RIF-S/Rfb-S） and in 1 reference strain ofH37Rv.Results No mutations of rpoB full-length gene were found in H37Rv reference strain and RIF-S/Rfb-S clinical isolates.In RIF/Rfb-R clinical isolates,531 （70.5% ） and 526 （20.9% ） were the most frequent mutation codons.223 （80.2% ） isolates possesed single mutations as S531L,S531W,H526D,H526Y,H526R,Q513K,Q513P,Q510H,V176F,P287R,Y395C and H404Y.55 （19.8%） isolates had multiple mutations,and among these the S531L,H526 R,H526Y,H526D,D516G and Q513K were the main substitutions which were in combination with other points.In RIF-R/Rfb-S clinical isolates,516 （65.0%）,526 （ 17.5% ） and 533 （ 10.0% ） were the most frequent mutation codons.21 （52.5% ） isolates possesed single mutations as L533P,H526L,H526S,S522L,D516V,D516Y and D516F.19 （47.5%）isolates had multiple mutations and among these the D516V and L533P were the main substitutions which were in combination with other points.Condusions In our study,100% rifamycin-resistant clinical isolates of Mycobacterium tuberculosis had rpoB mutations,but the mutations in RIF/Rfb-R clinical isolates were sharply different from RIF-R/Rfb-S clinical isolates in mutation positions or amino acids substitutions of single mutations strains,mutation positions or combination types and the most frequently mutation codons or amino acids substitutions of multiple mutations strains.Thus,DNA sequencing is instructive and meaningful to choose rifampicin or rifabutin for tuberculosis treatment.