Giannoni Federico, Iona Elisabetta, Sementilli Federica, Brunori Lara, Pardini Manuela, Migliori Giovanni Battista, Orefici Graziella, Fattorini Lanfranco
Dipartimento di Malattie Infettive, Parassitarie e Immunomediate, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.
Antimicrob Agents Chemother. 2005 Jul;49(7):2928-33. doi: 10.1128/AAC.49.7.2928-2933.2005.
Resistance of Mycobacterium tuberculosis to fluoroquinolones (FQ) results mostly from mutations in the gyrA gene. We developed a reverse hybridization-based line probe assay in which oligonucleotide probes carrying the wild-type gyrA sequence, a serine-to-threonine (S95T) polymorphism, and gyrA mutations (A90V, A90V-S95T, S91P, S91P-S95T, D94A, D94N, D94G-S95T, D94H-S95T) were immobilized on nitrocellulose strips and hybridized with digoxigenin-labeled PCR products obtained from M. tuberculosis strains. When a mutated PCR product was used, hybridization occurred to the corresponding mutated probe but not to the wild-type probe. A panel of M. tuberculosis complex strains including 19 ofloxacin-resistant (OFL-R) and 9 ofloxacin-susceptible (OFL-S) M. tuberculosis strains was studied for detection and identification of gyrA mutations by the line probe assay and nucleotide sequencing, in comparison with testing of in vitro susceptibility to FQ. Results were 100% concordant with those of nucleotide sequencing. The S95T polymorphism, which is not related to FQ resistance, was found in 5 OFL-S and 2 OFL-R strains; the other 17 OFL-R strains harbored single mutations associated with serine or threonine at codon 95. No mutations were found in the other OFL-S strains. Overall, on the basis of the MICs on solid medium, the new line probe assay correctly identified all OFL-S and 17 out of 19 (89.5%) OFL-R strains. A nested-PCR protocol was also evaluated for the assay to amplify PCR products from M. tuberculosis-spiked sputa, with a good specificity and a sensitivity of 2 x 10(3) M. tuberculosis CFU per ml of sputum.
结核分枝杆菌对氟喹诺酮类药物（FQ）的耐药性主要源于gyrA基因突变。我们开发了一种基于反向杂交的线性探针检测法，其中携带野生型gyrA序列、丝氨酸到苏氨酸（S95T）多态性以及gyrA突变（A90V、A90V - S95T、S91P、S91P - S95T、D94A、D94N、D94G - S95T、D94H - S95T）的寡核苷酸探针被固定在硝酸纤维素膜条上，并与从结核分枝杆菌菌株获得的地高辛标记的PCR产物杂交。当使用突变的PCR产物时，杂交发生在相应的突变探针上，而不是野生型探针上。通过线性探针检测法和核苷酸测序，对一组包括19株耐氧氟沙星（OFL - R）和9株对氧氟沙星敏感（OFL - S）的结核分枝杆菌复合群菌株进行了gyrA突变的检测和鉴定，并与体外对FQ的敏感性测试进行了比较。结果与核苷酸测序结果100%一致。在5株OFL - S和2株OFL - R菌株中发现了与FQ耐药性无关的S95T多态性；其他17株OFL - R菌株在密码子95处存在与丝氨酸或苏氨酸相关的单突变。在其他OFL - S菌株中未发现突变。总体而言，基于固体培养基上的最低抑菌浓度，新的线性探针检测法正确鉴定了所有OFL - S菌株和19株OFL - R菌株中的17株（89.5%）。还对该检测法的巢式PCR方案进行了评估，以从接种了结核分枝杆菌的痰液中扩增PCR产物，其具有良好的特异性，每毫升痰液对2×10³个结核分枝杆菌CFU的灵敏度。
