Turkmani Aun, Psaroulaki Anna, Christidou Athanasia, Chochlakis Dimosthenis, Tabaa Darem, Tselentis Yannis
Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, WHO Collaborating Center, Department of Social Medicines, Faculty of Medicine, University of Crete, P.O. Box 1393, TK 71409 Heraklion, Crete, Greece.
Int J Antimicrob Agents. 2008 Sep;32(3):227-32. doi: 10.1016/j.ijantimicag.2008.03.012. Epub 2008 Jun 26.
In the present study, Brucella melitensis biovar Abortus 2308 and Brucella abortus 3196 biotype 5 reference strains, which are susceptible to fluoroquinolones, became in vitro-resistant to fluoroquinolones by culture in trypticase soy agar. The quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes of the two reference strains were analysed by polymerase chain reaction sequencing analysis to obtain the wild-type sequence. These sequences were then compared with the corresponding sequences of four in vitro-selected fluoroquinolone-resistant mutants to characterise mutations associated with resistance. Sequencing of the ofloxacin-selected resistant mutant 2308 revealed a transition of GAT to AAT (corresponding to position 87 of Escherichia coli gyrA), leading to substitution of Asp91-->Asn, whilst at the same position the ciprofloxacin-selected resistant mutant 2308 revealed a transition of GAT to TAT (corresponding to the same position of E. coli as above), leading to substitution of Asp91-->Tyr. The ofloxacin-selected resistant mutant 3196 had a transition of GCT to GTT, generating an amino acid change of Ala87-->Val. Amino acid changes were detected in the portion of the Brucella gyrA gene (Ala71 to Gln110) corresponding to the E. coli gyrA QRDR region (Ala67 to Gln110). Amino acid changes were also detected in Ser83, corresponding to the region where fluoroquinolone-associated amino acid changes are most commonly found in other bacterial species.
在本研究中，对氟喹诺酮敏感的羊种布鲁氏菌生物变种流产型2308株和牛种布鲁氏菌3196生物型5参考菌株，通过在胰蛋白胨大豆琼脂中培养而在体外对氟喹诺酮产生耐药性。通过聚合酶链反应测序分析对这两种参考菌株的gyrA和parC基因的喹诺酮耐药决定区（QRDRs）进行分析以获得野生型序列。然后将这些序列与四个体外筛选的氟喹诺酮耐药突变体的相应序列进行比较，以鉴定与耐药性相关的突变。对氧氟沙星筛选的耐药突变体2308进行测序，结果显示GAT突变为AAT（对应于大肠杆菌gyrA的第87位），导致Asp91替换为Asn，而在同一位置，环丙沙星筛选的耐药突变体2308显示GAT突变为TAT（对应于上述大肠杆菌的相同位置），导致Asp91替换为Tyr。氧氟沙星筛选的耐药突变体3196有GCT突变为GTT，产生Ala87替换为Val的氨基酸变化。在布鲁氏菌gyrA基因对应于大肠杆菌gyrA QRDR区域（Ala67至Gln110）的部分（Ala71至Gln110）检测到氨基酸变化。在Ser83也检测到氨基酸变化，该区域对应于在其他细菌物种中最常发现的与氟喹诺酮相关的氨基酸变化区域。
