Izumi S, Ouchi S, Kuge T, Arai H, Mito T, Fujii H, Aranishi F, Shimizu A
Stock Assessment Division, National Institute of Fisheries Science, Yokohama, Japan.
J Fish Dis. 2007 Mar;30(3):141-7. doi: 10.1111/j.1365-2761.2007.00797.x.
A novel genotyping method for epizootiological studies of bacterial cold-water disease caused by Flavobacterium psychrophilum and associated with quinolone resistance was developed. Polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP) was performed on 244 F. psychrophilum isolates from various fish species. PCR was performed with primer pair GYRA-FP1F and GYRA-FP1R amplifying the A subunit of the DNA gyrase (GyrA) gene, which contained the quinolone resistance determining region. Digestion of PCR products with the restriction enzyme Mph1103I showed two genotypes, QR and QS. The difference between these genotypes was amino acid substitutions at position 83 of GyrA (Escherichia coli numbering). The genotype QR indicated an alanine residue at this position associated with quinolone resistance in F. psychrophilum isolates. Of the 244 isolates tested in this study, the number of QR genotype isolates was 153 (62.7%). In isolates from ayu (n=177), 146 (82.5%) were genotype QR. With combination of this technique and previously reported PCR-RFLP genotyping, eight genotypes were observed in F. psychrophilum isolates. Using this genotyping system, the relationships between genotype and host fish species, or locality of isolation, were analysed and are discussed.
开发了一种用于嗜冷黄杆菌引起的细菌性冷水病的流行病学研究且与喹诺酮耐药性相关的新型基因分型方法。对来自各种鱼类的244株嗜冷黄杆菌分离株进行了聚合酶链反应，随后进行限制性片段长度多态性分析（PCR-RFLP）。使用引物对GYRA-FP1F和GYRA-FP1R进行PCR，扩增包含喹诺酮耐药决定区的DNA促旋酶（GyrA）基因的A亚基。用限制性内切酶Mph1103I消化PCR产物显示出两种基因型，即QR和QS。这些基因型之间的差异是GyrA第83位氨基酸的替换（以大肠杆菌编号）。基因型QR表明该位置的丙氨酸残基与嗜冷黄杆菌分离株中的喹诺酮耐药性相关。在本研究中测试的244株分离株中，QR基因型分离株的数量为153株（62.7%）。在香鱼分离株（n = 177）中，146株（82.5%）为基因型QR。结合该技术和先前报道的PCR-RFLP基因分型，在嗜冷黄杆菌分离株中观察到8种基因型。使用该基因分型系统，分析并讨论了基因型与宿主鱼类物种或分离地点之间的关系。
