鸡源性沙门氏菌DNA旋转酶与耐氟喹诺酮类药物关系研究

取临床分离的、对5种氟喹诺酮类药物（环丙沙星、氧氟沙星、恩诺沙星、单诺沙星和沙拉沙星）均耐药的9株鸡源性沙门氏菌耐药株,提取其染色体DNA。设计引物gyrAF和gyrAR、gyrBF和gyrBR,分别扩增菌株DNA旋转酶gyrA基因和gyrB基因的氟喹诺酮类耐药决定区（QRDR）,对PCR扩增产物进行测序及序列分析。与质控菌株相比,9株临床分离耐药株中只有菌株38和60的gyrA基因发生单碱基突变,菌株38的gyrA基因第371位碱基发生C→T突变,菌株60的gyrA基因第350位碱基发生A→C突变,两处突变均位于QRDR内,其余菌株的核苷酸未发生任何突变。菌株38的碱基突变导致gyrA基因第99位氨基酸发生R→C取代,即Arg→Cys;菌株60的碱基突变导致gyrA基因第92位氨基酸发生M→L取代,即Met→Leu。9株临床分离鸡源性沙门氏菌氟喹诺酮类耐药株gyrB基因QRDR的核苷酸序列与质控菌株完全相同;只有菌株42的gyrB基因第1592位碱基发生C→A突变,但其位于gyrB基因QRDR之外,且菌株42的gyrB基因的碱基突变并没有导致相应氨基酸的改变。上述结果提示,DNA旋转酶gyrA基因和gyrB基因QRDR突变可能并非沙门氏菌耐药性产生的主要原因。

Fluoroquinolone resistance and DNA gyrase mutations in Salmonella Isolated from Chickens

The QRDR regions of gyrA and gyrB genes were amplified by PCR from 9 clinically isolated Salmonella strains showing resistant to 5 fluoroquinolones(Ciprofloxacin,Ofloxacin,Enrofloxacin,Danofloxacin and Sarafloxacin).Sequence comparison with NCTC5776 reference strain showed a site mutation from C to T at 371 bp in gyrA of strain 38 and a mutation from A to C at 350 bp in gyrA of strain 60.However no mutation was found in gyrA of other isolates.The mutation resulted in an amino acid substitution of Arg99Cys in strain 38 and an amino acid substitution of Met92Leu in strain 60.A single mutation from C to A was found at 1 592 bp in gyrB of strain 42,which was located outside of gyrB QRDR and led to no amino acid change. These results indicated that mutations in QRDR of gyrA and gyrB of DNA gyrase may play an insignificant role in fluoroquinolone resistance in Salmonella.