Detection Of The Mutation In Gyra Gene Of The Pathogenic Salmonella Isolated From Swines By PCR-RFLP And The Gyra Genes Sequencing

The serotypes of 16 pathogenic salmonella stains isolated from different swine farms of Sichuan proveince were detected,and there were 6 strains of Salmonella typhimurium,5 strains of Salmonella choleraesuis ,3 strains of Salmonella enteritidis and 2 strains of Salmonella derby.16 strains of pathogenic salmonella were investigated by drug suspectivity.The results showed that the resistant rate of the 12 types of the prodominent antibiotics from 31.3% to 93.8%.The MICs of Norfloxacin and Ciprofloxacin of the sixteen strains of pathogenic Salmonella were detected,and there were nine strains of the salmonella resistant to the Norfloxacin ,seven to the ciprofloxacin.According to the published sequences of gyrA gene ,a pair of primers were disigned for ampilified the gyrA gene of Salmonella. After plasmid and cherosome DNA extraction and purification,the gyrA of 16 isolates of salmonella gene was amplified by Polymerase chain reaction (PCR) . PCR fragments of 668bp,containing the quinolone resistance-determining region of the gyrA gene got from the whole strains in the cherosome,and the one in the plasmid which were the first found in the Salmonella typhimurium that reported in P.R.China.To determine the incidence of quinolone resistance associated mutations in the gyrA gene,16 strains were examined for mutation in the gyrA gene at Ser 83 by PCR-RFLP. The Hinf I restriction enzyme digestion gave rise to restriction fragment length polymorphism (RFLP) of the PCR products. When there were two DNA fragments of 363bp and 305bp were produced from a PCR product,the strains were assumed to have a mutation at Ser83,when three fragments of 363bp,206bp and 99bp were produced ,the strains were assumed to have no mutation at the 83or 116.The results indicated that certain mutation of Ser 83 abolished a Hinf I restriction enzyme site and may be detected as a RFLP. The RFLP styles showed thatthe moderate and the resistant strains which the value of MIC of CIP2mg/Lwere examined for mutations in the QRDR in the cherosome,the PCR productes of the suspectie strains which the value of MIC of CIP 0.5mg/L were digested in three fragments revealed no mutation in the gyrA.The PCR products of gyrA of a resistant strain (SLl-3) was sequenced and analysed.when compared to the corresponding sequence of the gyrA of salmonella typhimurium from the nucleotide sequences data of the gyrA reported by Griggs,5 mutants sites were found in the QRDR. DMA sequencing identified in the strain SLl-3 Ser to Phe change at codon 83 of gyrA gene. The sequence of quinolone resistant determing regions (QRDR) of gyrase subuint A of salmonella analysis indicated that the gyrA gene of strain SLl-3 share 95.8% respectively identities with the sequence that Griggs reported which showed a high homology between the strains.These findings indicated that the alternation in gyrA was responsible for the resistance of the FQNs of the Salmonella. And PCR-RFLP analysis was a sensitive ,rapid and simple assay for the detection of nucleotide sequence change in Ser 83.And our data sugget that PCR-RFLP may be a useful device for detecting the mutation in gyrA gene associated the resistance to FQNs.