Research Of Relationship Between Levofloxacin Resistance And Mutations Of GyrA In Helicobacter Pylori

Objective:To isolate and assess levofloxacin resistance of Helicobacter pylori;Examine the difference of gyrA between the resistance-strains and susceptive-strains by sequencing, and probe the role of the mutation of gyrA gene in the development of resistance;and construct the gene chip for detecting the mutations of gyrA.Methods:Helicobacter pylori were cultured on H.pylori selective medium,and were cultured under microaerobic conditions at 37℃for 3-5 days.The traditional methods and UreA PCR were used to identify the Helicobacter pylori.The E-test was used to determine the resistance and susceptibility of H.pylori strains to levofloxacin.Three segments of gyrA gene were amplified by PCR from resistance and susceptive strains.These PCR-products were sequenced,and the nucleotide sequences were analyzed.Design and synthetic oligonucleotide and primers labeled with fluorescence,fabricate the chip and detect the mutations of gyrA.Results:38 strains of H.pylori were isolated from gastric mucosa;Among 38 isolates,12 strains 31.58%(12/38) were resistant to levofloxacin,and other stains were sensitive by E-test.The 38 strains of whole gyrA gene were sequenced,10 of which existed three mutant points(261,271,272).In the above results,there were 2 strains having C261A mutation,1 strains having C261G mutation,2 strains having G271A mutation,2 strains having A272G mutation,2 strains having C261A,G271T,A272G mutations,1 strain having G271A,A272G mutations.However,26 strains sensitive to levofloxacin had no mutant points.The results obtained by gene chip were identical to that of direct sequencing method.Conclusions:The rate of drug-resistance for levofloxacin obtained from this region was high. The HP strain of drug-resistance was associated with mutant points of 261,271 and 272. It was a rapid,accurate assay for constructing gene chip for detecting mutations of gyrA gene.This assay provided integrated data for individual therapy in clinic and then guided doctors to utilize suitable drugs to heal various patients.The study described here provided reliable data for investigating the relationship between mechanism of drug-resistance and mutant points of gyrA gene.