Identification Of Helicobacter Pylori Clarithromycin Resistance And Study On Bioinformatics Of The Whole Gene Of Helicobacter Pylori 23S RRNA

Objective:To isolate and assess clarithromycin resistance of Helicobacter pylori;Examine the difference between the resistancestrains and susceptive-strains by bioinformatics,and probe the role of the mutation in 23S rRNA gene to the development of resistance.Methods:Helicobacter pylori were cultured on fresh 7%-10%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 be identified.The E-test was used to determine the resistance and susceptibility of H.pylori strains to clarithromycin.Three segments of 23S rRNA gene were amplified by PCR from resistance and susceptive strains.These PCR-products were sequenced,and the nucleotide sequences were analyzed by Vector NIT Suite 9.0,and the 23S rRNA gene Secondary structure of wild and mutational strain were predicted.Results:67(30.45%)strains of H.pylori were isolated from gastric mucosa;The positive of H.pylori rate was 20.15%(27/134),48.72% (38/78)and 25.00%(2/8)in gastritis,gastric ulcer and gastric cancer respectively.Among 67 isolates,23 strains 34.33%(23/67), were resistant to clarithromycin,and other stains were sensitive;No intermedial sensitive strain was found in these isolates.The resistance rate of clarithromycin was 29.63%(8/27),36.84%(14/38)and 50.00%(1/2) in gastritis,gastric ulcer and gastric cancer respectively. 5(31.25%)strains of H.pylori were found to have an A-to-G mutation in 23S rRNA domain V at position 2143 among 16 strains of clarithromycin resistance strains,and the C2182T mutation was detected in 2(12.50%) strains.A new mutation C588T was found in 6(37.50%)strains in which no other known hot spot mutation occurred.Conclusion:The positive rate and clarithromycin resistance rate of H.pylori in gastric ulcer are higher than in gastritis.Clarithromycin resistance H.pylon isolated from wenling of Zhejiang Province have A2144G and C2182T mutation in 23S rRNA domain V.The new found mutation C588T may be another spot mutation related to clarithromycin resistance.