An Analysis Of Drug Resistance Of Helicobacter Pylori Isolates In Fujian And The Mechanism Of Metronidazole Resistance

Objective: 1. To analyse drug resistance of H. pylori isolates from FuJian in China. 2.To investigate the role of the rdxA gene in H. pylori resistant to metronidazole (Mtz).3.To identify the relationship between the state of cagA gene at Pathogenity Island and metronidazole resistance in H. pylori.Methods: 1. All the strains were isolated from patients living in different areas in Fujian of China who underwent a gastroduodenoscopy in Fujian provincial hospital because of epigastric complaints. Mtz susceptibility was determined by both E-test method and Breakpoint susceptibility test (MIC>8mg/L). Clarithromycin > amoxycillin and tetracycline were determined only by E-test method. 2. The rdxA gene fragment (886bp) in H. pylori was amplified by PCR and then was sequenced. At last, polymorphism analysis was performed in the rdxA gene sequence from both strains (resistant or susceptible to Mtz) and standard strains H.pylori26695.3.Five strains with high levels Mtz resistance (MIC > 256 mg/L) were chosen at random and then werecultured with 16mg/L Mtz or without Mtz on Skirrow agar plates. The total RNA was extracted from the Mtz resistant H. pylori isolates. The rdxA gene expression for the proteins was examined using a method optimized for semi-quantitative reverse transcription polymerase chain reaction (RT-PCR).The galE gene was used as the control.4.The cagA gene fragment (349bp) in H. pylori isolates resistant or susceptible to Mtz was amplified by PCR.Results: 1. A total of 47 H. pylori strains were isolated from 51 patients positive for rapid urease test . Mtz resistance rate were 34% by E-test method and 31.9% by Breakpoint susceptibility testing(P>0.05). Clanthromycin resistance rate was 10.6%. NO resistance to amoxicillin and tetracycline was found. 2. The rdxA genes of 15 strains resistant to Mtz and 8 isolates susceptible to Mtz were sequenced. Compared with standard strain H.pylori26695, the rdxA gene nucleotide similarity of 15 H. pylori isolates resistant to Mtz ranged from 90% to 95% and was 92.5% on average, The similarity of susceptible strains ranged from 94% to 95% and was 94.5% on average (rdxA genes from unrelated strains differed in DNA sequence by 5% on average). The rdxA gene mutation was due to point mutations in several bases > insertional mutations or deletion mutations of one base, not large fragment. There was no stable mutation position.3.The level of transcripts from rdxA genes was significantly reduced in the total RNA prepared from H. pylori grown with Mtzcompared to that prepared without Mtz. The expression of the galE gene used as the control was no change.4.The positive rate of the cagA gene in H. pylori susceptible to Mtz was 80.6%(25/31), compared with 80%(12/15) in H. pylori resistant to Mtz (P>0.05).Conclusions: 1.Resistance rates of H. pylori isolates from Fujian were 34% (metronidazoie), 10.6%(clarithromycin). No resistance was found to tetracycline or amoxycillin.2.The rdxA gene mutation in H. pylori isolates resistant to Mtz was due to point mutations in several bases> insertional mutations or deletion mutations of one base, not large fragment and no stable mutation position. S.The low expression of the rdxA gene may play a role in H. pylori isolates with high levels of Mtz resistance.4.H.pylori resistant to metronidazoie is not related to the state of the cagA gene.