| Aims:Helicobacter pylori is a curved, microaerophilic, Gram staining negative bacterium. Eradication of this pathogen can alleviate profoundly inflammatory responses of gastric mucosa, particularly the activity of inflammation, heal ulcers and prevent the relapse of peptic ulcer. Eradication may effectively alleviate gastric MALT lymphoma. It has been demonstrated that eradication of the bacterium in the early age is capable of decreasing the gastric cancer risk. There is a great variation in the resistant rate of Hp to antibiotics. This study was aimed to analyze the single and multiple resistance of Hp strains isolated from Qingdao to metronidazole, clarithromycin and/or levofloxacin, to explore the association of antibiotic resistance with chronic gastric diseases or bacterial virulence. The mutation features and mechanisms of resistance related genes including rdx A, 23 S r RNA and gyr A were to be investigated as well in this study.Methods:Strains were isolated from antral biopsies of gastric mucosa. Hp was identified based on typical colony appearances, biochemical tests, Gram staining and sequence analyses of 16 S r RNA gene. Agar dilution test was used to determine thesusceptibility of Hp to metronidazole, clarithromycin and levofloxacin. Genotypes of cag A and vac A determined with a PCR method. The relationship of drug resistance to bacterial virulence was then investigated. Involvement of Hp rdx A in metronidazole resistance, 23 S r RNA in clarithromycin resistance and gyr A in levofloxacin was investigated through sequencing their PCR products. Sequence analyses were carried out using DNAMAN software.Results:1. A total of 101 Hp strains were isolated. Identification of Hp has been validated through typical colony appearances, biochemical tests, Garm staining and sequence analyses of the 16 S r RNA gene.2. Results of antibiotic susceptibility tests showed that only 17.9% Hp isolates were susceptible to all three antibiotics tested. The resistant rate of Hp to metronidazole, clarithromycin and levofloxacin were 43.6%, 40.6% and 34.6%, respectively. Of 101 strains tested, 30.1% were resistant to two or more antibiotics. In the case of double resistance, strains resistant to both clarithromycin and levofloxacin were most frequent, with a rate of 10.9%. In contrast, resistant to both levofloxacin and metronidazole was least common, with a rate of 3.0%. There was a statistical difference(χ2 = 4.91,P = 0.027). These results suggested that Hp isolated from Qingdao was most susceptible to levofloxacin plus metronidazole. It was also found that 8.9% Hp isolates were multiple resistant to three antibiotics. These results demonstrated that resistance of Hp to antibiotics was frequent in Qingdao, which could affect the clinical eradication of Hp.3. Analyses of the association of drug resistance with diseases showed that the resistant rate of strains from gastric cancer were significant lower(33.3%) compared to that from chronic gastritis(55.3%)(χ2 = 4.94,P = 0.029). This indicated that gastric cancer was probably involved in the resistance of Hp to metronidazole. It also showed that there was no significant difference in resistance rate to clarithromycin or levofloxacin between gastric cancer and chronic gastritis4. Analyses of virulent genotypes of Hp found there were no statistically significant differecens in the distribution of susceptible strains in different cag A and vac A types. This suggested that virulence of Hp was not related to the antibiotic resistance.5. Sequence analyses showed that the most common mutation of rdx A of Hp from Qingdao was a insertion of A at the 20 th position. This led to the occurrence of a stop codon at 23 th position of amino acid sequence, resulting in inactivation of rdx A. This happened in 43.2% strains isolated. It also found that the majority of insertion/deletion occurred at the nucleotide repeats, which reached to 59.1%. Of them, tandem repeat A was the most frequent locution. For 23 S r RNA and gyr A genes, the most frequent mutation was A2143G(77.3%) and N87K(80.0%), respectively.Conclusions1. In Qingdao, it appeared that the resistance rate of Hp was higher to clarithromycin, to multiple drugs. These indicated that guidance with antibiotic susceptibility test is necessary for the choice of eradication regimes. Levofloxacin could be the first line drug for the treatment of Hp infection in this area.2. This study first demonstrated that resistance rate of Hp to metronidazole was lower in gastric cancer. This possibly contributed to the increased nitrites concentration in gastric cancer.3. Analyses of mutation features of rdx A demonstrated thast insertion/deletion occurred most frequently at nucleotide repeats. These findings would benefit for the clinical managements, providing new ways for the exploration of mechanisms of drug resistance.
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