| BackgroundsHelicobacter pylori (H.pylori) is the main cause of chronic gastritis, peptic ulcer, gastric cancer and mucosa associated lymphoid tissue (MALT) lymphoma. Now due to widely use of antibiotic in clinic, the phenomena of resistance become more and more prominent, which is the important factor leading to treatment failure. Metronidazole (MTZ) is the important factor for successful treatment to H.pylori infection and is the main ingredient of new triple therapy. But MTZ combined therapy is also restricted to the resistance. It is very significant to elucidate the mechanism of MTZ resistance in H.pylori.It's difficult and complex task to clinically isolate and culture the H.pylori, which prevent susceptibility test being carried out clinically. It's necessary to detect the resistance mechanism, only based on which can we carry out the susceptibility test using biochemistry method and instruct the clinical description. In addition, detecting the resistance mechanism may be helpful in reverting the MTZ resistance.Mechanism for MTZ resistance in H.pylori was very complicated, So far, several possible mechanism were proposed: (1) reduced activity of the nitroreductases; (2) bacterial metabolism and the change of enzyme activities; (3) reduced uptake or increased efflux; (4)increased activity of the DNA repair enzymes; and (5) increased activity of the scavenger system. Goodwin et al. reported that the mutation of rdxAgene, which encode an oxygen-insensitive NADPH nitroreductase, played main role in the evolution of MTZ resistance in H.pylori. In addition, the mutation of frxA and fdxB gene that encode oxygen-sensitive NADPH nitroreductase and the mutation of recA that encode DNA repair enzymes were also associated with MTZ resistance. In recent years, some researcher have cloned some genes that associated with MTZ resistance, such as rdxA, frxA, fdxB, recA, et al, however these were not enough to elucidate the mechanism of MTZ resistance in Helicobacter pylori and other genes related MTZ resistance maybe exist, which need further investigating.Suppression subtractive hybridization (SSH), one of new methods of phenotypic cloning, has been developed for identifying differences between the genomes of close relatives. It is a powerful approach for screening gene associated with drug resistance. We obtained some DNA fragments specific to the resistant strains against susceptible strain by SSH, and identified them by dot blotting. The indentified DNA fragments were sequenced and compared with GenBank database for searching for some gene relative to drug resistance.Methods1. Culture of H.pylori: All samples used in this study were clinical isolates from patients through endoscope biopsy. Each the sample was smeared on Hpylori selective Columbia agar plate containing 8% sheep blood. H.pylori cultures were incubated at 37 under microaorobic conditions(5%O2, 10%CO2, 85%N2). NCTC11637 was reference strain.2. Determination of minimal inhibition concentration (MIC): The MIC was determined by paper test and agar dilution method of NCCLS. Agar dilution plates were prepared with two fold serial dilution of MTZ, ranging from 0.25 to 128 g/ml. The young exponentially growing H.pylori strains were prepared in sterile saline and adjusted to No.2 McFarland standard. About 1 to 5 l of adjusted inocula was delivered to agar dilution plate. Results were read after 72h incubation and the MIC determined as the lowest concentration of MTZ in which no visible growth occurred.Strains that of MIC valued 8 g/ml were classified as resistant.3. Preparation of H.pylori Genomic DNA: Phenol-Chloroform and DNase-free RNase digestion were used for preparation and purification of H.pylori genomic DNA. Then the genomic DNA was prepared and submitted to Rsal digestion.4. Suppression subtractive hybridization: The subtractive DNA library was made according to Clontech PCR-Select鈩?Bacterial Genome Subtraction Kit (PT3170-1) User Mannual. The adaptors and primers were provided by kit. The genomic DNA... |
