Screening for metronidazole-resistance associated gene fragments of H pylori by suppression subtractive hybridization.

Aim: To screen for metronidazole (MTZ)-resistance associated gene fragments of H pylori by suppression subtractive hybridization (SSH).

Methods: Five MTZ-resistant (tester, T) and 1 MTZ-susceptible (driver, D) clinical H pylori isolates were selected. Genomic DNAs were prepared and submitted to Rsa I digestion. Then two different adaptors were ligated respectively to the 5'-end of two aliquots of the tester DNA fragments and SSH was made between the tester and driver DNAs. The specific inserts of tester strains were screened and MTZ-resistance related gene fragments were identified by dot blotting.

Results: Among the randomly selected 120 subtractive colonies, 37 DNA fragments had a different number of DNA copies (> or = 2 times) in resistant and susceptible strains and 17 of them had a significantly different number of DNA copies (> or = 3 times). Among the sequences obtained from the 17 DNA fragments, new sequences were found in 10 DNA fragments and duplicated sequences in 7 DNA fragments, representing respectively the sequences of depeptide ABC transporter periplasmic dipeptide-binding protein (dppA), permease protein (dppB), ribosomal protein S4 (rps4), ribonuclease III (rnc), protease (pqqE), diaminopimelate epimerase (dapF), acetatekinase (ackA), H pylori plasmid pHP51 and H pylori gene 1334.

Conclusion: Gene fragments specific to MTZ-resistant H pylori strains can be screened by SSH and may be associated with MTZ-resistant H pylori.