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


CONCLUSIONS
Gene fragments specific to MTZ-resistant H pylori strains can be screened by SSH and may be associated with MTZ-resistant H pylori.
Li
Ai-Qing
AQ
Department of Gastroenterology, Sir Run Run Shaw Affiliated Hospital, College of Medcine, Zhejiang University, Hangzhou 310016, Zhejiang Province, China. li_aq@hotmail.com
Dai
Ning
N
Yan
Jie
J
Zhu
Yong-Liang
YL
D000890:Anti-Infective Agents / Q000494:pharmacology*; D053938:DNA Fragmentation; D004269:DNA, Bacterial / Q000235:genetics*; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D018628:Gene Dosage; D005820:Genetic Testing; D016481:Helicobacter Infections / Q000188:drug therapy; D016480:Helicobacter pylori / Q000235:genetics*; D006801:Humans; D015151:Immunoblotting; D008795:Metronidazole / Q000494:pharmacology*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D017422:Sequence Analysis, DNA
D000890:Anti-Infective Agents; D004269:DNA, Bacterial; D008795:Metronidazole