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World J Gastroenterol. Mar 28, 2007; 13(12): 1847-1850
Published online Mar 28, 2007. doi: 10.3748/wjg.v13.i12.1847
Screening for metronidazole-resistance associated gene fragments of H pylori by suppression subtractive hybridization
Ai-Qing Li, Ning Dai, Jie Yan, Yong-Liang Zhu
Ai-Qing Li, Ning Dai, Department of Gastroenterology, Sir Run Run Shaw Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310016, Zhejiang Province, China
Jie Yan, Department of Medical Microbiology and Parasitology, College of Medicine, Zhejiang University, Hangzhou 310031, Zhejiang Province, China
Yong-Liang Zhu, Department of Gastroenterology, Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310009, Zhejiang Province, China
Author contributions: All authors contributed equally to the work.
Supported by the Natural Science Foundation of Zhejiang Province, No. 29801
Correspondence to: Dr. Ai-Qing Li, Sir Run Run Shaw Affiliated Hospital, College of Medcine, Zhejiang University, Hangzhou 310016, Zhejiang Province, China. li_aq@hotmail.com
Telephone: +86-571-86090073 Fax: +86-571-86006788
Received: September 25, 2006
Revised: December 30, 2006
Accepted: January 25, 2007
Published online: March 28, 2007
Abstract

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 RsaIdigestion. 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 (≥ 2 times) in resistant and susceptible strains and 17 of them had a significantly different number of DNA copies (≥ 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 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.

Keywords: H pylori; Metronidazole; Drug resistance; Gene; Suppression subtractive hybridization