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Journal of Bacteriology, September 2000, p. 5082-5090, Vol. 182, No. 18
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Sequential Inactivation of rdxA (HP0954) and frxA (HP0642) Nitroreductase Genes Causes Moderate and High-Level Metronidazole Resistance in Helicobacter pylori

Jin-Yong Jeong,1 Asish K. Mukhopadhyay,1 Daiva Dailidiene,1 Yipeng Wang,1 Billie Velapatiño,1,2 Robert H. Gilman,2 Alan J. Parkinson,3 G. Balakrish Nair,4 Benjamin C. Y. Wong,5 Shiu Kum Lam,5 Rajesh Mistry,1,6 Isidore Segal,6 Yuan Yuan,1,7 Hua Gao,1,7 Teresa Alarcon,8 MaNuel Lopez Brea,8 Yoshiyuki Ito,1 Dangeruta Kersulyte,1 Hae-Kyung Lee,1 Yan Gong,1 Avery Goodwin,9 Paul S. Hoffman,9 and Douglas E. Berg1,*

Department of Molecular Microbiology and Department of Genetics, Washington University Medical School, St. Louis, Missouri 631101; Department of Pathology, Universidad Peruana Cayetano Heredia, Lima, Peru2; Arctic Investigations Program, Centers for Disease Control and Prevention, National Center for Infectious Diseases, Anchorage, Alaska 995083; National Institute of Cholera and Enteric Diseases, Calcutta 700010, India4; Department of Medicine, Queen Mary Hospital, University of Hong Kong, Hong Kong5; Division of Gastroenterology, Chris Hani Baragawanath Hospital, Johannesburg 2013, South Africa6; Cancer Institute, China Medical University, Shenyang, China7; Department of Microbiology, Hospital Universitario de la Princesa, Madrid, Spain8; and Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada9

Received 22 March 2000/Accepted 28 June 2000

Helicobacter pylori is a human-pathogenic bacterial species that is subdivided geographically, with different genotypes predominating in different parts of the world. Here we test and extend an earlier conclusion that metronidazole (Mtz) resistance is due to mutation in rdxA (HP0954), which encodes a nitroreductase that converts Mtz from prodrug to bactericidal agent. We found that (i) rdxA genes PCR amplified from 50 representative Mtzr strains from previously unstudied populations in Asia, South Africa, Europe, and the Americas could, in each case, transform Mtzs H. pylori to Mtzr; (ii) Mtzr mutant derivatives of a cultured Mtzs strain resulted from mutation in rdxA; and (iii) transformation of Mtzs strains with rdxA-null alleles usually resulted in moderate level Mtz resistance (16 µg/ml). However, resistance to higher Mtz levels was common among clinical isolates, a result that implicates at least one additional gene. Expression in Escherichia coli of frxA (HP0642; flavin oxidoreductase), an rdxA paralog, made this normally resistant species Mtzs, and frxA inactivation enhanced Mtz resistance in rdxA-deficient cells but had little effect on the Mtz susceptibility of rdxA+ cells. Strains carrying frxA-null and rdxA-null alleles could mutate to even higher resistance, a result implicating one or more additional genes in residual Mtz susceptibility and hyperresistance. We conclude that most Mtz resistance in H. pylori depends on rdxA inactivation, that mutations in frxA can enhance resistance, and that genes that confer Mtz resistance without rdxA inactivation are rare or nonexistent in H. pylori populations.

* Corresponding author. Mailing address: Department of Molecular Microbiology, Campus Box 8230, Washington University Medical School, 4566 Scott Ave., St. Louis, MO 63110. Phone: (314) 362-2772. Fax: (314) 362-1232 or (314) 362-3203. E-mail berg{at}borcim.wustl.edu.

Journal of Bacteriology, September 2000, p. 5082-5090, Vol. 182, No. 18
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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