Insertion of mini-IS605 and deletion of adjacent sequences in the nitroreductase (rdxA) gene cause metronidazole resistance in Helicobacter pylori NCTC11637

Abstract

We found that NCTC11637, the type strain of Helicobacter pylori, the causative agent of peptic ulcer disease and an early risk factor for gastric cancer, is metronidazole resistant. DNA transformation, PCR-based restriction analysis, and DNA sequencing collectively showed that the metronidazole resistance of this strain was due to mutation in rdxA (gene HP0954 in the full genome sequence of H. pylori 26695) and that resistance did not depend on mutation in any of the other genes that had previously been suggested: catalase (katA), ferredoxin (fdx), flavodoxin (fldA), pyruvate:flavodoxin oxidoreductase (porgammadeltaalphabeta), RecA (recA), or superoxide dismutase (sodB). This is in accord with another recent study that attributed metronidazole resistance to point mutations in rdxA. However, the mechanism of rdxA inactivation that we found in NCTC11637 is itself also novel: insertion of mini-IS605, one of the endogenous transposable elements of H. pylori, and deletion of adjacent DNA sequences including 462 bp of the 851-bp-long rdxA gene.

FIG. 1

Ethidium bromide-stained agarose gel displaying the restriction endonuclease digestion pattern of the sodB amplicon. (A) Digestion with Sau3AI; (B) digestion with MseI. Lane 1, H. pylori NCTC11638 (Mtz^s); lane 2, H. pylori NCTC11637 (Mtz^r); lanes 3 to 14, H. pylori NCTC11638 transformants (Mtz^r). The positions of the molecular size markers (1-kb ladder; Stratagene) are indicated on the left.

FIG. 2

Ethidium bromide-stained agarose gel of the rdxA amplicon. Lane 1, H. pylori NCTC11638 (Mtz^s); lane 2, H. pylori NCTC11637 (Mtz^r); lanes 3 to 14, H. pylori NCTC11638 transformants (Mtz^r). The positions of the molecular size markers (1-kb ladder; Stratagene) are indicated on the left.

FIG. 3

Alignment of the DNA sequences and the predicted encoded proteins of the rdxA amplicons. Mtz^s, H. pylori NCTC11638; Mtz^r, H. pylori NCTC11637 and the metronidazole-resistant transformants of NCTC11638. The sequence of the donor NCTC11637 amplicon is identical to that of all the metronidazole-resistant NCTC11638 transformants. The primers used to amplify the rdxA region (and amino acids derived therefrom) are indicated in boldface; the inverted repeats of the IS605 (7) are in boldface and underlined. The amino acids directly above the DNA sequence correspond to H. pylori NCTC11638; the ones directly below the DNA sequence correspond to NCTC11637 and the transformants. Gaps in the DNA sequence are marked with dashes, and identical nucleotides are indicated by dots.

FIG. 4

Alignment of the mini-IS605 sequence present in the rdxA gene from strain NCTC11637 (see Fig. 3); the cag pathogenicity island (cag PAI) of strain NCTC11638 (7), GenBank accession no. U60176 bp 22476 to 22735; one of the eight partial copies of IS605 present in strain 26695 (27), GenBank accession no. HPAE000537 bp 2867 to 2606; and one of the five partial copies present in strain J99 (4), GenBank accession no. HPAE000537 bp 2633 to 2891. The inverted repeats of IS605 (7) are indicated in boldface and underlined. For definitions of the dots and dashes, please see the legend to Fig. 3.