a Department of Medicine, Baylor College of Medicine, Houston, TX; b Department of Medical Laboratory Science, Northeastern University, Boston, MA; c Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA; d Division of Laboratory Medicine, Department of Pathology, Massachusetts General Hospital, Boston, MA, USA
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Abstract |
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Introduction |
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Materials and methods |
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H. pylori isolates were cultivated from gastric biopsies isolated by oesophagogastroduodenal endoscopy at the Veterans' Affairs Medical Center in Houston, TX, USA. MIC values for H. pylori isolates were determined by two-fold agar dilution. Plates were prepared using Mueller Hinton (MH) agar as the basal medium supplemented with 5% aged sheep blood (2 weeks old). Serial concentrations ranging from 0.015 to 256 mg/L were achieved by addition of Mtz (Sigma Chemical Co., St Louis, MO, USA) prepared in sterile distilled water and added to 5% sheep bloodMH basal medium. Fresh H. pylori cultures (23 days old) were suspended in saline at an optical density (OD625) between 0.38 and 0.4. Using a Steers-type replicating device (Cathra, St Paul, TN, USA), 15 µL of adjusted inocula were delivered to the agar plates. Plates were incubated in microaerobic jars with CampyPak Plus sachets (Becton Dickinson BBL, Cockeysville, MD, USA) at 37°C for 3 days, without anaerobic pre-incubation. Mtz-resistant H. pylori ATCC 43504 (NCTC 11637) was used as control. H. pylori ATCC 43504 was evaluated by agar dilution antimicrobial susceptibility testing and found to possess an MIC > 256 mg/L. A breakpoint MIC (8 mg/L) was defined in accordance with published studies.4
Amplification and sequencing of rdxA alleles
Genomic DNA was isolated from H. pylori isolates by phenol/choroform extraction and ethanol precipitation. Genomic DNA specimens were suspended in TE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0).
Primers complementary to regions flanking the 630 bp coding sequence of rdxA were used to amplify an 882 bp product. PCRs (50 µL final volume) included 0.5 U Taq DNA polymerase (Roche Molecular Biochemicals, Indianapolis, IN, USA), 1 x reaction buffer supplemented with 2.2 mM MgCl2 (Roche Molecular Biochemicals), 5 ng bovine serum albumin (Roche Molecular Biochemicals), 50 nmol dNTP (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and 100 pmol each primer RdxA-19 (5'-GCCAGATAGCCAAATGGGGG, forward) and RdxA-900R (5'-GAAACGCTTGAAAACACCCC, reverse).
PCR was performed in a DNA Thermal Cycler 480 (Applied Biosystems, Foster City, CA, USA) with the following conditions: an initial denaturation (94°C, 6 min), followed by 35 cycles of denaturation (94°C, 30 s), annealing (48°C, 1 min) and extension (72°C, 3 min), and a final extension (72°C, 10 min). Amplification products were separated in 1% agarose by electrophoresis, visualized with 0.5 µg/mL ethidium bromide and purified using the GFX PCR DNA and Gel Band Purification Kit (Amersham Pharmacia Biotech).
RdxA amplicons were sequenced in the ABI Prism 310 Genetic Analyzer (Applied Biosystems) using ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit chemistry (Applied Biosystems) according to the manufacturer's recommendations. An internal sequencing primer, RdxA-255 (5'-AATCGCTGAAATCGCCAGGC) was used to determine and verify sequence data. Sequences obtained were aligned and translated by Megalign sequence analysis software (Lasergene, DNAStar, Madison, WI, USA). Newly obtained rdxA sequences were compared with published rdxA data of Mtz-susceptible H. pylori strain 500.2
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Results |
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DNA sequence analyses of rdxA alleles from Mtz- susceptible H. pylori isolates revealed intact reading frames (i.e. lacking nonsense mutations). Pairwise alignment of susceptible isolates against a reference Mtz-susceptible H. pylori strain 500 (Hp 500), using the MartinezNeedlemanWunsch method, yielded rdxA allelic identities ranging from 95.5 to 97%. Nucleotide substitutions varied in number (1929), with two to eight resultant amino acid substitutions. Peptide similarity indices ranged from 95.8 to 99.1 using the LipmanPearson method.
In contrast, most Mtz-resistant H. pylori isolates contained nonsense mutations (seven of nine) in rdxA, resulting in expression of putatively truncated nitroreductase, RdxA (Table). Insertion or deletion (indel) events were identified in six of seven rdxA alleles with nonsense mutations. Four of six Mtz-resistant H. pylori isolates contained single nucleotide insertions in rdxA, whereas two isolates contained polynucleotide deletion mutations. These indel mutations resulted in translational frameshifts, creating premature stop codons at downstream positions. Premature termination codons in the six frameshift mutants were distributed between codon positions 32 and 176 in the 209 amino acid RdxA polypeptide (Figure
). One of the nine Mtz-resistant isolates contained a transition mutation, C
T, at nucleotide position 148. This substitution mutation created a premature termination codon at that site, probably resulting in a truncated RdxA polypeptide containing 49 amino acids.
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Discussion |
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Mtz is inactive in its parent form and requires reductive activation by RdxA or other nitroreductases in H. pylori to exert antimicrobial activity. In contrast to macrolide resistance in H. pylori and associated drug target alteration of 23S ribosomal RNA,7 frameshift and point mutations in rdxA result in truncated nitroreductase and diminish the ability of H. pylori to activate Mtz. In addition to frameshift mutations caused by insertion or deletion events, substitution mutations in important structural or catalytic domains of rdxA may lead to functional inactivation of nitroreductase. Nucleotide analyses showed that in Mtz-susceptible isolates, transition substitutions were most frequently found in rdxA alleles, as expected (140 of 166 nucleotide substitutions, data not shown). Sequence divergence in susceptible isolates ranged from 3 to 4.5% and is consistent with previously reported (c. 5%) in allelic differences rdxA.2 Presumably, these nucleotide polymorphisms do not result in functionally important amino acid substitutions. Functionally important point mutations in rdxA are proposed in this study. Point mutations found only in resistant isolates are described in the Table. The A80T mutation (GA transition event), resulting in an alanine to threonine residue substitution (A80T) is intriguing due to its presence in both resistant strains (B1/18 and 52/16) lacking nonsense mutations. This amino acid is conserved in an Escherichia coli nitroreductase, nfsB,8 and lies adjacent to the mini-IS605 insertion in H. pylori NCTC 11637 (ATCC 43504).9 Mutations in nfsB may decrease reductive activation of nitrofuran compounds (e.g. furazolidone, nitrofurazone) leading to nitrofuran resistance in E. coli.9 As with Mtz, nitrofurans require reduction of nitro moieties to cause DNA breakage.10 We propose that this amino acid lies in a functionally important domain and structural alteration of this region inactivates RdxA. The identical mutation (A80T) has been reported in a resistant North African isolate, and substitution mutations at nearby codon positions 79 and 87 have recently been reported in resistant isolates from France.6
Metronidazole and related nitroimidazole compunds (e.g. tinidazole) represent important agents in H. pylori eradication regimens. The prevalence of resistant isolates in patients varies depending on the geographical region in question, but has reached alarming proportions in both developed and developing nations. This study is the first report describing the mutational mechanisms of Mtz resistance in H. pylori from the USA and corroborates the findings of previous studies.2,5 The panoply of mutations found in rdxA is consistent with the high level of genetic diversity of H. pylori. Mutations in rdxA and other nitroreductases may explain molecular mechanisms of nitroimidazole drug action and resistance and may facilitate the development of novel antimicrobial agents for future use in patients.
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Acknowledgments |
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Notes |
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References |
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Received 20 March 2000; returned 13 June 2000; revised 12 July 2000; accepted 4 August 2000