a Department of Medical Microbiology, Royal Free and University College Medical School, Rowland Hill Street, London, UK; b Unité de Pathogénie Bactérienne des Muqueuses, Institut Pasteur, 28 Rue du Dr Roux, Paris, France; c Institute of Infections and Immunity, University of Nottingham, Nottingham NG7 2UH, UK
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Abstract |
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Introduction |
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Materials and methods |
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Metronidazole-susceptible and -resistant derivatives of SS1 (Table) were cultured from the stomachs of mice that had been experimentally infected with the metronidazole-susceptible SS1 strain and then treated orally with metronidazole.7 H. pylori strains were routinely cultured on 10% horse blood medium supplemented with appropriate antibiotics as previously described.6 Susceptibility to metronidazole was assessed by agar dilution determination of the MIC.7 The MIC was defined as the lowest concentration of antibiotic inhibiting growth when the plates were read after 72 h incubation under microaerobic conditions at 37°C. Isolates were considered resistant to metronidazole if the MIC was
8 mg/L.
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Genomic DNA from individual H. pylori strains was extracted using the QIAamp Tissue Kit (Qiagen, Crawley, UK) according to the manufacturer's instructions. DNA sequencing of the rdxA gene was carried out using two pairs of oligonucleotide primers, rdxA-1 (CGTTAGGGATTTTATTGTATGCTAC) and rdxA-2 (CCCCACAGCGATATAGCATTGCTC), and rdxA-3 (GTTAGAGTGATCCCCTCTTTTGCTC) and rdxA-4 (CACCCCTAAAAGAGCGATTAAAACC). These were used to amplify two overlapping PCR products that contained the entire rdxA gene, and the nucleotide sequences of these products were determined on both strands using the four oligonucleotide primers described above.
Protein analysis by SDSPAGE and immunoblotting
Immunoblotting with polyclonal rabbit antisera against RdxA was carried out as previously described.6 Briefly, cell extracts prepared from 2 day cultures were analysed on slab gels, comprising a 4.5% acrylamide stacking gel and a 17.5% resolving gel. Proteins were transferred to nitrocellulose membranes and reacted with RdxA antisera that had been diluted 1:100 in 50% Escherichia coli TG1 extract, 5% milk powder and 0.2% Tween in phosphate-buffered saline and incubated for 4 h at room temperature to remove non-specific antibodies to E. coli. Immunoreactants were detected with anti-rabbit peroxidase-linked immunoglobulin (Amersham, Little Chalfont, UK) diluted 1:10 000 and reaction products were visualized on autoradiographic film by chemiluminescence using the ECL Western blotting detection system (Amersham).
Natural transformation
Metronidazole-susceptible H. pylori strain SS1 was transformed using a specific PCR fragment generated from resistant strains using the oligonucleotide primers rdxA-1 and rdxA-4. Bacteria were inoculated as 1 cm patches and grown for 5 h before addition of 30 ng of the PCR fragment. After further incubation for 18 h, the bacteria from each individual patch were harvested and plated directly on to a single plate containing metronidazole 0.5 µg/mL. After 4 days incubation, single colonies were obtained, and the MIC of metronidazole for three independent transformants from each transformation was determined.
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Results |
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Immunoblotting with RdxA antisera was carried out on whole bacterial cell lysates of 15 metronidazole-susceptible and 50 metronidazole-resistant isolates. The metronidazole-resistant strains included 10 in which the rdxA gene had previously been sequenced: in nine the rdxA gene contained one or more mutations (SS1-17 to SS1-25), and in one the gene sequence was identical to that of the parental SS1 strain (SS1-16).8 In all of the 15 metronidazole-susceptible strains a 24 kDa immunoreactive band corresponding to the RdxA protein was observed. The 24 kDa protein was also produced by five of the 50 metronidazole-resistant H. pylori isolates: SS1-17, SS1-18, SS1-19, SS1-52 and SS1-56 (Figure). In three of these isolates, which were isolated from the stomachs of different mice, the mutations in the rdxA gene had been identified in a previous study: SS1-17 contained Y47H and P51L amino acid substitutions; SS1-18 and SS1-19 contained P51L amino acid substitutions (Table).8 The nucleotide sequence of the rdxA gene of the other two isolates, SS1-52 (isolated from the same mouse as SS1-19) and SS1-56 (isolated from the same mouse as SS1-17), was determined and found to contain the missense point mutation (C to T at position 153) that resulted in the P51L amino acid substitution. In the remaining 45 metronidazole-resistant strains, including strain SS1-16, which had an intact rdxA gene, no equivalent immunoreactive band corresponding to the RdxA protein was observed.
Further analysis of metronidazole-resistant H. pylori isolates that produced the RdxA protein
In order to determine whether the mutations within the rdxA gene of the metronidazole-resistant H. pylori isolates that produced the RdxA protein were associated with inactivation of rdxA, the rdxA gene of SS1-17, SS1-18, SS1-19, SS1-52 and SS1-56 was amplified and transformed into the metronidazole-susceptible, parental SS1 strain (MIC 0.0625 mg/L). PCR fragments amplified from SS1 and SS1 rdxA mutant (MIC 1.0 mg/L) were used as controls. The MIC of metronidazole for colonies isolated after transformation with the rdxA allele of the SS1 isogenic deletion mutant in rdxA (SS1 rdxA-) and strains SS1-17, SS1-18, SS1-19, SS1-52 and SS1-56 was 0.52.0 mg/L. No colonies were isolated on plates containing metronidazole 0.5 mg/L after transformation with the rdxA allele of strain SS1.
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Discussion |
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The rdxA gene of many metronidazole-resistant H. pylori strains contain frameshift mutations that result in the creation of a translational stop codon in the region immediately downstream of the mutation, and such strains would be predicted to produce a truncated RdxA protein.2,8,10 However, this study confirmed that production of the RdxA protein is completely abrogated in the majority of resistant strains, including five (SS1-21 to SS1-25) that contained such frameshift mutations. The finding that the RdxA protein was not produced by strain SS1-16 was particularly interesting, since this strain is one of a handful of metronidazole-resistant strains that have been reported to contain an intact rdxA gene.8 This implies that this isolate contained a mutation within the promoter region of the rdxA gene and is the first good evidence that mutations within this region are associated with the resistant phenotype.
The exact contribution of other genes to the acquisition of metronidazole resistance by H. pylori is currently unclear. However, regardless of whether other resistance mechanisms are present or not, our results demonstrate that the vast majority of resistant strains contain mutations within the rdxA gene or its promoter that prevent production of the RdxA protein or result in production of an abnormal polypeptide that is subsequently degraded. This is an important finding, since it indicates that testing for the absence of the RdxA protein would identify the majority of isolates that will respond poorly to metronidazole-containing eradication regimens and has implications for the development of assays capable of detecting metronidazole resistance in H. pylori. One major advantage of this approach is that it identifies all resistant strains carrying mutations that affect expression of the rdxA gene, including those not yet identified by nucleotide sequence analysis.
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Acknowledgements |
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Notes |
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References |
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Received 13 August 2001; returned 6 December 2001; revised 2 January 2002; accepted 11 January 2002