Helicobacter Reference Unit, Laboratory of Enteric Pathogens, Central Public Health Laboratory, 61 Colindale Avenue, Colindale, London NW9 5HT, UK
Received 25 October 2002; returned 6 December 2002; revised 3 February 2003; accepted 3 February 2003
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Abstract |
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Keywords: Helicobacter pylori, metronidazole resistance, rdxA, mutation
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Introduction |
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In this study, the sequences of rdxA were examined in a unique collection of 46 clinical H. pylori isolates from 19 patients in the UK, to identify mutations that contribute to metronidazole resistance in this previously unexplored English population.
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Materials and methods |
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H. pylori from gastric biopsies of 19 patients in Ipswich (AG) and London (HS), UK, were examined. For patients AK, stored paired isolates were recovered before and after patients had received eradication regimens that included metronidazole. The remaining eight sets, recovered pre-treatment only, were either from single antral biopsies (patients LO) or from both the gastric antrum and corpus (patients PS).
Isolates were cultured from gastric biopsies, or recovered from storage (80°C) by microaerobic (86% N2, 4% O2, 5% CO2, 5% H2) incubation (37°C for 72 h) on blood agar (CBA) [Columbia agar base (Oxoid) containing 10% (v/v) defibrinated horse blood].
MIC determination
MICs of metronidazole were determined by Etest (AB Biodisk, Solna, Sweden) (concentration range 0.016256 mg/L) on CBA inoculated with an H. pylori lawn (107 cfu/mL). MICs were recorded after 48 h microaerobic incubation at 36°C. Isolates with MICs
8 mg/L were defined as metronidazole resistant.2
Separation of metronidazole-susceptible and metronidazole-resistant subpopulations
Metronidazole-resistant subpopulations were purified by subculture on CBA plates containing 8 mg/L metronidazole (MTZCBA; Sigma Ltd, UK) under microaerobic conditions. A velvet blotting technique was used to identify metronidazole-susceptible colonies that grew on CBA, but not on MTZCBA. The purity of metronidazole-susceptible strains subcultured on CBA was confirmed by their inability to grow on MTZCBA. MICs were determined for all purified populations, as described above.
DNA extraction
For all populations examined, a sweep of bacteria was subcultured for genomic DNA extraction by the cetyl-trimethylammonium-bromide (CTAB) method.8 Extracts were stored (20°C) until required.
Amplified fragment length polymorphism (AFLP)
AFLP analysis was performed as described previously.9 Ligated genomic fragments were amplified using primer HI-A (5'-GGTATGCGACAGAGCTTA-3').9 AFLP profiles with more than two band differences were considered distinct types.
rdxA amplification and sequencing
Fragments (686 bp) containing rdxA were amplified using primers rdxF863 (5'-TTAGGGATTTTATTGTATGCTA-3') and rdxR1544 (5'-TCACAACCAAGTAATTGCATCAA-3') (MWG Biotech Ltd, Ebersberg, Germany). Amplicons were sequenced in both directions either commercially (23 isolates) (MWG Biotech Ltd and Cytomyx, Cambridge, UK) or in-house (23 isolates), as described previously.8 Sequence chromatograms were examined and corrected in Chromas version 1.42 (Griffith University, Brisbane, Australia). Corrected sequences were aligned and translated in GeneBase version 1 (Applied Maths, Kortjivik, Belgium).
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Results |
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Pre-treatment, H. pylori isolates from patients AK were either predominantly metronidazole susceptible with metronidazole-resistant colonies (1050) growing in the Etest inhibition zone (n = 7), or fully metronidazole susceptible (n = 4) (Table 1). All patients but one (D) were infected with a uniformly metronidazole-resistant isolate only post-treatment. Mixed metronidazole-susceptible/metronidazole-resistant resistotype infections were found pre-treatment in the gastric antrum of patients LO, whereas isolates with different metronidazole resistotypes were identified in the antrum and in the body of the stomachs of patients PS (Table 1).
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All patients were infected with unique H. pylori AFLP genotypes (Table 1). Isolate pairs from the 16 patients with mixed (metronidazole-susceptible/metronidazole-resistant) infections (pre-treatment, except for patient D) were either identical (n = 14) or similar (n = 2), differing by only a single band. AFLP genotypes of matched pre- and post-treatment isolates (patients AK) were mainly identical (n = 7) or similar (n = 2), although different genotypes were identified post-treatment in two patients (Table 1).
Comparison of translated metronidazole-susceptible and metronidazole-resistant RdxA sequences
RdxA amino acid sequences of matched pre- and post-therapy metronidazole-susceptible and metronidazole-resistant strains of similar genotypes were different in 7/9 patients (AF, IK) (Table 2). Nucleotide point mutations (corresponding to a substitution of Arg-16His in three of five cases) occurred in metronidazole-resistant strains from patients A, B, E, F and J. Frameshifts leading to protein truncation occurred in metronidazole-resistant isolates from patients C and K. No mutations were found in the remaining two matched metronidazole-susceptible/metronidazole-resistant pairs (patients D and I) (Table 2).
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Discussion |
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In our patient sets, AFLP typing demonstrated that mixed metronidazole-susceptible and metronidazole-resistant isolates from individual patients were phenotypic variants of the same strain. Isolates recovered post-therapy were, in most cases, similar or identical to pre-therapy strains. Thus, as demonstrated previously,10 treatment failure was linked to persistence of the original metronidazole-resistant subpopulation during therapy. Re-infection with a novel strain was infrequent (only observed in two patients).
Mutations were demonstrated in 77.8% of translated rdxA sequences from metronidazole-resistant post-treatment isolates, compared with matched metronidazole-susceptible pre-treatment strains. The absence of a single universal mutation associated with metronidazole resistance in English isolates supports findings reported from other countries.1,3 Mutations causing Arg-16His substitutions and protein truncations (positions 50 and 74) reported previously could be critical to the metronidazole-resistant phenotype. Premature protein truncation would significantly reduce RdxA enzyme activity. However, these stop codon mutations were not observed in matched pre-treatment metronidazole-resistant variants and so were not essential for resistance. Furthermore, no differences were observed in rdxA sequences of five additional paired metronidazole-susceptible/metronidazole-resistant strains from patients PS or N before therapy. Although metronidazole-resistant strains with unaltered rdxA exist,1,3 this is the first report that has demonstrated mutations in rdxA of metronidazole-resistant strains post-therapy that are absent in matched metronidazole-resistant and metronidazole-susceptible strains pre-therapy; thus metronidazole-resistant strains with unaltered rdxA occur more frequently than has been hitherto indicated.
Evidence suggests that metronidazole is a highly mutagenic drug.4 The mutations reported in metronidazole-resistant H. pylori recovered post-treatment may be induced by metronidazole administered during therapy and may therefore occur coincidentally, rather than contribute to the metronidazole-resistant phenotype. Whereas mutationally inactivated rdxA genes in metronidazole-resistant subpopulations have been reported, the majority of metronidazole-resistant strains analysed were induced from progenitor metronidazole-susceptible strains by serial passage on metronidazole-containing media,4 so increasing concentrations of mutagenic metronidazole may have induced rdxA mutations. In contrast, our metronidazole-resistant subpopulations were naturally occurring and were observed on primary susceptibility testing.
One study of naturally occurring mixed, presumably pre-treatment, metronidazole-susceptible and metronidazole-resistant populations of French and North African isolates reported mutational differences in rdxA between resistotypes.3 In contrast, rdxA sequences were identical in most (73.3%) of our mixed metronidazole susceptibility populations, again suggesting that mutational inactivation of this gene is not necessary for a metronidazole-resistant phenotype. It is difficult to account for the differences between our findings and those reported earlier,3 particularly as relatively small numbers were investigated in both studies. Differences may reflect geographical variations, or even local differences in metronidazole usage and rdxA mutation rates.
Previous studies have provided compelling evidence to suggest an important role for rdxA in metronidazole metabolism and in development of resistance.1,4,5 Expression of RdxA protein is lower in metronidazole-resistant strains,7 so a potential role for rdxA in metronidazole resistance cannot be excluded. However, we propose that resistance resulting from altered RdxA expression does not necessarily result from functional inactivation of the gene by mutation. Control of RdxA expression may occur by an alternative regulatory mechanism, possibly at the transcriptional or translational level. Future investigation of this possibility in our patient strain sets could improve understanding of the role of rdxA in metronidazole resistance.
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Acknowledgements |
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Footnotes |
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References |
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