Mutations in Helicobacter pylori rdxA gene sequences may not contribute to metronidazole resistance

Stephanie A. Chisholm* and Robert J. Owen

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Metronidazole resistance in Helicobacter pylori reportedly occurs by mutational inactivation of the oxygen-insensitive nitroreductase gene rdxA. Nucleotide sequences of rdxA were determined in a set of 46 isolates from 19 dyspeptic patients from the UK. The study set comprised matched isolates that were either metronidazole susceptible (four) or mixed metronidazole susceptible and metronidazole resistant (15) before therapy and metronidazole resistant post-therapy (10) in the 11 patients that were followed up. Various mutation types were identified in rdxA of metronidazole-resistant strains (post-treatment) that were absent in matched metronidazole-susceptible strains (pre-treatment). However, rdxA sequences from pre-treatment metronidazole-resistant and metronidazole-susceptible subpopulations were identical in 11 of 15 patients. Thus, mutations in rdxA may not always be essential for metronidazole resistance. Future examination of rdxA expression at the transcription and translational level may provide further insight into the role of this locus in metronidazole action and resistance in H. pylori.

Keywords: Helicobacter pylori, metronidazole resistance, rdxA, mutation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A potential role of oxygen-insensitive NADPH nitroreductase encoded by the rdxA gene in resistance of Helicobacter pylori to metronidazole was proposed following presentation of evidence that rdxA inactivation was associated with the metronidazole-resistant phenotype.1 Subsequent studies have confirmed that inactivation of rdxA can occur by a range of mutation types,24 and mutated rdxA genes can transform metronidazole-susceptible strains to resistance. Conversely, lethal effects of metronidazole can be restored in resistant strains by introduction of functional rdxA.1,4 Introduction of metronidazole resistance by rdxA knockout mutagenesis,5 and the virtual absence of rdxA mRNA levels6 or of RdxA protein expression7 in metronidazole-resistant strains, provides further evidence of the importance of this gene in the resistance mechanism. However, some metronidazole-resistant strains possess an apparently wild-type rdxA,1 suggesting the involvement of other genes, including those encoding flavin oxidoreductase (frxA), ferridoxin-like proteins (fdxA, fdxB) and pyruvate oxidoreductase (porA, porB).2,6

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial cultures

H. pylori from gastric biopsies of 19 patients in Ipswich (A–G) and London (H–S), UK, were examined. For patients A–K, 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 L–O) or from both the gastric antrum and corpus (patients P–S).

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 0.016–256 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).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Resistotype characterization

Pre-treatment, H. pylori isolates from patients A–K were either predominantly metronidazole susceptible with metronidazole-resistant colonies (10–50) 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 L–O, whereas isolates with different metronidazole resistotypes were identified in the antrum and in the body of the stomachs of patients P–S (Table 1).


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Table 1.  Metronidazole resistotypes and AFLP genotypes of 46 isolates of H. pylori from 19 patients
 
Genotype characterization

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 A–K) 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 (A–F, I–K) (Table 2). Nucleotide point mutations (corresponding to a substitution of Arg-16->His 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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Table 2.  Sequence variations in rdxA identified by comparison of matched metronidazole-susceptible and -resistant strains of H. pylori recovered from patients before and after therapy or simultaneously as a mixed infection
 
RdxA was identical in 11/15 metronidazole-susceptible and metronidazole-resistant subpopulation sets. Mutations in the metronidazole-resistant subpopulation only (patients G, H, L and M) included stop codons (n = 3) and carboxy terminus sequence alteration (n = 1) (Table 2). RdxA was truncated in both metronidazole-susceptible and metronidazole-resistant subpopulations of patient O (Table 2). Comparison of all isolates with sequences held in GenBank identified novel mutations (Table 2).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We compared rdxA sequences in a unique H. pylori strain set of metronidazole-resistant as well as metronidazole-susceptible pre-treatment populations with matched post-treatment metronidazole-resistant strains, to establish whether treatment failures were the result of selection of a pre-existing mutated metronidazole-resistant strain, or de novo mutation of both subpopulations during eradication therapy.

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-16->His 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 P–S 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.


    Acknowledgements
 
We thank Drs S. M. Dobbs and R. J. Dobbs (King’s College Hospital, London, UK) for their kind provision of biopsy material for culture, and Miss Nicola Elviss for her useful advice on Etest susceptibility testing.


    Footnotes
 
* Corresponding author. Tel: +44-20-8200-4400; Fax: +44-20-8905-9929; E-mail: schisholm{at}phls.nhs.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Goodwin, A., Kersulyte, D., Sisson, G., Veldhuyzen van Zanten, S. J., Berg, D. E. & Hoffman, P. S. (1998). Metronidazole resistance in Helicobacter pylori is due to null mutations in a gene (rdxA) that encodes an oxygen-insensitive NADPH nitroreductase. Molecular Microbiology 28, 383–93.[CrossRef][ISI][Medline]

2 . Mendz, G. L. & Megraud, F. (2002). Is the molecular basis of metronidazole resistance in microaerophilic organisms understood? Trends in Microbiology 10, 370–5.[CrossRef][ISI][Medline]

3 . Tankovic, J., Lamarque, D., Delchier, J. C., Soussy, C. J., Labigne, A. & Jenks, P. J. (2000). Frequent association between alteration of the rdxA gene and metronidazole resistance in French and North African isolates of Helicobacter pylori. Antimicrobial Agents and Chemotherapy 44, 608–13.[Abstract/Free Full Text]

4 . Wang, G., Wilson, T. J., Jiang, Q. & Taylor, D. E. (2001). Spontaneous mutations that confer antibiotic resistance in Helicobacter pylori. Antimicrobial Agents and Chemotherapy 45, 727–33.[Abstract/Free Full Text]

5 . Kwon, D. H., El-Zaatari, F. A., Kato, M., Osato, M. S., Reddy, R., Yamaoka, Y. et al. (2000). Analysis of rdxA and involvement of additional genes encoding NAD(P)H flavin oxidoreductase (FrxA) and ferredoxin-like protein (FdxB) in metronidazole resistance of Helicobacter pylori. Antimicrobial Agents and Chemotherapy 44, 2133–42.[Abstract/Free Full Text]

6 . Kwon, D. H., Osato, M. S., Graham, D. Y. & El-Zaatari, F. A. (2000). Quantitative RT-PCR analysis of multiple genes encoding putative metronidazole nitroreductases from Helicobacter pylori. International Journal of Antimicrobial Agents 15, 31–6.[CrossRef][ISI][Medline]

7 . Latham, S. R., Owen, R. J., Elviss, N. C., Labigne, A. & Jenks P. J. (2001). Differentiation of metronidazole-sensitive and -resistant clinical isolates of Helicobacter pylori by immunoblotting with antisera to the RdxA protein. Journal of Clinical Microbiology 39, 3052–5.[Abstract/Free Full Text]

8 . Owen, R. J. & Xerry, J. (2003). Tracing clonality of Helicobacter pylori infecting family members from analysis of DNA sequences of three housekeeping genes (ureI, atpA and ahpC), deduced amino acid sequences, and pathogenicity-associated markers (cagA and vacA). Journal of Medical Microbiology 52, in press.

9 . Gibson, J. R., Slater, E., Xerry, J., Tompkins, D. S. & Owen, R. J. (1998). Use of an amplified-fragment length polymorphism technique to fingerprint and differentiate isolates of Helicobacter pylori. Journal of Clinical Microbiology 36, 2580–5.[Abstract/Free Full Text]

10 . Adamsson, I., Edlund, C., Seensalu, R. & Engstrand, L. (2000). The use of AP-PCR and flaA-RFLP typing to investigate treatment failure in Helicobacter pylori infection. Clinical Microbiological Infections 6, 265–7.[CrossRef][ISI][Medline]