J Med Microbiol 53 (2004), 1123-1128; DOI: 10.1099/jmm.0.45701-0
© 2004 Society for General Microbiology
ISSN 0022-2615
Role of the rdxA and frxA genes in oxygen-dependent metronidazole resistance of Helicobacter pylori
Monique M Gerrits1,
Egbert-Jan van der Wouden2,
Dorine A Bax1,
Anton A van Zwet3,
Arnoud HM van Vliet1,
Albertine de Jong3,
Johannes G Kusters1,
Jaap C Thijs2 and
Ernst J Kuipers1
1Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center, Rotterdam, The Netherlands 2Department of Internal Medicine, Bethesda Hospital, Hoogeveen, The Netherlands 3Regional Public Health Laboratory Groningen/Drenthe, Groningen, The Netherlands
Correspondence Arnoud H. M. van Vliet a.h.m.vanvliet{at}erasmusmc.nl
Received April 15, 2004
Accepted July 23, 2004
Almost 50 % of all Helicobacter pylori isolates are resistant to metronidazole, which reduces the efficacy of metronidazole-containing regimens, but does not make them completely ineffective. This discrepancy between in vitro metronidazole resistance and treatment outcome may partially be explained by changes in oxygen pressure in the gastric environment, as metronidazole-resistant (MtzR) H. pylori isolates become metronidazole-susceptible (MtzS) under low oxygen conditions in vitro. In H. pylori the rdxA and frxA genes encode reductases which are required for the activation of metronidazole, and inactivation of these genes results in metronidazole resistance. Here the role of inactivating mutations in these genes on the reversibility of metronidazole resistance under low oxygen conditions is established. Clinical H. pylori isolates containing mutations resulting in a truncated RdxA and/or FrxA protein were selected and incubated under anaerobic conditions, and the effect of these conditions on the MICs of metronidazole, amoxycillin, clarithromycin and tetracycline, and cell viability were determined. While anaerobiosis had no effect on amoxycillin, clarithromycin and tetracycline resistance, all isolates lost their metronidazole resistance when cultured under anaerobic conditions. This loss of metronidazole resistance also occurred in the presence of the protein synthesis inhibitor chloramphenicol. Thus, factor(s) that activate metronidazole under low oxygen tension are not specifically induced by low oxygen conditions, but are already present under microaerophilic conditions. As there were no significant differences in cell viability between the clinical isolates, it is likely that neither the rdxA nor the frxA gene participates in the reversibility of metronidazole resistance.
Abbreviations: MtzR, metronidazole-resistant; RAPD, random amplified polymorphic DNA.
The GenBank accession numbers for the rdxA and frxA gene sequences of seven metronidazole-resistant H. pylori strains are AY568322–AY568328 and AY568330–AY568336.
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INTRODUCTION
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Helicobacter pylori is a spiral-shaped, Gram-negative bacterium that colonizes the stomach of approximately half the world's population (Blaser & Berg, 2001). Colonization with H. pylori is the most common cause of chronic active gastritis and peptic ulcer disease, and is strongly associated with the development of gastric cancer and gastric lymphoma. Unless treated with antibiotics, H. pylori colonization tends to persist for life. Cure of H. pylori infection results in ulcer healing and may reduce the risk of gastric cancer and gastric lymphoma development (Sugiyama et al., 2002; Wilhelmsen & Berstad, 1994). In vitro, H. pylori is susceptible to the majority of antibiotics, but for effective treatment a combination of drugs is required (Debets-Ossenkopp et al., 1999b). Currently used anti-H. pylori therapies often consist of two antibiotics with a proton pump inhibitor and/or a bismuth component (Malfertheiner et al., 2002). Metronidazole [1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole] is a key component of such combination therapies (Malfertheiner et al., 2002; van der Hulst et al., 1996).
In Western Europe it has been estimated that 20–45 % of the H. pylori isolates are metronidazole-resistant (MtzR) (Glupczynski et al., 2001; Lopez-Brea et al., 2001). This percentage is even higher in developing countries and immigrant populations (Falsafi et al., 2004; Loffeld & Fijen, 2003). Although there are conflicting reports concerning the clinical relevance of metronidazole resistance in H. pylori, metronidazole resistance reduces the efficacy of metronidazole-containing regimens significantly (Jenks et al., 1999a; van der Wouden et al., 1999), but surprisingly does not render them inactive.
Metronidazole is a prodrug that needs to be activated by a reduction of the nitro group that is attached to the imidazole ring. This reduction step leads to the production of DNA-damaging nitroso- and hydroxylamine-compounds. Exposure to these toxic compounds causes DNA damage, and subsequently results in the death of the bacterium. In H. pylori, it is believed that reduction of metronidazole is mainly mediated by an oxygen-insensitive NADPH nitroreductase (RdxA) (Goodwin et al., 1998; Sisson et al., 2002), but recently it has been shown that the NADPH-flavin-oxidoreductase (FrxA) also participates in the reduction of metronidazole (Jeong et al., 2000).
In H. pylori, metronidazole resistance is primarily associated with mutational inactivation of the rdxA gene (Debets-Ossenkopp et al., 1999a; Goodwin et al., 1998; Jenks et al., 1999b). However, recently it has been demonstrated that inactivation of the frxA gene also confers metronidazole resistance, either alone or in association with the rdxA gene (Jeong et al., 2000; Kwon et al., 2000a, b, 2001). Whether mutational inactivation of these two enzymes accounts for metronidazole resistance in all clinical isolates is still being debated (Bereswill et al., 2003; Chisholm & Owen, 2004; Kwon et al., 2000a), but they most likely reflect the two major contributing factors.
The discrepancy between the in vitro resistance to metronidazole and treatment outcome may be explained by the antimicrobial activity of other components in the regimens and/or duration and doses of the therapy (van der Wouden et al., 1999). Apart from these factors, it is likely that low oxygen tension in the gastric environment may also be involved (Smith & Edwards, 1995), since low oxygen conditions affect the activity of metronidazole-reducing enzymes (Jenks & Edwards, 2002). As in vitro MtzR H. pylori isolates become susceptible to metronidazole after a short period of anaerobic incubation (Cederbrant et al., 1992; Smith & Edwards, 1995), it has been suggested that the FrxA protein and/or other ferredoxin and flavin reductases may contribute to the activation of metronidazole under these conditions (Goodwin et al., 1998; Kaihovaara et al., 1998).
In this study the role of null mutations in the rdxA and frxA genes on the reversibility of metronidazole resistance under low oxygen conditions was determined.
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METHODS
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Strains and growth conditions.
H. pylori isolates used in this study and their respective rdxA and frxA gene status inferred from DNA sequences are listed in Table 1. The H. pylori isolates were routinely grown on Dent plates as described previously (Gerrits et al., 2002b). Broth cultures were grown in Brucella broth supplemented with 3 % newborn calf serum (BBN). All cultures were incubated either under microaerophilic (5 % O2, 10 % CO2 and 85 % N2) or anaerobic conditions (10 % H2, 5 % CO2 and 85 % N2) at 37 °C. The anaerobic culture condition was created using the Anoxomat (Mart) in combination with a catalyst. Escherichia coli strain DH5
MCR (Life Technologies) was grown on Luria–Bertani agar plates (Sambrook et al., 1989) for 24 h at 37 °C in an aerobic environment. Selection of E. coli transformed with pGEM-T Easy clones was performed on LB-agar plates containing ampicillin to a final concentration 100 µg ml–1 (Sigma-Aldrich).
DNA manipulation.
DNA manipulations were performed according to standard protocols (Sambrook et al., 1989). Oligonucleotides (Table 2; Isogen), PCR-core system I (Promega) and pGEM-T Easy vector (Promega) were used according to the manufacturer's recommendations. Plasmid DNA was isolated with Wizard Plus SV Minipreps DNA Purification System (Promega) according to the manufacturer's instructions. Sequencing of the obtained plasmid and PCR products was performed by Baseclear (Leiden, The Netherlands).
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Table 2. Oligonucleotides used in this study Oligonucleotides used for amplification were based on the published genome sequence of H. pylori strain 26695 (Tomb et al., 1997).
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Random amplified polymorpic DNA (RAPD) fingerprinting.
RAPD analysis of chromosomal DNA was performed with three independent primers, 1254, D11344 and D9355 as described before by Akopyanz et al. (1992). RAPD products were separated in 2 % agarose gels containing 0.5 µg ethidium bromide ml–1 (Promega).
The influence of the length of anaerobic incubation on antibiotic resistance.
MIC values were routinely analysed by E-test (AB Biodisk) (Gerrits et al., 2002b) or agar dilution (Trieber & Taylor, 2002). The plates were incubated under anaerobic conditions for 0, 0.25, 0.5, 1, 2, 4 and 8 h and subsequently incubated for 3 days at 37 °C under microaerophilic conditions, according to the guidelines of the National Committee for Clinical Laboratory Standards. The isolates were considered resistant when the MICs of amoxycillin 
8 µg ml–1, clarithromycin 2 µg ml–1, metronidazole 8 µg ml–1and tetracycline 4 µg ml–1 (Gerrits et al., 2002b). As controls, resistant strains were included for each tested antibiotic (Debets-Ossenkopp et al., 1998; Gerrits et al., 2002a, b). All MIC determinations were performed in triplicate.
The influence of chloramphenicol on metronidazole resistance during anaerobic incubation.
Bacteria freshly grown on Dent plates were harvested and inoculated in BBN to a cell density of 
1x107 c.f.u. ml–1, and incubated overnight with gentle agitation under microaerophilic conditions. Fumaric acid (Sigma-Aldrich, final concentration 0.1 %) was then added to the overnight culture to facilitate the generation of anaerobic conditions (Jenks et al., 1999b). The culture was then split into 10 ml portions and when indicated supplemented with metronidazole (Sigma-Aldrich, final concentration 16 µg ml–1) and/or chloramphenicol (Sigma-Aldrich, final concentration 10 µg ml–1). Subsequently, the cultures were incubated under microaerophilic or anaerobic conditions for 0, 2, 4, 6 and 8 h. At each time point 1 ml of the broth was taken and washed twice with PBS to remove the antibiotics. In order to determine the number of viable bacteria, 50 µl of undiluted suspension and 10–1, 10–2, 10–3 and 10–4 dilutions were plated on Columbia agar plates containing 7 % lysed horse blood and incubated under microaerophilic conditions. When present, colonies were counted and data are expressed as c.f.u. ml–1.
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RESULTS AND DISCUSSION
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Effect of anaerobic incubation on MIC
To evaluate the effect of anaerobic incubation on the MIC of metronidazole, seven MtzR H. pylori clinical isolates and the MtzR H. pylori reference strain ATCC 43504T were selected for this study. To ensure that these strains represented different isolates, RAPD fingerprinting was performed with primers D11344 (Fig. 1), 1254 and D9355 (data not shown). All strains gave different profiles with each of the three primers, indicating they represent unrelated isolates. These seven MtzR isolates and reference strain ATCC 43504T were incubated in microaerophilic and anaerobic conditions, and the MIC of metronidazole was determined by E-test and agar dilution. Under standard microaerophilic culture conditions, the MIC of metronidazole for the eight isolates, as determined by E-test, ranged from 24 to >256 µg ml–1, (Table 3). These MICs for metronidazole decreased under anaerobic conditions. After 4 h of anaerobic incubation, the MIC values for metronidazole dropped below the breakpoint (8 µg ml–1) for three of the eight isolates, and after 8 h, all MtzR isolates had become metronidazole-susceptible (Table 3). In contrast to metronidazole, the MICs for amoxycillin, clarithromycin and tetracycline were stable during anaerobic incubation (data not shown). There were no clear differences found in the MIC values between the E-test and agar dilution.
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Fig. 1. The H. pylori isolates used in this study represent unrelated isolates, as shown by RAPD-PCR. DNA isolated from the MtzR isolates was amplified with primer D11344 according to standard procedures (Akopyanz et al., 1992). The RAPD-PCR products were separated on a 2.0 % agarose gel and stained with ethidium bromide. Lane 1, H. pylori reference strain ATCC 43504T, lanes 2–8, BH9711-176, BH9713-141, BH9714-19, DM9735-58, DM9727-179, DM9642-108 and DM9716-140, respectively. M, 1 kb marker (Promega).
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Table 3. The effect of anaerobic incubation on metronidazole resistance MICs shown are means of results from three independent experiments. The isolates were considered to be resistant when the MIC of metronidazole was 8 µg ml–1 (Gerrits et al., 2002b). T0, time point zero, start point; T2, T4 and T8, after 2, 4 and 8 h of anaerobic incubation, respectively.
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Effect of metronidazole and anaerobic incubation on cell viability
To determine the effect of metronidazole and anaerobic incubation on cell viability, all seven MtzR H. pylori isolates and the MtzR reference strain ATCC 43504T were cultured in broth under microaerophilic and anaerobic conditions either in the presence or absence of 16 µg metronidazole ml–1, and at different time intervals the amount of viable bacteria (c.f.u. ml–1) was determined. Under standard microaerophilic conditions, the amount of viable cells for all tested MtzR isolates varied between 106 and 107 c.f.u. ml–1, and there were no significant differences observed in c.f.u. ml–1 between the cultures with and without metronidazole (Fig. 2). Similar data were obtained for the cultures without metronidazole that were incubated anaerobically (Fig. 2). This suggests that neither the incubation with metronidazole nor anaerobic growth conditions alone affect the cell viability of the MtzR isolates.
However, under anaerobic conditions in the presence of metronidazole, the amount of viable cells dropped more than 1000-fold when the MtzR isolates were incubated for 4 h anaerobically, and after 8 h of anaerobic incubation in the presence of metronidazole there were no viable cells present (Fig. 3). Since there were no differences in cell viability or time-course observed between the MtzR isolates containing mutations that resulted in either a truncated RdxA protein or FrxA protein, or a truncation in both, this suggests that neither the rdxA gene nor the frxA gene is involved in the reversibility of metronidazole resistance in H. pylori.
Effect of de novo protein synthesis on reversibility of metronidazole resistance during anaerobic incubation
To determine whether H. pylori requires de novo protein synthesis for the reversibility of metronidazole resistance, all experiments were repeated in broth in the presence of the bacterial protein synthesis inhibitor chloramphenicol (10 µg ml–1). This concentration of chloramphenicol was optimized previously, and its effects on the inhibition of the protein synthesis and cell viability are known (Kusters et al., 1997). When the MtzR isolates were incubated anaerobically in the presence of metronidazole and chloramphenicol, cell viability was reduced (Fig. 4). As there was no significant difference in cell viability between the cultures with and without chloramphenicol these results indicate that factors that are involved in the reversibility of metronidazole resistance are already present under microaerophilic conditions. De novo protein synthesis is not required for this phenomenon.
Implications of experimental data
Metronidazole, a nitroimidazole, is administered as a prodrug that is activated by the reduction of the nitro group that is attached to an imidazole ring (Edwards, 1986). Since oxygen has a higher reduction potential than metronidazole, this reduction step works out most effectively in an environment with low oxygen tension, such as anaerobic cells and protozoa (Jenks & Edwards, 2002). Surprisingly, the drug was also found to be active against the microaerophilic pathogen H. pylori (Lacey et al., 1993). In many strictly anaerobic bacteria, the activation of metronidazole is mediated by the pyruvate : ferredoxin oxidoreductase complex (Smith et al., 1998). In H. pylori, this function might be fulfilled by the electron carriers, RdxA (HP0954), FrxA (HP0642), ferredoxin (FdxA, HP0277), flavodoxin (FldA, HP1161), pyruvate : ferredoxin oxidoreductase (PorD, HP1109) and 2-oxoglutarate ferredoxin oxidoreductase (OorD, HP0588). As mutations of the latter four nitroreductases were found to be lethal (Jeong et al., 2000; Kwon et al., 2000a), we only tested the involvement of the rdxA and frxA genes. In contrast with the findings under normal microaerophilic conditions (Kwon et al., 2000b; Sisson et al., 2002), we showed that neither the rdxA nor the frxA gene is required for the activation of metronidazole under low oxygen conditions, since strains with one or both genes inactivated still become susceptible to metronidazole under anaerobic conditions.
As MtzR H. pylori isolates lose their resistance to metronidazole after exposure to short periods of anaerobiosis in vitro (Cederbrant et al., 1992; Smith & Edwards, 1995), it has been suggested that this reversibility is mediated by compensatory metabolic pathways that are induced under anaerobic conditions (Jenks & Edwards, 2002; Smith & Edwards, 1997). This hypothesis is not supported by our data obtained using the protein synthesis inhibitor chloramphenicol. The loss of metronidazole resistance is mediated by a pre-existing mechanism that functions under anaerobic conditions, and is not dependent on de novo protein synthesis when H. pylori is exposed to these conditions. Since our data excluded the role of the RdxA and FrxA proteins in this process, we assume that in H. pylori metronidazole is reduced by one of the other known nitroreductases.
In summary, MtzR H. pylori isolates become fully metronidazole-susceptible at low oxygen conditions, and this does not require de novo protein synthesis. This reversibility in metronidazole resistance also occurred in H. pylori isolates that contained mutations in the rdxA and/or frxA genes. Exposure of H. pylori to such low oxygen conditions in the gastric mucosa or gastric pit is likely to induce reduction of metronidazole, and thus assist in the eradication of MtzR H. pylori.
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ACKNOWLEDGEMENTS
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The authors would like to thank Andy van Oosterhout for technical assistance.
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INTRODUCTION
METHODS
) and absence (
) of 16 µg metronidazole ml–1, and anaerobically (
). At different time points the c.f.u. ml–1 was determined. Results shown are a representative example of one of the MtzR isolates, BH9714-19, and are the means (±SD) of two independent experiments performed in duplicate.
, DM9735-58 (containing mutations resulting in a truncated RdxA); •/
, BH9713-141 (containing mutations resulting in a truncated FrxA). Results shown are the means (±SD) of two independent experiments performed in duplicate.
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, with chloramphenicol; •/