The role of the rdxA gene in the evolution of metronidazole resistance in Helicobacter pylori

P. J. Jenks*, R. L. Ferrero and A. Labigne

Unité de Pathogénie Bactérienne des Muqueuses, Institut Pasteur, 28 Rue du Dr Roux, 75724 Paris Cedex 15, France


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It was recently demonstrated that inactivation of the rdxA gene, which encodes an oxygen-insensitive NADPH nitroreductase, is associated with the development of resistance to metronidazole by Helicobacter pylori. In order to further evaluate the contribution of rdxA to metronidazole resistance, the sequence of the rdxA gene was determined for a series of metronidazole-sensitive and -resistant isolates derived from a single, metronidazole-sensitive strain using an H. pylori mouse model. These strains were cultured from the stomachs of mice experimentally infected with H. pylori strain SS1 and then treated orally with metronidazole. The sequence of the rdxA gene of all 10 sequenced metronidazole-sensitive and two (7%) of the 27 metronidazole-resistant isolates was identical to that of the parental strain. In contrast, the rdxA gene of the other 25 metronidazole-resistant isolates contained between one and three frameshift or missense mutations. This suggests that while the development of metronidazole resistance in H. pylori is frequently associated with mutational inactivation of the rdxA gene, other mechanisms of resistance are likely to exist in this bacterium.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Helicobacter pylori is the aetiological agent for peptic ulceration, and eradication of H. pylori from the stomachfacilitates duodenal ulcer healing and reduces ulcer relapse rate. 1,2 The 5-nitroimidazole, metronidazole, is an important component of many currently used H. pylori eradication regimens. 3,4,5, 6 Resistance of H. pylori to this class of antibiotics reduces the efficacy of metronidazole-containing eradication regimens, and is hence recognized as an important cause of treatment failure. 3,4,7 It has been estimated that 10- 30% of clinical strains isolated in western Europe and the USA are resistant to metronidazole, and this incidence is far higher in developing countries and in certain immigrant populations. 8,9,10 Goodwin et al. recently demonstrated that the loss of oxygen-insensitive NADPH nitroreductase activity resulted in the development of resistance to metronidazole in H. pylori. 11 This enzyme reduces metronidazole to an active metabolite that is directly toxic to the bacterium, and it was proposed that resistance arose from mutational inactivation of the underlying gene, rdxA (H0954 in the H. pylori genome database), 12 which encodes a protein of 210 amino acids.

We have previously used the mouse model of infection with H. pylori 13 to generate a series of metronidazole- resistant H. pylori isolates derived from strain SS1 (unpublished data). Mice colonized with the metronidazole- sensitive H. pylori strain SS1 were treated orally with various metronidazole-containing treatment regimens. After treatment, the stomachs of the majority of the animals contained a mixed population of metronidazole-resistant and -sensitive bacteria. Interestingly, despite originating from an isogenic parental strain, the degree of susceptibility to metronidazole of the resistant isolates varied from 8 to 64 mg/L. The aim of this study was to examine further the evolution of metronidazole resistance in H. pylori in vivo. Specifically, we wanted to examine the contribution of the rdxA gene to the development of resistance to metronidazole and to evaluate if other potential resistancemechanisms might exist in H. pylori.


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

The mouse-adapted H. pylori strain SS1, originally isolated from a patient with peptic ulcer disease, 13 was grown on blood agar plates at 37°C under microaerobic conditions. Six week-old specific-pathogen-free Swiss mice (Centre d' Elevage R Janvier, Le-Genest-St-Isle, France) were housed in polycarbonate cages in isolators and fed a commercial pellet diet with water ad libitum. All animal experimentation was performed in accordance with institutional guidelines. Mice were administered a single 100 µL aliquot of a suspension of H. pylori SS1 (10 5 cfu/mL), equivalent to 100 times the ID 100. 14 After the animals had been infected for at least 1 month they were treated intragastrically with various combinations of peptone trypsin broth, metronidazole and a recommended metronidazole-containing H. pylori eradication regimen (Table I). 6 Mice in Group 2 were treated for 7 days with the mouse equivalent of 400 mg metronidazole (Rhône-Poulenc Rorer, Vitry sur Seine, France) tds. The animals in Groups 1, 3, 4 and 5 were administered two treatment regimens. Treatment 1 consisted of either peptone trypsin broth (Groups 1 and 4) or the mouse equivalent of 400 mg metronidazole tds (Groups 3 and 5). Treatment 2 was administered 1 month after the completion of treatment 1 and consisted of either peptone trypsin broth (Groups 1 and 3) or the mouse equivalent of 20 mg omeprazole (Astra Hässle AB, Mölndal, Sweden), 250 mg clarithromycin (Abbott Laboratories, Saint-Rémy-sur-Avre, France) and 400 mg metronidazole (Rhône-Poulenc Rorer) bd for 1 week (Groups 4 and 5). One month after the completion of treatments the mice were killed and their stomachs cultured for H. pylori. Stomach homogenates were serially diluted in sterile saline and plated directly onto blood and serum plates for enumeration, and onto a selective plate containing 8 mg/L metronidazole. The susceptibility of isolates to metronidazole was assessed by agar dilution of the MIC. Isolates were considered resistant to metronidazole if they had an MIC >= 8 mg/L. 15


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Table I. Treatments for isolation of metronidazole-resistant H. pylori in a mouse model and metronidazole MICs for the resulting isolates
 
For the purposes of this study, a total of 38 isolates were examined further: the inoculating parental SS1 strain (MIC 0.064 mg/L); two metronidazole-resistant strains (20A and 20B) with different metronidazole MICs (8 and 64 mg/L) isolated from the same mouse (mouse 20); one metronidazole-resistant strain isolated from each of the other 25 mice (mice 11 to 19 and 21 to 36) treated with metronidazole (MICs 16- 64 mg/L); one metronidazole-sensitive strain isolated from each of the 10 mice (mice 1- 10) not treated with metronidazole (MICs of 0.064 mg/L).

Polymerase chain reaction (PCR) amplification of H. pylori rdxA sequence

Target chromosomal DNA was extracted from H. pylori strains using the QIAamp Tissue Kit (Qiagen, Courtaboeuf, France). Two pairs of oligonucleotide primers (5'[position 1014242 in the H. pylori genome database]-CGTTA-GGGATTTTATTGTATGCTAC-[position 1014217]3' and 5'[position 1013751]-CCCCACAGCGATATAGCATTGCTC-[position 1013775]3'), and (5'[position 1013856]-GTTAGAGTGATCCCCTCTTTTGCTC-[position 1013831]3' and 5'[position 1013451]-CACCCCTAAAAGAGCGATTAAAACC-[position 1013476]3') were used to amplify two overlapping PCR products (of 491 bp and 405 bp respectively) that constituted a total of 789 bp that contained the entire rdxA gene. After heat-denaturation of chromosomal DNA, gene amplification was carried out through 30 consecutive cycles consisting of a denaturation step of 95°C for 2 min, a primer annealing step of 48°C for 2 min and an extension step at 72°C for 2 min, with a single final extension step of 72°C for 10 min. Nucleotide sequences of the PCR products obtained were determined on both strands using the four oligonucleotide primers described above. Computer-aided sequence alignments were performed with the PILEUP program by using the Genetics Computer Analysis Software Package. Sequencing of the rdxA gene of the 10 metronidazole-sensitive strains, isolated from mice not treated with metronidazole, was used as a control of PCR fidelity. The sequence of rdxA was determined for one metronidazole-sensitive strain isolated from each of the 10 mice in treatment Group 1 (Table I), two metronidazole-resistant strains isolated from Group 2 (mouse 20) and one metronidazole-resistant strain isolated from the mice in Groups 2- 5.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In all the isolates studied, two PCR-amplified rdxA- containing fragments of the expected size were obtained. The sequence of the rdxA gene of strain SS1 differed from that of strain 26695 12 by 28 of 630 bp (4.4%) and this resulted in seven amino acid substitutions (positions 31, 58, 88, 97, 107, 112, 132). The sequence of the rdxA gene of the 10 metronidazole-sensitive isolates (strains 1- 10) and two of the metronidazole-resistant strains (strains 11 and 33) was identical to that of the parental SS1 strain. The rdxA genes of the other 25 metronidazole-resistant strains differed from that of the parental SS1 strain by one to three bp (Table II). When considered together, there were 17 distinct mutational changes in the rdxA gene of these strains (Table II), of which 12 (71%) were frameshift and five (29%) were missense mutations.

Nineteen of the metronidazole-resistant strains contained single frameshift mutations within their rdxA gene that were the result of the loss or gain of one or two nucleotides (Table II). In all cases this frameshift resulted in the creation of a translational stop codon in the region immediately downstream of the mutation. Frameshift mutations at positions 186, 187, 263, 425 and 576 were present in multiple strains. Seven of the 12 frameshift mutations were the result of the loss or gain of adenine (A) or thymine (T) nucleotides in polyA or polyT tracts.

The rdxA gene of four of the metronidazole-resistant strains (strains 17, 18, 19, 20A) contained one or two missense point mutations that resulted in amino acid substitutions (Table II). These strains were all isolated from mice that had received the mouse equivalent of 400 mg metronidazole tds for 1 week (Table I). Three of these strains contained the same amino acid substitution: substitution of proline by leucine at position 51. The other amino acid changes were tyrosine to histidine (position 46) and alanine to valine (position 67). Four of the substitutions were at positions within a region that is highly conserved in classical oxygen-insensitive NADPH nitroreductases (position 43- 57). 11


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Table II. Types of point mutation in rdxA and amino acid substitutions in metronidazole-resistant H. pylori
 
One strain (strain 21) contained a single frameshift mutation and two missense point mutations, and one strain (strain 32), isolated from a mouse that received both metronidazole-containing treatment regimens, contained two frameshift mutations. There was no correlation between the MIC for the metronidazole-resistant isolates and either the type or position of mutation in the rdxA gene.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The finding that the sequence of the rdxA gene of the metronidazole-sensitive parental strain SS1 was dissimilar to that of strain 26695 is consistent with the observation that unrelated rdxA genes of metronidazole-sensitive strains of H. pylori differ by approximately 5% at the nucleotide level. 11 The natural polymorphism of the rdxA gene observed between susceptible strains has meant that previous assessment of the role of this gene in the acquisition of metronidazole resistance has required comparison of the nucleotide sequence of paired sensitive and resistant clinical isolates. 11 The H. pylori infection model we used is well validated and has been standardized for antimicrobial studies, vaccine development and studies in pathogenesis. 13,14 We have previously used this model to demonstrate that mice infected with the metronidazole-sensitive strain SS1 develop isolates with varying degrees of resistance to metronidazole after oral treatment with metronidazole-containing regimens (unpublished data). Such a system provides a convenient model to study the evolution of metronidazole resistance in vivo, and is particularly useful for examining underlying resistance mechanisms, such as inactivation of the rdxA gene. The advantage of this system is that a large number of different in-vivo-generated metronidazole-resistant isolates can be compared with the same metronidazole-sensitive, parental strain.

The metronidazole-sensitive strains, isolated from mice that were not treated with metronidazole, all contained a rdxA gene that was identical to that of the parental strain. The presence of an unchanged rdxA gene in these strains is supportive of the conclusion that intact nitroreductase activity is associated with susceptibility to metronidazole, and validates the approach used and the fidelity of the Taq polymerase. It also suggests that the nucleotide sequence of this gene is relatively stable when the organism is not exposed to metronidazole. Mutational changes in the rdxA gene were observed exclusively in strains that had been exposed to metronidazole, implying that mutational inactivation of this gene does confer a selective advantage in the presence of this antibiotic through the development of the resistant phenotype. Changes in the rdxA gene were present after treatment with the mouse equivalent of either 400 mg metronidazole tds (as used to treat anaerobic and protozoal infections), a recommended metronidazole- containing eradication regimen, 6 or a combination of both treatments. Modifications of this gene may therefore be associated with the acquisition of either primary or secondary resistance by H. pylori. The MIC for each resistant isolate was unchanged after three consecutive subcultures on non-selective medium, suggesting that the resistant phenotype is relatively stable.

In 25 of the 27 isolates the development of metronidazole resistance in vivo was associated with modification of the rdxA gene. In total, 17 distinct mutations were observed, none of which have been described before; clearly resistance does not develop as the result of a conserved alteration in this gene. Despite this, six mutations were present in multiple isolates, which suggests that certain regions of the gene are particularly susceptible to mutational modification. The two metronidazole-resistant strains that were isolated from the same mouse (strains 20A and 20B) had different mutations in their rdxA gene, which demonstrates that individual bacteria within the same stomach may develop resistance independently.

In contrast to the findings of Goodwin et al. 11 the majority of changes in the rdxA gene were due to frameshift rather than missense mutations. All of the frameshift mutations resulted in a translational stop codon immediately downstream of the mutation, and hence a truncated RdxA protein. A significant proportion (58%) of the frameshift mutations occurred within polyA or polyT tracts, which suggests that slipped-strand mispairing may be an important mechanism in the regulation of the expression of this gene. 16 Missense mutations were less common and were only present in strains isolated from mice treated with the equivalent of 400 mg metronidazole tds. The same amino acid substitution was observed in three isolates (position 51) and four substitutions occurred in a region likely to be essential for enzyme activity (position 43- 57). 11

Two strains had multiple mutations in their rdxA gene. Strain 21 had one frameshift and two missense mutations, the latter occurring downstream of the translation stop codon. Strain 32 had two frameshift mutations and was isolated from a mouse that had been treated with both metronidazole-containing regimens. The presence of more than one inactivating mutation in the rdxA gene would not appear to confer any additional selective advantage to the bacterium. However, repeated exposure to the mutagenic effects of this antibiotic could result in the accumulation of multiple mutations and suggests that metronidazole may directly cause some of the nucleotide changes that were observed.

It is evident from our results that inactivation of the rdxA gene of H. pylori is highly associated with the development of resistance to metronidazole. It is not currently known whether inactivation of the rdxA gene is the sole mechanism of metronidazole resistance in H. pylori. Our finding that two metronidazole-resistant strains contained an rdxA gene that was identical to that of the parental strain SS1 provides strong evidence that other mechanisms conferring resistance to metronidazole are likely to exist in this organism. These other mechanisms remain to be determined, but may include metronidazole efflux or reduced uptake, deficiency of other enzymes involved in reduction of metronidazole to its active form, target modification or increased DNA repair.


    Acknowledgments
 
P. J. Jenks is supported by a Research Training Fellowship in Medical Microbiology from the Wellcome Trust (Ref 044330). We are grateful to J. O' Rourke for helpful advice and also thank Rhône-Poulenc Rorer, Vitry sur Seine, France, Astra Hässle AB, Mölndal, Sweden and Abbott Laboratories, Saint-Rémy-sur-Avre, France for the gift of the pharmaceutical agents used in the treatment protocols.


    Notes
 
* Corresponding author. Tel: +33-1-40613272; Fax: +33-1-40613640; E-mail: pjenks{at}pasteur.fr Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 27 August 1998; returned 15 December 1998; revised 12 January 1999; accepted 12 February 1999