Abstract

Metronidazole (Mtz) resistance in the gastric pathogen Helicobacter pylori is closely associated with inactivation of the nitroreductase gene rdxA. In order to identify respective mutations for diagnostic purposes we analyzed the rdxA gene in a collection of high-level Mtz-resistant clinical H. pylori isolates. Size alterations in the rdxA gene region were found in only two out of 45 and one out of 40 isolates showing lower-level (minimal inhibitory concentrations (MICs) 32–192 µg ml−1) and high-level (MIC≥256µg ml−1) Mtz resistance, respectively. Point mutations that interrupt the rdxA reading frame were detected in two out of eight high-level resistant isolates (MICs≥256µg ml−1). Most remarkably, the rdxA gene sequence was found to be identical in four out of five high-level Mtz-resistant and -susceptible paired H. pylori isolates from the same patients each. Taken together, these results demonstrate that although some isolates carry classical resistance-associated rdxA mutations, as described earlier, the use of rdxA mutations as a marker for prediction of Mtz resistance is limited.

Introduction

The nitroimidazole antibiotic metronidazole (Mtz) is widely used in eradication therapy of the gastric pathogen Helicobacter pylori[1–3] which causes gastritis and peptic ulcer disease [4]. Resistance to Mtz has been reported to increase in the worldwide H. pylori population and it was observed in a recent study that secondary resistance to Mtz is frequently associated with resistance to clarithromycin, which is often used in combination therapy [2]. The H. pylori oxygen-insensitive nitroreductase RdxA, annotated HP0954 in the genome sequence of the reference strain 26695 [5] activates Mtz by reduction [6,7]. The antimicrobial activity of the drug results in the formation of toxic products causing chemical modifications of macromolecules including DNA damage [8]. Various studies demonstrated that inactivation of the rdxA gene by allelic exchange mutagenesis confers Mtz resistance to susceptible H. pylori strains [6,9–12]. Single nucleotide transitions introducing frameshift mutations or stop codons [13] as well as larger DNA insertions or deletions [9] both destroying or altering the rdxA reading frame, were found to be associated with the Mtz-resistant phenotype [6].

Recent investigations have demonstrated that rdxA mutations in H. pylori reference strains are induced upon exposure to sublethal Mtz concentrations in vitro [13] and are an essential prerequisite for development of high-level Mtz resistance [11,12]. It was shown recently that besides rdxA other genes like the gene for the flavin oxidoreductase FrxA (HP0642) are involved in causing Mtz resistance [11,12,14]. However, a detailed study on the roles of frxA and rdxA genes in causing Mtz resistance showed that inactivation of frxA alone does not render H. pylori Mtz-resistant, whereas development of high-level Mtz resistance can be caused by rdxA/frxA double mutations [12]. However, the molecular mechanisms causing H. pylori Mtz resistance are not well understood [15]. In order to identify defined rdxA mutations associated with Mtz resistance, we analyzed the rdxA gene in a collection of clinical H. pylori isolates showing a high Mtz resistance level. Specific aims were (i) to determine the frequency of larger insertions and deletions within the rdxA gene, (ii) to identify rdxA mutations suitable for the development of rapid molecular approaches for prediction of Mtz susceptibility in clinical materials (e.g. biopsies).

Materials and methods

Bacterial strains, growth conditions and resistance testing

The strain collection analyzed consisted of isolates showing lower-level (MIC 32 µg ml−1, 18 isolates; MIC 48 µg ml−1, one isolate; MIC 64 µg ml−1, 22 isolates; MIC 96 µg ml−1, two isolates; MIC 128 µg ml−1, one isolate; MIC 192 µg ml−1, one isolate), and high-level (MIC≥256, 40 isolates) Mtz resistance. The H. pylori isolates showing lower-level Mtz resistance and the paired Mtz-resistant and -susceptible isolates from antrum and corpus originated from patients with peptic ulcer disease, which did not receive pretreatment against H. pylori. The corresponding biopsy material was submitted to our reference center during two multicenter studies [1,16] from gastroenterologists all over Germany. The H. pylori isolates showing high-level resistance were collected routinely by our diagnostics laboratory. Single H. pylori colonies cultivated from biopsy specimen were subcultivated, tested for Mtz resistance by E-test as described earlier [1,16], and then stored frozen at −80°C for further examinations. Isolates with lower-level and high-level resistance to the drug were chosen for rdxA analysis because our own in-house studies and studies of other investigators [17] have shown that the E-test is reliable only for resistance levels at or above MICs of 16 µg ml−1. To verify the high-level Mtz resistance status of the strain collection, H. pylori isolates displaying low resistance levels (MICs<32µg ml−1), and nearby the breakpoint (MIC 8 µg ml−1), were not included in the study. For a subset of the isolate collection analyzed here, results from E-test were controlled by agar dilution assays. In case of 18 isolates showing MICs of 32 µg ml−1 the agar dilution assay confirmed the E-test results, demonstrating that the standardized E-test protocol applied is well suited for identification of H. pylori isolates showing lower-level and high-level Mtz resistance.

Genetic analysis of the rdxA gene and genotyping of H. pylori isolates

The rdxA gene was analyzed with primers RDXA-L1 (5′-TTTACAGAGAGCCGGAC-3′), -L2 (5′-GCTTCAGCGTTAATGGT-3′), -R1 (5′-TAGCGCTTAATGAAACG-3′), and -R2 (5′-CCAATCCCATTAAGCTC-3′) (Fig. 1). The isolation of H. pylori from gastric biopsies, DNA amplification by polymerase chain reaction (PCR), and DNA sequencing (on both strands) were performed as described earlier [16]. Genotyping of H. pylori isolates by PCR, ERIC- and RAPD-PCR was performed according to published standard protocols [18,19]. The rdxA nucleotide sequences referred to in this publication were made available in the EMBL database and have been assigned accession numbers AJ305346–AJ305358.

Figure 1

Overview on the genetic organization of the rdxA gene (A) and results from detection of rdxA size variants by PCR (B). A: Schematic presentation of the rdxA region in H. pylori strain 26695. Genes, and primer oligonucleotides used for PCR and for sequence analysis are marked by gray and black arrows, respectively. B: Ethidium bromide-stained agarose gel displaying the results of PCR analysis of selected Mtz-resistant H. pylori isolates with (lanes 1–3) and without (lanes 4–7) size variations in the rdxA region. The PCR product amplified with primers RDXA-L1 and -R1 is marked by the arrow at the right. Size variants 1 (isolate 7639), 2 (isolate 9768), and 3 (isolate 5885) are indicated at the top. Lane M, marker DNA; lane C, control without target DNA.

Figure 1

Overview on the genetic organization of the rdxA gene (A) and results from detection of rdxA size variants by PCR (B). A: Schematic presentation of the rdxA region in H. pylori strain 26695. Genes, and primer oligonucleotides used for PCR and for sequence analysis are marked by gray and black arrows, respectively. B: Ethidium bromide-stained agarose gel displaying the results of PCR analysis of selected Mtz-resistant H. pylori isolates with (lanes 1–3) and without (lanes 4–7) size variations in the rdxA region. The PCR product amplified with primers RDXA-L1 and -R1 is marked by the arrow at the right. Size variants 1 (isolate 7639), 2 (isolate 9768), and 3 (isolate 5885) are indicated at the top. Lane M, marker DNA; lane C, control without target DNA.

Results and discussion

Detection of size alterations in the rdxA gene of Mtz-resistant H. pylori isolates

To determine the frequency of larger size variations in the rdxA gene region, the 85 Mtz-resistant H. pylori isolates, including 40 isolates showing high-level resistance, from our collection (see Section 2), were analyzed by PCR with primers RDXA-L1 and -R1, which amplify a 903 bp product carrying the 630 bp rdxA coding region and flanking sequences (Fig. 1A). Specific PCR products with significant size alterations were detected only in isolates 7639, 9768, and 5885, which displayed high-, and lower-level Mtz resistance, respectively (Fig. 1B; Table 1). Sequence analysis revealed that in all three isolates the coding region of the rdxA gene is modified by DNA deletions or insertions which interrupt the reading frame (summarized in Table 1). In case of isolate 5885 the gene is inactivated by deletion of the 5′-region including the ATG start codon and the ribosome binding site. These results show that modifications of the rdxA gene by larger insertions or deletions are rare within the Mtz-resistant strain population and that the PCR screening approach is not suited for the detection of Mtz-resistant isolates.

Table 1

Frameshift mutations detected in rdxA of Mtz-resistant H. pylori isolates

Isolate MIC for Mtz (µg ml−1Changes in the rdxA gene 
7639 ≥256 His53→Ile; Phe54→Stop (frameshift caused by deletion of nt 155–179) 
9768 96 correct start codon removed (ATG→CTG); multiple frameshifts caused by nt insertions at different sites and by duplication of nt 629–703) 
5885 32 start codon and RBS missing (deletion of nt −12 to +58) 
1013 ≥256 Asn73→Stop (frameshift caused by insertion of A behind nt 192) 
9918 ≥256 Glu175→Stop (nt 523 GAG→TAG) 
Isolate MIC for Mtz (µg ml−1Changes in the rdxA gene 
7639 ≥256 His53→Ile; Phe54→Stop (frameshift caused by deletion of nt 155–179) 
9768 96 correct start codon removed (ATG→CTG); multiple frameshifts caused by nt insertions at different sites and by duplication of nt 629–703) 
5885 32 start codon and RBS missing (deletion of nt −12 to +58) 
1013 ≥256 Asn73→Stop (frameshift caused by insertion of A behind nt 192) 
9918 ≥256 Glu175→Stop (nt 523 GAG→TAG) 

Positions refer to the rdxA coding region in the reference strain 26695 [5] RBS, ribosome binding site.

Identification of rdxA point mutations associated with Mtz resistance

To find putative resistance-associated point mutations altering the rdxA reading frame, we sequenced the gene of eight out of the 40 H. pylori isolates displaying high-level Mtz resistance (MIC≥256, see Section 2). Among these, nucleotide transitions introducing missense stop codons were found only in two isolates 1013 and 9918 in which the rdxA reading frame is interrupted by stop codons at different positions (Table 1). In the other six isolates 1009, 1031, 826, 8966, 9171 and 1040 (summarized in Table 3) the rdxA reading frame was not interrupted. Alignments of rdxA from Mtz-resistant isolates with sequences from Mtz-susceptible reference strains from databases and from this study did not highlight any clustering of amino acid substitutions in RdxA of Mtz-resistant isolates (Table 3), which is well in agreement with findings of other investigators (reviewed in [15]). Interestingly, a number of different amino acid substitutions were exclusively found in Mtz-resistant isolates with an MIC of ≥256 µg ml−1. Such residues (Table 3) are suspected to be involved in the catalytic activity of the enzyme and exchanges can be proposed to be causative for resistance via inhibiting or modifying the enzymatic activity [6,13]. However, because the substitutions are arbitrarily distributed in the protein, it was not possible to draw any conclusion concerning their functional relevance. The diagnostic value of the amino acid exchanges His53→Arg, Met56→Ile, Leu62→Val, Ala68→Val, and Gly98→Ser present in Mtz-resistant isolates (Table 3) is put in perspective by the fact that identical substitutions were also found in RdxA proteins from Mtz-susceptible H. pylori isolates in other studies [15,20,21]. The recently discovered RdxA amino acid substitutions Cys19→Tyr and Thr49→Lys, which were experimentally confirmed to be causative for Mtz resistance in H. pylori reference strains in vitro [13], were not observed in RdxA of high-level Mtz-resistant clinical isolates analyzed here (Table 3).

Table 3

Overview on amino acid substitutions in RdxA proteins from H. pylori isolates

Strain or isolate Mtz MIC (µg ml−1Amino acid positions: 
  16 31 44 53 56 59 62 68 88 90 97 98 118 131 159 178 
Susceptible to Mtz 
26695  
NCTC11638 0.049           
40C 0.75              
53A 0.5              
190A 0.25               
242C              
345A                
Resistant to Mtz 
40A ≥256               
53C ≥256              
190C ≥256               
242A >256              
345C 128                
1009 ≥256             
1031 ≥256            
826 ≥256             
8966 ≥256             
9171 ≥256             
1040 ≥256               
Strain or isolate Mtz MIC (µg ml−1Amino acid positions: 
  16 31 44 53 56 59 62 68 88 90 97 98 118 131 159 178 
Susceptible to Mtz 
26695  
NCTC11638 0.049           
40C 0.75              
53A 0.5              
190A 0.25               
242C              
345A                
Resistant to Mtz 
40A ≥256               
53C ≥256              
190C ≥256               
242A >256              
345C 128                
1009 ≥256             
1031 ≥256            
826 ≥256             
8966 ≥256             
9171 ≥256             
1040 ≥256               

, strain 26695 is susceptible to Mtz as determined earlier [11].

Amino acids positions refer to the RdxA protein of the reference strain 26695 [5].

Analysis of the rdxA gene sequence in paired high-level Mtz-resistant and -sensitive H. pylori isolates

Changes in the amino acid sequence can be caused by the natural genetic diversity of H. pylori, which is in case of rdxA with about 5–8% [15,20] high enough to interfere with the identification of resistance-associated nucleotide transitions, the influence of the natural genetic variability of H. pylori was minimized by direct comparison of rdxA sequences from paired high-level Mtz-resistant and -susceptible isolates from different gastric sites (antrum and corpus) of the same patients. The natural genetic variability was shown to be lower in such isolate pairs [21]. Patients carrying both, high-level resistant (MIC≥128µg ml−1) and susceptible (MIC≤4µg ml−1) H. pylori isolates (Table 2) were found to be very rare (about 1%). However, five such isolate pairs could be identified in an isolate collection containing antrum and corpus isolates from 500 patients. After reassessment of Mtz resistance by E-test (MICs are given in Table 2), these isolates were further analyzed. Sequencing of the complete rdxA coding region including about 30 nucleotides in front of the gene (Fig. 1A) revealed complete identity of the rdxA coding sequence in case of four isolate pairs (from patients 53, 190, 242, and 345). In one isolate pair (from patient 40), the sequences show differences which do not destroy the rdxA reading frame and are with 2% clearly in the range of the natural genetic variability of H. pylori.

Table 2

Properties and genotypes of high-level Mtz-resistant and -susceptible isolate pairs

Isolate pairs Resistance MIC for Mtz (µg ml−1vacA genotype and cag status RibAP genotype VacAP genotype RAPD-PCR pattern rdxA DNA sequence 
40A R, ≥256 s1b/m1, cag+   
40C S, 0.75 s1b/m1, cag+ different different 
53A S, 0.5 s1a/m2, cag+   
53C R, ≥256 s1a/m2, cag+ identical identical 
190A S, 0.25 s2/m2, cag−   
190C R, ≥256 s2/m2, cag− identical identical 
242A R, ≥256 s1a/m1a, cag+   
242C S, 4 s1a/m1a, cag+ identical identical 
345A S, 1 s2/m2, cag−   
345C R, 128 s2/m2, cag− identical identical 
Isolate pairs Resistance MIC for Mtz (µg ml−1vacA genotype and cag status RibAP genotype VacAP genotype RAPD-PCR pattern rdxA DNA sequence 
40A R, ≥256 s1b/m1, cag+   
40C S, 0.75 s1b/m1, cag+ different different 
53A S, 0.5 s1a/m2, cag+   
53C R, ≥256 s1a/m2, cag+ identical identical 
190A S, 0.25 s2/m2, cag−   
190C R, ≥256 s2/m2, cag− identical identical 
242A R, ≥256 s1a/m1a, cag+   
242C S, 4 s1a/m1a, cag+ identical identical 
345A S, 1 s2/m2, cag−   
345C R, 128 s2/m2, cag− identical identical 

Isolates were cultivated from antrum (A) and corpus (C) gastric biopsies as indicated.

Isolates are resistant (R) and susceptible (S) to Mtz as indicated.

Signal sequence (s) and mid-region (m) subtypes of vacA and the cagA status were determined as described [16].

Genotypes were analyzed as previously described [18].

The rdxA sequence identity suggests that the antrum and corpus isolate pairs from patients 56, 190, 242, and 345 are genetically more related than the isolate pair from patient 40. This was further confirmed by molecular fingerprinting. Results obtained by RAPD-PCR, by ERIC-PCR, and with a recently developed combination of PCR assays for detection of genetic variability in noncoding DNA [18] revealed that the isolates from patient 40 are genetically less related than the other four isolate pairs (Tables 2 and 3, Fig. 2). This provides evidence that in case of isolates with identical rdxA sequences, the Mtz-resistant organisms have evolved from a Mtz-susceptible ancestor population, whereas in case of patient 40 a double infection with both, susceptible and resistant strains cannot be excluded. The identity of the rdxA gene in four out of five sensitive and resistant isolate pairs from the same patients indicates that resistance to metronidazole is not always associated with inactivation of rdxA. This is well in agreement with the results of earlier studies, in which rdxA mutations in paired isolates were found to be more frequently but not in all cases associated with resistance [15,21]. The fact that the mutations identified here do also not cluster in a defined gene region underlines that rdxA mutations can not be used to generally predict Mtz resistance in clinical H. pylori isolates.

Figure 2

Genotyping of Mtz-resistant and -susceptible H. pylori isolate pairs by using RAPD-PCR. DNA isolated from Mtz-resistant (R) and -susceptible (S) H. pylori isolates from antrum (A) and corpus (C) of the same patients (isolate and patient numbers indicated on the top) were analyzed by RAPD-PCR with primer M13-FP (5′-TGTAAAACGACGGCCAGT-3′) according to standard procedures [19]. The RAPD-PCR products were separated on a 1.6% agarose gel and stained with ethidium bromide. The sizes of selected marker DNA fragments are given at the right.

Figure 2

Genotyping of Mtz-resistant and -susceptible H. pylori isolate pairs by using RAPD-PCR. DNA isolated from Mtz-resistant (R) and -susceptible (S) H. pylori isolates from antrum (A) and corpus (C) of the same patients (isolate and patient numbers indicated on the top) were analyzed by RAPD-PCR with primer M13-FP (5′-TGTAAAACGACGGCCAGT-3′) according to standard procedures [19]. The RAPD-PCR products were separated on a 1.6% agarose gel and stained with ethidium bromide. The sizes of selected marker DNA fragments are given at the right.

Conclusions

The finding that rdxA sequence variations detected in high-level Mtz-resistant isolates did not cluster in a defined gene region limits the use of rdxA as a marker gene for Mtz resistance. Thus, rdxA-based molecular diagnostic approaches for determination of Mtz susceptibility of H. pylori isolates will be difficult to establish. In agreement with results from earlier studies [15,20], this conclusion is supported here by the absence of rdxA mutations in high-level Mtz-resistant clinical H. pylori isolates and by the fact that in four Mtz-susceptible and -resistant isolate pairs originating from antrum and corpus of the same patients the rdxA sequences were identical. The fact that in a significant number of clinical H. pylori isolates the Mtz-resistant phenotype is not associated with mutations in rdxA raises the question if besides rdxA other genes are involved in causing Mtz resistance, which is currently controversely discussed [21]. Together with earlier studies, the observations made here support a role of other genes in Mtz resistance as some but not all isolates carry resistance-associated rdxA mutations. This necessitates investigations that combine the genetic investigation of rdxA with gene expression analysis and determination of RdxA activity [15]. The possible role for the frxA gene in causing Mtz resistance in the isolates showing high-level resistance is put in perspective by the fact that extensive investigations focussed on the molecular basis of Mtz resistance have demonstrated that frxA mutations only cause high-level Mtz resistance in combination with rdxA mutations [11,12].

Acknowledgements

The authors thank Tanja Tanbouze for excellent technical assistance. We are also grateful to Klaus Melchers (ALTANA Research Institute, Waltham, USA) for critical comments on the manuscript.

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