Unité de Pathogénie Bactérienne des Muqueuses, Institut Pasteur, 28 Rue du Dr Roux, 75724 Paris Cedex 15, France
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Abstract |
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Introduction |
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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with H. pylori 13 to generate a series of metronidazole- resistant H. pylori isolates derived from strain SS1 (unpublished data).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.
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Materials and methods |
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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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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.
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Results |
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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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Discussion |
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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.
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Acknowledgments |
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Notes |
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References |
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Received 27 August 1998; returned 15 December 1998; revised 12 January 1999; accepted 12 February 1999