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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, October 2000, p. 2623-2629, Vol. 44, No. 10
Unité de Pathogénie
Bactérienne des Muqueuses, Institut Pasteur, 75724 Paris Cedex
15,1 and Service de
Bactériologie-Virologie, Hôpital Henri Mondor,
Creteil,3 France, and Department of
Medical Microbiology, Royal Free and University College Medical
School, London, United Kingdom2
Received 7 February 2000/Returned for modification 1 June
2000/Accepted 27 June 2000
The main objectives of this study were to determine whether the
nitroreductase enzyme encoded by the rdxA gene of
Helicobacter pylori was responsible for reductive
activation of nitrofurantoin and whether a triple-therapy regimen with
nitrofurantoin was able to eradicate metronidazole-sensitive and
-resistant H. pylori infections from mice. The
susceptibilities to nitrofurantoin of parent and isogenic
rdxA mutant strains (three pairs), as well as a series of
matched metronidazole-sensitive and -resistant strains isolated from
mice (30) and patients (20), were assessed by
agar dilution determination of the MIC. Groups of mice colonized with
the metronidazole-sensitive H. pylori SS1 strain or a
metronidazole-resistant rdxA SS1 mutant were treated with
either metronidazole or nitrofurantoin as part of a triple-therapy
regimen. One month after the completion of treatment the mice were
sacrificed and their stomachs were cultured for H. pylori.
The nitrofurantoin MICs for all strains tested were between 0.5 and 4.0 µg/ml. There was no significant difference between the susceptibility
to nitrofurantoin of the parental strains and those of respective
rdxA mutants or between those of matched
metronidazole-sensitive and -resistant H. pylori isolates.
The regimen with metronidazole eradicated infection from all eight
SS1-infected mice and from one of eight mice inoculated with the
rdxA mutant (P Helicobacter pylori is a
gram-negative, microaerobic, spiral bacterium that colonizes the
stomachs of approximately one-half the world's population
(54). Infection with H. pylori is associated with
chronic gastritis and peptic ulceration, and the bacterium is also
considered a risk factor for the development of gastric adenocarcinoma
and mucosa-associated lymphoid tissue lymphoma (3, 41, 42).
Although the 5-nitroimidazole metronidazole is an important component
of many currently used H. pylori eradication regimens,
resistance to this class of antibiotics is relatively common. It has
been estimated that 10 to 30% of clinical strains isolated in western
Europe and the United States are metronidazole resistant, and this
prevalence is far higher in developing countries and in certain
immigrant populations (11, 13). Although there have been
conflicting reports concerning the clinical impact of metronidazole
resistance in H. pylori, many studies have now demonstrated that resistance to the 5-nitroimidazoles reduces the efficacy of
eradication regimens involving metronidazole and is therefore an
important predictor of treatment failure (5, 23, 27, 43,
51). Several reports also suggest that the prevalence of
metronidazole resistance is rising and is likely to become an
increasingly important problem in the clinical management of H. pylori infection (34, 52). This, combined with the
expense of currently used antimicrobial regimens, means that there is a
need to evaluate alternative antibiotics for combination therapy of
H. pylori infections. We have previously used the H. pylori SS1 mouse model to characterize the evolution of
metronidazole resistance by H. pylori in vivo and to examine
the contribution of underlying resistance mechanisms (26,
27). This model system may also be used to assess the efficacy of
novel anti-H. pylori agents in vivo and to determine optimal
regimens for the eradication of resistant strains.
Recently it was demonstrated that loss of oxygen-insensitive NADPH
nitroreductase activity resulted in the development of resistance to
metronidazole in H. pylori (18). It was proposed that this enzyme reduces the nitro group of metronidazole to active metabolites that are toxic to the bacterium and that resistance arose
from mutational inactivation of the underlying gene, rdxA (HP0954 in the H. pylori genome database
[49]). Subsequent studies have suggested that, while
the development of metronidazole resistance in H. pylori is
highly associated with mutational inactivation of the rdxA
gene, other mechanisms of resistance are likely to exist in this
bacterium (26, 48; D. H. Kwon, D. Y. Graham, and F. A. K. El-Zaatari, Gut 43(Suppl.
2):A6).
The nitrofuran group of compounds, which includes furazolidone and
nitrofurantoin, appears a particularly promising source of alternative
agents for metronidazole in H. pylori eradication regimens
(7, 46, 57). Like that of the 5-nitroimidazoles, the
biological activity of these nitroaromatic compounds is largely derived
from reductive metabolism of the parent compound's nitro moiety by
oxygen-insensitive nitroreductases (2, 36, 55). A number of
recent clinical trials have demonstrated that short-term triple
therapies with furazolidone are effective in the treatment of H. pylori infection (10, 33, 46, 57). Based on
nitrofurantoin's in vitro activity, there is evidence that it might
also be a suitable alternative agent in combination antimicrobial
therapy, particularly against metronidazole-resistant H. pylori strains (7). Although nitrofurantoin has failed
to eradicate H. pylori in a limited number of clinical
trials, in all cases the agent was given either as a monotherapy or in
combination with bismuth subsalicylate and not as part of a
triple-agent regimen (4, 21, 24, 39, 44).
The aims of this study were to (i) determine whether the
rdxA gene of H. pylori is responsible for
reductive activation of nitrofurantoin, (ii) evaluate the in vitro
activity of nitrofurantoin against a series of matched
metronidazole-sensitive and -resistant H. pylori isolates,
and (iii) determine the efficacy of a triple-therapy regimen with
nitrofurantoin in eradicating established metronidazole-sensitive and
-resistant H. pylori infections from mice.
Bacteria and growth conditions.
Escherichia coli
strain MC1061 (6) was used as the host for plasmid cloning
experiments and was grown at 37°C in L broth (10 g of tryptone,
5 g of yeast extract, and 5 g of NaCl per liter, pH 7.0) or
on L agar plates (1.5% agar) at 37°C. Antibiotics were used at the
following final concentrations: 100 µg of spectinomycin (Upjohn
Laboratories, Paris, France) and 25 µg of kanamycin (Serva, Frankfurt, Germany)/ml.
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Evaluation of Nitrofurantoin Combination Therapy of
Metronidazole-Sensitive and -Resistant Helicobacter pylori
Infections in Mice
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
0.001). The regimen with
nitrofurantoin failed to eradicate infection from any of the six
SS1-infected mice (P 0.001) and cleared infection
from one of seven mice inoculated with the rdxA mutant.
These results demonstrate that, despite the good in vitro activity of
nitrofurantoin against H. pylori and the lack of
cross-resistance between metronidazole and nitrofurantoin, eradication
regimens involving nitrofurantoin are unable to eradicate either
metronidazole-sensitive or -resistant H. pylori infections
from mice.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
H. pylori strains used in this study
General molecular-biology techniques and construction of a defined mutation in the H. pylori rdxA gene. The alkaline lysis procedure was used for small-scale plasmid preparation (45). MIDI columns (Qiagen, Courtaboeuf, France) were used for large-scale plasmid preparation. Genomic DNA from individual H. pylori strains was extracted using the QIAamp tissue kit (Qiagen) according to the manufacturer's instructions. Standard procedures for cloning and DNA analysis were used (45).
H. pylori strains with a defined mutation in the rdxA gene were generated by allelic exchange. For this purpose, a recombinant plasmid was constructed in E. coli MC1061 as follows. Oligonucleotide primers were designed to amplify a fragment of 510 bp from the 5' end (HP0954-1 and HP0954-2) and 490 bp from the 3' end (HP0954-3 and HP094-4) of the rdxA gene (Table 2). The two generated fragments were restricted with EcoRI and BamHI and with PstI and BamHI, respectively, and were cloned simultaneously into the plasmid vector pILL570-1 (30) linearized with EcoRI and PstI. The resulting plasmid was restricted with BamHI, and the BamHI-digested kanamycin cassette from pILL600 (31) was introduced to generate the final construct.
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Susceptibility testing.
Susceptibility to metronidazole and
nitrofurantoin was assessed by agar dilution determination of the MIC.
Inoculates yielding 104 CFU/spot were inoculated onto
plates of IsoSensitest agar (Oxoid) enriched with 10% horse blood
containing doubling dilutions of metronidazole or nitrofurantoin. The
MIC was defined as the lowest concentration of antibiotic inhibiting
growth when the plates were read after 72 h of incubation under
microaerobic conditions (generated as described above) at 37°C.
Isolates were considered resistant to nitrofurantoin or metronidazole
if the MIC of either was 
8 µg/ml (56).
Infection of mice with H. pylori SS1. 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 inoculated intragastrically with a suspension of either H. pylori SS1 (n = 24; Table 4) or the rdxA mutant, SS1-11 (n = 25), which had been harvested directly from 48-h plate cultures into peptone-trypsin broth (Organotéchnique, La Courneuve, France). SS1-11 is a mouse-derived H. pylori SS1 isolate that is resistant to metronidazole (MIC of 32 µg/ml) and whose rdxA gene contains frameshift mutations at positions 90 and 159, resulting in the creation of two translational stop codons within the gene (26, 27). Each animal was administered a single 100-µl aliquot of an inoculating suspension of 105 CFU/ml (equivalent to 100 times the 100% infectious dose [14]) on two consecutive days. This was administered with polyethylene catheters (Biotrol, Paris, France) attached to 1-ml disposable syringes. A control group of mice (n = 10) was given peptone-trypsin broth alone.
Antimicrobial chemotherapy. Mice were administered antimicrobial chemotherapy 7 weeks after infection (Table 4). All solutions were administered intragastrically in a final volume of 100 µl via polyethylene catheters as previously described. The H. pylori SS1-colonized mice in group 1 (n = 10) and the H. pylori SS1-11-colonized mice in group 2 (n = 10) were treated for 7 days with peptone-trypsin broth. The H. pylori SS1-colonized mice in group 3 (n = 8) and the H. pylori SS1-11-colonized mice in group 4 (n = 8) were treated for 7 days with the mouse body weight equivalent of a recommended H. pylori eradication regimen of 20 mg of omeprazole (0.0086 mg; Astra Hassle AB, Mölndal, Sweden), 250 mg of clarithromycin (0.107 mg; Abbott Laboratories, Saint-Rémy-sur-Avre, France), and 400 mg of metronidazole (0.171 mg; Rhône-Poulenc Rorer, Vitry sur Seine, France) twice daily for 1 week (12). The H. pylori SS1-colonized mice in group 5 (n = 6) and the H. pylori SS1-11-colonized mice in group 6 (n = 7) were treated for 7 days with the mouse body weight equivalent of 20 mg of omeprazole (0.0086 mg), 250 mg of clarithromycin (0.107 mg), and 200 mg of nitrofurantoin (0.086 mg; Proctor & Gamble Pharmaceuticals, Staines, United Kingdom) twice daily for 1 week.
Assessment of H. pylori infection in mice. Colonization with H. pylori was assessed 1 month after the completion of each treatment regimen as recommended by recent guidelines (56). The animals were sacrificed, the stomach of each mouse was removed, and serum was recovered in microtubes (Sarstedt France, Orsay, France). The presence of H. pylori infection was determined by biopsy urease, quantitative culture, and serology. Stomachs were washed in physiological buffered saline and divided longitudinally into tissue fragments so that each fragment contained the cardia, body, and antrum. For each stomach, one fragment was immediately placed in urea-indole medium and another was immediately placed in peptone-trypsin broth. The presence of urease activity in tissue fragments was detected in urea-indole medium incubated for 24 h at room temperature (14). For the performance of quantitative bacterial cultures on stomach samples, tissue fragments were homogenized in peptone-trypsin broth using disposable plastic grinders and tubes (PolyLabo, Strasbourg, France). The homogenates were serially diluted in sterile saline and plated directly onto blood and serum plates for enumeration and onto selective plates containing 8 µg of either metronidazole or nitrofurantoin/ml. To increase the sensitivity of detection of metronidazole- and nitrofurantoin-resistant strains, all colonies that grew on the two enumeration plates were pooled and subcultured onto plates containing 8 µg of either metronidazole or nitrofurantoin/ml, respectively. H. pylori colonies were identified using standard criteria and were enumerated as described above.
Serum samples were tested for the H. pylori antigen-specific immunoglobulin antibody by a previously described enzyme-linked immunosorbent assay technique (14). Briefly, 96-well Maxisorb plates (Nunc, Kamstrup, Denmark) were coated with 25 µg of a sonicated whole-cell extract of H. pylori SS1. Serum samples were diluted 1:100 and were added in 100-µl aliquots to coated microtiter wells. To allow for nonspecific antibody binding, samples were also added to uncoated wells. Bound H. pylori-specific antibodies were detected by using biotinylated goat anti-mouse immunoglobulin and streptavidin-peroxidase conjugate (Amersham, Les Ulis, France). The readings for uncoated wells were subtracted from those for the respective test samples. A cutoff value was determined from the mean optical density value ± 2 standard deviations for the corresponding samples from naive uninfected mice. Samples with optical density readings greater than this cutoff value were considered positive for H. pylori-specific antibodies.Statistical analysis.
Differences in the eradication rates
between the groups of mice were determined by Fisher's exact
probability test. Differences in bacterial loads were determined by the
Mann-Whitney U test (two-sided). A P value of 0.05 was
considered significant.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Construction of the H. pylori rdxA mutant.
In
order to determine whether the oxygen-insensitive NADPH nitroreductase
of H. pylori, RdxA, was responsible for susceptibility to
nitrofurantoin as well as metronidazole, an isogenic mutant in the
rdxA gene was constructed in three strains. To do this, a
plasmid with the aphA3 kanamycin resistance gene
(50) inserted in rdxA was constructed in E. coli. H. pylori rdxA mutants derived from strains SS1, G27, and
HAS-141 were then produced by allelic exchange following natural
transformation with a concentrated preparation of the recombinant
plasmid. The genotypes of the constructed mutants were verified by
performing PCR with primers that flanked the point of insertion of the
antibiotic resistance cassette (HP0954-1 and HP0954-4). The PCR
products obtained with genomic DNA of these strains were of the correct
size and consistent with insertion of the kanamycin cassette (1,400 bp)
and an engineered deletion of 114 bp within rdxA: for
parental strains SS1, G27, and HAS-141, 1,114 bp; for mutant strains
SS1-rdxA, G27-rdxA, and HAS-141-rdxA, 2,400 bp (Fig. 1).
|
In vitro activity of nitrofurantoin against metronidazole-sensitive
and -resistant H. pylori.
To determine if there was
cross-resistance between nitrofurantoin and metronidazole in H. pylori, the MICs of nitrofurantoin and metronidazole for the
rdxA mutants and a series of matched metronidazole-sensitive
and -resistant strains were determined using an agar dilution method
(Table 3). The metronidazole MICs for
rdxA mutant strains SS1-rdxA,
G27-rdxA, and HAS-141-rdxA were significantly
higher than those for the respective parental strains (Table 3).
Although the MIC for SS1 rose from 0.0625 µg/ml for the parent strain
to 2 µg/ml for the mutant, the MIC for the mutant was not
sufficiently high for this strain to be considered resistant by
standard criteria (56). The susceptibilities to
nitrofurantoin of the rdxA mutants and respective parental
strains were identical (Table 3).
|
In vivo activity of nitrofurantoin against metronidazole-sensitive
and -resistant H. pylori.
In the control group, none of the
10 mice inoculated with peptone-trypsin broth were infected with
H. pylori 1 month after the completion of treatment (Table
4). In contrast, all 10 SS1-inoculated mice in group 1 that were treated with peptone-trypsin broth (Table 4)
were infected, with bacterial counts of between 2.2 × 104 and 7.0 × 106 CFU/g of tissue. In
group 2, quantitative cultures of gastric tissue samples taken from all
10 rdxA mutant-inoculated mice 1 month after completion of
treatment were positive for H. pylori (Table 4). The
bacterial counts obtained varied from 2.9 × 103 to
9.1 × 105 CFU/g of tissue. Although the bacterial
loads recovered from mice infected with the rdxA mutant
(group 2) were approximately 10-fold lower than those recovered from
mice infected with SS1, this difference was not statistically
significant.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Currently the most effective regimens for the eradication of H. pylori combine a proton pump inhibitor with two of the following antibiotics: metronidazole, clarithromycin, and amoxicillin (11, 12). It is, however, increasingly recognized that the rising prevalence of resistant H. pylori strains, particularly those resistant to metronidazole, threatens to compromise the efficacy of these regimens. Although there has been controversy regarding the clinical relevance of metronidazole resistance, it is now generally accepted that there is a global decrease in the efficacies of treatment regimens involving metronidazole when strains are resistant to this agent (37). This problem has led to the evaluation of a number of compounds with properties similar to those of metronidazole but without the problems of resistance (7, 33, 38). Like that of metronidazole, the bactericidal mechanism of action of the nitrofurans involves enzymatic reduction of the parent compound to generate electrophilic radicals (2, 36). These compounds have good in vitro activity against H. pylori (16, 40, 47), and antimicrobial combinations that included nitrofurantoin have been shown to have a greater in vitro bactericidal effect against a metronidazole-sensitive and a metronidazole-resistant strain of H. pylori than those with metronidazole (7). In addition, H. pylori does not appear to readily acquire resistance to this group of antimicrobial agents (22).
In E. coli, resistance to the nitrofuran derivatives occurs in a stepwise manner and results from mutations in genes encoding oxygen-insensitive nitroreductases (nfsA and nfsB) (35). First-step resistance results from an nfsA mutation, while the increased resistance associated with second-step mutants is due to mutation of nfsB (55). While there is no homolog of NfsA in the genome sequences of H. pylori, NfsB has 22.9% amino acid sequence identity with RdxA, the nitroreductase responsible for reductive activation of metronidazole in H. pylori (1, 18, 49, 55). The identity between NfsB and RdxA is particularly high (71%) in a conserved 14-residue region corresponding to Ser-37 to Val-50 (1, 49, 55). As well as being responsible for susceptibility to metronidazole, the activity of the oxygen-insensitive NADPH nitroreductase encoded by the rdxA gene might also be associated with reduction of, and hence susceptibility to, nitrofurantoin in H. pylori. In order to test this hypothesis, we constructed an isogenic rdxA deletion mutant from three different strains of H. pylori. Although the metronidazole MICs for strains SS1, G27, and HAS-141 carrying the mutant rdxA were significantly higher than those for the respective parental strains, the MIC for mutant SS1-rdxA was not sufficiently raised for this strain to be considered resistant by standard criteria (56). This observation is unlikely to result from residual RdxA activity and provides indirect evidence for additional mechanisms of metronidazole resistance in H. pylori which may increase the degree of resistance in an additive, stepwise fashion. The nitrofurantoin MICs for the rdxA mutants and respective parental strains were identical, suggesting that this enzyme is not responsible for the reductive activation of nitrofurantoin in H. pylori and that inactivation of rdxA does not result in resistance to this antimicrobial agent. Whether nitrofurantoin is reduced by one of the other putative nitroreductases identified in H. pylori remains to be determined. Alternatively, the mechanism of action in this organism may not require production of reactive nitrofurantoin metabolites by a bacterial reductase.
To confirm these observations and to determine whether other mechanisms of cross-resistance to metronidazole and nitrofurantoin might exist in H. pylori, we examined the MICs of metronidazole and nitrofurantoin for a series of well-characterized strains of H. pylori. These included a group of 10 metronidazole-sensitive and 20 metronidazole-resistant isolates generated in vivo by treating mice infected with the metronidazole-sensitive SS1 H. pylori strain with various regimens involving metronidazole (27). Of the 20 resistant isolates, 9 were known to contain mutations within the rdxA gene. In one, the rdxA gene was intact, suggesting that other mechanisms were responsible for the resistant phenotype of this isolate (26). In addition a series of 10 paired metronidazole-sensitive and -resistant clinical strains (48) were also tested for susceptibility to nitrofurantoin. The nitrofurantoin MICs for all strains tested were within a range (0.5 to 4 µg/ml) that would be considered susceptible for a comparative antimicrobial, such as metronidazole. There were no significant differences between the nitrofurantoin MICs for the metronidazole-sensitive and -resistant SS1 isolates, regardless of whether the rdxA gene was intact or not, or between MICs for the sensitive and resistant isolates of each individual pair of clinical strains. These data suggest that nitrofurantoin has comparable in vitro activities against metronidazole-sensitive and -resistant strains of H. pylori and that there is no cross-resistance between metronidazole and nitrofurantoin in this organism.
Clinical trials have demonstrated that triple therapies involving furazolidone (including omeprazole, clarithromycin, and furazolidone regimens) are able to achieve a high cure rate of H. pylori, and such regimens may prove particularly useful in areas where the prevalence of metronidazole-resistant strains is high (10, 33, 46, 57). The clinical evaluation of nitrofurantoin has been limited to studies in which the agent was given either as monotherapy or in combination with bismuth subsalicylate and not as part of a triple-agent regimen (4, 21, 24, 39, 44). Similarly, an assessment of the ability of various antimicrobial agents to eradicate H. pylori-infected gnotobiotic piglets only examined nitrofurantoin monotherapy (29). We therefore wanted to compare the abilities of two regimens to eradicate metronidazole-sensitive and -resistant strains from mice: the first regimen was a standard triple therapy involving metronidazole (12), and the second was the same regimen with nitrofurantoin substituted for metronidazole. To establish the infections, mice were inoculated with H. pylori SS1 and an SS1-derived rdxA mutant (27). Although the bacterial loads recovered from mice infected with the rdxA mutant were approximately 10-fold lower than those from mice infected with SS1, this difference was not statistically significant. The apparent ability of the rdxA mutant to colonize mice at levels similar to those for the parental strain was observed despite a reported decreased fitness of the mutant in the stationary phase of in vitro growth (J. Y. Jeong, W. W. Su, P. S. Hoffmann, and D. E. Berg, Abstr. 99th Gen. Meet. Am. Soc. Microbiol., abstr. D/B-182, 1999).
In previous work, we have demonstrated that prior exposure of H. pylori to metronidazole had a considerable negative influence on eradication of the organism by a regimen involving metronidazole (27). In this study, the efficacy of the regimen with metronidazole was significantly reduced in mice infected with the metronidazole-resistant rdxA mutant (eradicated in one of eight mice) compared to its efficacy in mice infected with the susceptible SS1 strain (eradicated in all mice). The magnitude of this effect is likely to reflect the fact that the stomachs of the rdxA mutant-infected mice contained a population of bacteria entirely made up of resistant isolates rather than the mixed population of sensitive and resistant strains that is frequently observed in clinical practice (20, 28). This observation provides compelling evidence for the role of metronidazole resistance in determining the successful outcome of regimens with metronidazole. When metronidazole was replaced by nitrofurantoin, the regimen failed to eradicate infection from any of the SS1-inoculated mice and eradicated infection in only one of seven mice inoculated with the rdxA mutant. Nitrofurantoin therefore appears to be similar to a number of antimicrobial agents that have been found to be ineffective in eradicating H. pylori in clinical practice despite good in vitro activity (19). The failure of the regimen with nitrofurantoin to eradicate H. pylori may be due to poor delivery of the drug, resulting in an insufficient concentration of the antimicrobial to exert an effective antibacterial activity in vivo (17). It is also possible that therapy was unsuccessful because of intrinsic differences between the activity of nitrofurantoin in mice and in humans. However, the limited data available suggest that nitrofurans are capable of therapeutic activity in murine models of infection (9, 15). Alternatively, it is possible that H. pylori has a low level of metabolic activity within the stomach and is thus relatively resistant to certain bactericidal agents.
These data demonstrate that, despite good in vitro activity and lack of induction of resistance (and particularly cross-resistance to metronidazole), nitrofurantoin is unable to eradicate H. pylori from mice when included as a component of a triple-therapy regimen. The H. pylori SS1 mouse model appears to be a suitable system for assessing novel anti-H. pylori agents and for determining the ability of new regimens to eradicate resistant strains.
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ACKNOWLEDGMENTS |
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P. J. Jenks was supported by a Research Training Fellowship in Medical Microbiology from the Wellcome Trust, United Kingdom (Ref. 044330). Financial support was provided in part by Pasteur-Mérieux-Connaught (Lyon, France) and OraVax Inc. (Boston, Mass.).
We are grateful to Proctor & Gamble Pharmaceuticals, Rhône-Poulenc Rorer, Astra Hassle AB, and Abbott Laboratories for the gift of the pharmaceutical agents used in the treatment protocols.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Medical Microbiology, Royal Free and University College Medical School, Royal Free Campus, Rowland Hill St., London NW3 2PF, United Kingdom. Phone: 44-20-77940500, ext. 4111. Fax: 44-20-77940433. E-mail: pjenks2{at}hotmail.com.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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| 1. | Alm, R. A., L.-S. L. Ling, D. T. Moir, B. L. King, E. D. Brown, P. C. Doig, D. R. Smith, B. Noonan, B. C. Guild, B. L. deJonge, G. Carmel, P. J. Tummino, A. Caruso, M. Uria-Nickelsen, D. M. Mills, C. Ives, R. Gibson, D. Merberg, S. D. Mills, Q. Jiang, D. E. Taylor, G. F. Vovis, and T. J. Trust. 1999. Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature 397:176-180[CrossRef][Medline]. |
| 2. | Asnis, R. E. 1957. The reduction of furacin by cell-free extracts of furacin-resistant and parent-susceptible strains of Escherichia coli. Arch. Biochem. Biophys. 66:208-216. |
| 3. | Blaser, M. J. 1993. Helicobacter pylori: microbiology of a `slow' bacterial infection. Trends Microbiol. 1:255-260[CrossRef][Medline]. |
| 4. | Börsch, G., U. Mai, and K. M. Müller. 1988. Monotherapy or polychemotherapy in the treatment of Campylobacter pylori-related gastroduodenal disease. Scand. J. Gastroenterol. 23:101-106. |
| 5. | Buckley, M. J. M., H. X. Xia, D. M. Hyde, C. T. Keane, and C. A. O'Morain. 1997. Metronidazole resistance reduces efficacy of triple therapy and leads to secondary clarithromycin resistance. Dig. Dis. Sci. 42:2111-2115[CrossRef][Medline]. |
| 6. | Casadaban, M., and S. N. Cohen. 1980. Analysis of gene control signals by DNA fusions and cloning in E. coli. J. Mol. Biol. 138:179-207[CrossRef][Medline]. |
| 7. |
Coudron, P. E., and C. W. Stratton.
1998.
In-vitro evaluation of nitrofurantoin as an alternative agent for metronidazole in combination antimicrobial therapy against Helicobacter pylori.
J. Antimicrob. Chemother.
42:657-660 |
| 8. |
Covacci, A.,
S. Censini,
M. Bugnoli,
R. Petracca,
D. Burroni,
G. Macchia,
A. Massone,
E. Papini,
Z. Xiang,
N. Figura, and R. Rappuoli.
1993.
Molecular characterization of the 128-kDa immunodominant antigen of Helicobacter pylori associated with cytotoxicity and duodenal ulcer.
Proc. Natl. Acad. Sci. USA
90:5791-5795 |
| 9. | Cruz, C. C., L. Ferrari, and R. Sogayar. 1997. A therapeutic trial in Giardia muris infection in the mouse with metronidazole, tinidazole, secnidazole and furazolidone. Rev. Soc. Bras. Med. Trop. 30:223-228[Medline]. |
| 10. | Dani, R., D. M. Queiroz, M. G. Dias, J. M. Franco, L. C. Magalhaes, G. S. Mendes, L. S. Moreira, L. D. Castro, N. H. Toppa, G. A. Rocha, M. M. Cabral, and P. G. Salles. 1999. Omeprazole, clarithromycin and furazolidone for the eradication of Helicobacter pylori in patients with duodenal ulcer. Aliment. Pharmacol. Ther. 13:1647-1652[CrossRef][Medline]. |
| 11. | Dunn, B. E., H. Cohen, and M. J. Blaser. 1997. Helicobacter pylori. Clin. Microbiol. Rev. 10:720-741[Abstract]. |
| 12. |
European Helicobacter pylori Study Group.
1997.
Current European concepts in the management of Helicobacter pylori infection. The Maastricht Consensus Report.
Gut
41:8-13 |
| 13. | European Study Group on Antibiotic Susceptibility of Helicobacter pylori. 1992. Results of a multicentre European survey in 1991 of metronidazole resistance in Helicobacter pylori. Eur. J. Clin. Microbiol. Infect. Dis. 11:777-781[CrossRef][Medline]. |
| 14. |
Ferrero, R. L.,
J. M. Thiberge,
M. Huerre, and A. Labigne.
1998.
Immune responses of specific-pathogen-free mice to chronic Helicobacter pylori (strain SS1) infection.
Infect. Immun.
66:1349-1355 |
| 15. | Foster, R., G. Pringle, D. F. King, and J. Paris. 1969. The therapeutic activity of some nitrofurans in experimental filariasis and trypanosomiasis. Ann. Trop. Med. Parasitol. 63:95-107[Medline]. |
| 16. | Glupczynski, Y., M. Delmee, C. Bruck, M. Labbe, V. Avesani, and A. Burette. 1988. Susceptibility of clinical isolates of Campylobacter pylori to 24 antimicrobial and anti-ulcer agents. Eur. J. Epidemiol. 4:154-157[CrossRef][Medline]. |
| 17. |
Goddard, A. F.
1998.
Getting to the route of Helicobacter pylori treatment.
J. Antimicrob. Chemother.
42:1-3 |
| 18. | Goodwin, A., D. Kersulyte, G. Sisson, S. J. O. V. van Zanten, D. E. Berg, and P. S. Hoffman. 1998. Metronidazole resistance in Helicobacter pylori is due to null mutations in a gene (rdxA) that encodes an oxygen-insensitive NADPH nitroreductase. Mol. Microbiol. 28:383-393[CrossRef][Medline]. |
| 19. | Goodwin, C. S. 1997. Antimicrobial treatment of Helicobacter pylori infection. Clin. Infect. Dis. 25:1023-1026[Medline]. |
| 20. | Graham, D. Y., W. A. de Boer, and G. N. Tytgat. 1996. Choosing the best anti-Helicobacter pylori therapy: effect of antimicrobial resistance. Am. J. Gastroenterol. 91:1072-1076[Medline]. |
| 21. | Graham, D. Y., P. D. Klein, D. G. Evans, D. J. Evans, L. C. Alpert, A. Opekun, G. R. Jerdack, and D. R. Morgan. 1991. Simple noninvasive method to test efficacy of drugs in the eradication of Helicobacter pylori infection: the example of combined bismuth subsalicylate and nitrofurantoin. Am. J. Gastroenterol. 86:1158-1162[Medline]. |
| 22. |
Haas, C. E.,
D. E. Nix, and J. J. Schentag.
1990.
In vitro selection of resistant Helicobacter pylori.
Antimicrob. Agents Chemother.
34:1637-1641 |
| 23. | Harris, A. W., D. I. Pryce, S. M. Gabe, Q. N. Karim, M. M. Walker, H. Langworthy, J. H. Baron, and J. J. Misiewicz. 1996. Lansoprazole, clarithromycin and metronidazole for seven days in Helicobacter pylori infection. Aliment. Pharmacol. Ther. 10:1005-1008[CrossRef][Medline]. |
| 24. | Hunter, F. M., P. Correa, E. Fontham, B. Ruiz, M. Sobhan, and I. M. Samloff. 1993. Serum pepsinogens as markers of response to therapy for Helicobacter pylori gastritis. Dig. Dis. Sci. 38:2081-2086[CrossRef][Medline]. |
| 25. | Janvier, B., B. Grignon, C. Audibert, L. Pezennec, and J. L. Fauchère. 1999. Phenotypic changes of Helicobacter pylori components during experimental infection in mice. FEMS Immunol. Med. Microbiol. 24:27-33[CrossRef][Medline]. |
| 26. |
Jenks, P. J.,
R. L. Ferrero, and A. Labigne.
1999.
The role of the rdxA gene in the evolution of metronidazole resistance in Helicobacter pylori.
J. Antimicrob. Chemother.
43:753-758 |
| 27. |
Jenks, P. J.,
A. Labigne, and R. L. Ferrero.
1999.
Exposure to metronidazole in vivo readily induces resistance in Helicobacter pylori and reduces the efficacy of eradication therapy in mice.
Antimicrob. Agents Chemother.
43:777-781 |
| 28. | Jorgensen, M., G. Daskalopoulos, V. Warburton, H. M. Mitchell, and S. L. Hazell. 1996. Multiple strain colonization and metronidazole resistance in Helicobacter pylori-infected patients: identification from sequential and multiple biopsy specimens. J. Infect. Dis. 174:631-635[Medline]. |
| 29. |
Krakowka, S.,
K. A. Eaton, and R. D. Leunk.
1998.
Antimicrobial therapies for Helicobacter pylori infection in gnotobiotic piglets.
Antimicrob. Agents Chemother.
42:1549-1554 |
| 30. |
Labigne, A.,
V. Cussac, and P. Courcoux.
1991.
Shuttle cloning and nucleotide sequence of Helicobacter pylori genes responsible for urease activity.
J. Bacteriol.
173:1920-1931 |
| 31. |
Labigne-Roussel, A.,
P. Courcoux, and L. Tompkins.
1988.
Gene disruption and replacement as a feasible approach for mutagenesis of Campylobacter jejuni.
J. Bacteriol.
170:1704-1708 |
| 32. | Lee, A., J. O'Rourke, M. C. De Ungria, B. Robertson, G. Daskalopoulos, and M. F. Dixon. 1997. A standardized mouse model of Helicobacter pylori infection: introducing the Sydney strain. Gastroenterology 112:1386-1397[CrossRef][Medline]. |
| 33. | Liu, W. Z., S. D. Xiao, Y. Shi, S. M. Wu, D. Z. Zhang, W. W. Xu, and G. N. Tytgat. 1999. Furazolidone-containing short-term therapies are effective in the treatment of Helicobacter pylori infection. Aliment. Pharmacol. Ther. 13:317-322[CrossRef][Medline]. |
| 34. |
Lopez-Brea, M.,
D. Domingo,
I. Sanchez, and T. Alarcon.
1997.
Evolution of resistance to metronidazole and clarithromycin in Helicobacter pylori clinical isolates from Spain.
J. Antimicrob. Chemother.
40:279-281 |
| 35. |
McCalla, D. R.,
C. Kaiser, and M. H. L. Green.
1978.
Genetics of nitrofurazone resistance in Escherichia coli.
J. Bacteriol.
133:10-16 |
| 36. | McOsker, C. C., and P. M. Fitzpatrick. 1994. Nitrofurantoin: mechanism of action and implications for resistance development in common uropathogens. J. Antimicrob. Chemother. 33(Suppl. A):23-30. |
| 37. |
Mégraud, F., and H. P. Doermann.
1998.
Clinical relevance of resistant strains of Helicobacter pylori: a review of current data.
Gut
43(Suppl. 1):S61-S65 |
| 38. |
Mégraud, F.,
A. Occhialini, and J. F. Rossignol.
1998.
Nitazoxanide, a potential drug for eradication of Helicobacter pylori with no cross-resistance to metronidazole.
Antimicrob. Agents Chemother.
42:2836-2840 |
| 39. | Morgan, D., W. Kraft, M. Bender, and A. Pearson. 1988. Nitrofurans in the treatment of gastritis associated with Campylobacter pylori. Gastroenterology 95:1178-1184[Medline]. |
| 40. | Morgan, D. R., P. M. Fitzpatrick, K. L. David, and W. G. Kraft. 1987. Susceptibility patterns of Campylobacter pyloridis. FEMS Microbiol. Lett. 42:245-248. |
| 41. | Parsonnet, J., G. D. Friedman, D. P. Vandersteed, Y. Chang, J. H. Vogelman, N. Orentreich, and R. K. Sibley. 1991. Helicobacter pylori infection and the risk of gastric cancer. N. Engl. J. Med. 325:1127-1129[Abstract]. |
| 42. |
Parsonnet, J.,
S. Hansen,
L. Rodriguez,
A. B. Gelb,
R. A. Warnke,
E. Jellum,
N. Orentreich,
J. H. Vogelman, and G. D. Friedman.
1994.
Helicobacter pylori infection and gastric lymphoma.
N. Engl. J. Med.
330:1267-1271 |
| 43. |
Rautelin, H.,
K. Seppala,
O. V. Renkonen,
U. Vainio, and T. U. Kosunen.
1992.
Role of metronidazole in therapy of Helicobacter pylori infections.
Antimicrob. Agents Chemother.
36:163-166 |
| 44. | Ruiz, B., J. C. Rood, E. T. H. Fontham, G. T. Malcom, F. M. Hunter, M. Sobhan, W. D. Johnson, and P. Correa. 1994. Vitamin C concentration in gastric juice before and after anti-Helicobacter pylori treatment. Am. J. Gastroenterol. 89:533-539[Medline]. |
| 45. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 46. | Segura, A. M., O. Gutierrez, W. Otero, A. Angel, R. M. Genta, and D. Y. Graham. 1997. Furazolidone, amoxycillin, bismuth triple therapy for Helicobacter pylori infection. Aliment. Pharmacol. Ther. 11:529-532[CrossRef][Medline]. |
| 47. |
Simor, A. E.,
S. Ferro, and D. E. Low.
1989.
Comparative in vitro activities of six new fluoroquinolones and other oral antimicrobial agents against Campylobacter pylori.
Antimicrob. Agents Chemother.
33:108-109 |
| 48. |
Tankovic, J.,
D. Lamarque,
J. C. Delchier,
C. J. Soussy,
A. Labigne, and P. J. Jenks.
2000.
Frequent association between alteration of the rdxA gene and metronidazole resistance in French and North African isolates of Helicobacter pylori.
Antimicrob. Agents Chemother.
44:608-613 |
| 49. | Tomb, J. F., O. White, A. R. Kervalage, R. A. Clayton, G. G. Sutton, R. D. Fleischmann, K. A. Ketchum, H. P. Klenk, S. Gill, B. A. Dougherty, K. Nelson, J. Quackenbush, L. Zhou, E. F. Kirkness, S. Peterson, B. Loftus, D. Richardson, R. Dodson, H. G. Khalak, A. Glodek, K. McKenney, L. M. Fitzegerald, N. Lee, M. D. Adams, E. K. Hickey, D. E. Berg, J. D. Gocayne, T. R. Utterback, J. D. Peterson, J. M. Kelley, M. D. Cotton, J. M. Weidman, C. Fujii, C. Bowman, L. Watthey, E. Wallin, W. S. Hayes, M. Borodovsky, P. D. Karp, H. O. Smith, C. M. Fraser, and J. C. Ventor. 1997. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388:539-547[CrossRef][Medline]. |
| 50. | Trieu-Cout, P., G. Gerbaud, T. Lambert, and P. Courvalin. 1985. In vivo transfer of genetic information between gram-positive and gram-negative bacteria. EMBO J. 4:3583-3587[Medline]. |
| 51. |
van de Hulst, R. W. M.,
A. van der Ende,
A. Homan,
P. Roorda,
J. Dankert, and G. N. J. Tytgat.
1998.
Influence of metronidazole resistance on efficacy of quadruple therapy for Helicobacter pylori eradication.
Gut
42:166-169 |
| 52. | van der Wouden, E. J., M. van Zwet, G. D. Vosmaer, J. A. Oom, A. de Jong, and J. H. Kleibeuker. 1997. Rapid increase in the prevalence of metronidazole-resistant Helicobacter pylori in the Netherlands. Emerg. Infect. Dis. 3:385-389[Medline]. |
| 53. | Wang, Y., K. P. Roos, and D. E. Taylor. 1993. Transformation of Helicobacter pylori by chromosomal metronidazole resistance and by a plasmid with a selectable chloramphenicol resistance marker. J. Gen. Microbiol. 39:2485-2493. |
| 54. | Warren, J. R. 1983. Unidentified curved bacilli on gastric epithelium in chronic active gastritis. Lancet i:1273. |
| 55. |
Whiteway, J.,
P. Koziarz,
J. Veall,
N. Sandhu,
P. Kumar,
B. Hoecher, and I. B. Lambert.
1998.
Oxygen-insensitive nitroreductases: analysis of the roles of nfsA and nfsB in development of resistance to 5-nitrofuran derivatives in Escherichia coli.
J. Bacteriol.
180:5529-5539 |
| 56. | Working Party of the European Helicobacter pylori Study Group. 1997. Guidelines for clinical trials in Helicobacter pylori infection. Gut 41(Suppl. 2):S1-S23. |
| 57. | Xiao, S. D., W. Z. Liu, P. J. Hu, D. H. Xia, and G. N. Tytgat. 1999. High cure rate of Helicobacter pylori infection using tripotassium bismuthate, furazolidone and clarithromycin triple therapy for 1 week. Aliment. Pharmacol. Ther. 13:311-315[CrossRef][Medline]. |
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