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Antimicrobial Agents and Chemotherapy, April 1999, p. 777-781, Vol. 43, No. 4
Unité de Pathogénie
Bactérienne des Muqueuses, Institut Pasteur, 75724 Paris
Cedex 15, France
Received 9 October 1998/Returned for modification 25 November
1998/Accepted 25 January 1999
The Helicobacter pylori SS1 mouse model was used to
characterize the development of resistance in H. pylori
after treatment with metronidazole monotherapy and to
examine the effect of prior exposure to metronidazole
on the efficacy of a metronidazole-containing eradication regimen. Mice colonized with the
metronidazole-sensitive H. pylori
SS1 strain were treated for 7 days with either peptone trypsin broth or
the mouse equivalent of 400 mg of metronidazole once a
day or three times per day (TID). In a separate experiment, H. pylori-infected mice were administered either
peptone trypsin broth or the mouse equivalent of 400 mg of
metronidazole TID for 7 days, followed 1 month later by
either peptone trypsin broth or the mouse equivalent of 20 mg
of omeprazole, 250 mg of clarithromycin, and 400 mg of
metronidazole twice a day for 7 days. At least 1 month
after the completion of treatment, the mice were sacrificed and their
stomachs were cultured for H. pylori. The
susceptibilities of isolates to metronidazole were
assessed by agar dilution determination of the MICs. Mixed
populations of metronidazole-resistant and -sensitive
strains were isolated from 70% of mice treated with 400 mg of
metronidazole TID. The ratio of resistant to sensitive strains was 1:100, and the MICs for the resistant strains varied from 8 to 64 µg/ml. In the second experiment, H. pylori was
eradicated from 70% of mice treated with eradication therapy alone,
compared to 25% of mice pretreated with metronidazole
(P < 0.01). Mice still infected after
treatment with metronidazole and eradication therapy
contained mixed populations of metronidazole-resistant and
-sensitive isolates in a ratio of 1:25. These results demonstrate that
H. pylori readily acquires resistance to
metronidazole in vivo and that prior exposure of the
organism to metronidazole is associated with failure of
eradication therapy. H. pylori-infected mice provide a
suitable model for the study of resistance mechanisms in
H. pylori and will be useful in determining optimal
regimens for the eradication of resistant strains.
Helicobacter pylori is a
gram-negative, microaerobic, spiral bacterium that colonizes the
stomachs of approximately half the world's population (44).
Infection with H. pylori is associated with severe
gastrointestinal disease, including peptic ulceration, and
eradication of the organism from the stomach facilitates duodenal ulcer
healing and reduces ulcer relapse (16, 28). 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 (9, 11, 13, 36), and this prevalence is far higher
in developing countries and in certain immigrant populations (2,
9, 11, 13). Metronidazole is commonly used to treat anaerobic and
parasitic infections, and there is epidemiological evidence that
metronidazole resistance in H. pylori
is associated with prior use of this antibiotic in certain patient
groups (2, 4, 11, 13, 37). However, previous use of
metronidazole is frequently not reported by patients (11), and consequently the development of resistance in
H. pylori after metronidazole
monotherapy is poorly characterized.
The relationship between previous exposure to
metronidazole, the development of resistance to this
antibiotic, and the eventual outcome of H. pylori
eradication regimens remains unclear (30, 36). Many studies
have demonstrated that infection with
metronidazole-resistant strains is an important
predictor of failure of metronidazole-containing eradication regimens, even when quadruple therapy is used (3, 5-7, 17, 19, 22, 31, 34, 37, 40-42, 47). However, there is also
evidence that resistance to metronidazole can be partly
overcome when antisecretory drug-based triple or quadruple therapies are used, and such regimens may achieve eradication in around
75% of patients infected with resistant strains (3, 6, 7, 17, 19,
22, 30, 32, 37, 40, 41, 48).
The aims of this study were to use a well-validated mouse model of
H. pylori infection (24) to (i) characterize
the development of resistance in H. pylori after
treatment with metronidazole monotherapy at doses
normally used to treat anaerobic and parasitic infections and (ii)
examine the impact of prior exposure of H. pylori to
metronidazole on the efficacy of a
metronidazole-containing eradication regimen.
Bacteria and growth conditions.
H. pylori SS1 is
a mouse-adapted strain originally isolated from a patient with peptic
ulcer disease (24). H. pylori SS1 was
routinely cultured on a blood agar medium (Blood Agar Base no. 2;
Oxoid, Lyon, France) supplemented with 10% horse blood (bioMérieux, Marcy L'Etoile, France) and the following
antibiotics: 10 µg of vancomycin (Dakota Pharmaceuticals, Creteil,
France)/ml, 2.5 IU of polymyxin (Pfizer Laboratories, Orsay,
France)/liter, 5 µg of trimethoprim (Sigma Chemicals, Saint-Quentin
Fallavier, France)/ml, and 4 µg of amphotericin B (Bristol-Myers
Squibb, Paris, France)/ml. The plates were incubated at 37°C under
microaerobic conditions in an anaerobic jar (Oxoid) with a carbon
dioxide generator (CampyGen; Oxoid) without a catalyst. For the
selection of metronidazole-resistant colonies and their
subsequent subculture, the medium was additionally supplemented with
metronidazole at 8 µg/ml (Sigma).
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Exposure to Metronidazole In Vivo Readily Induces
Resistance in Helicobacter pylori and Reduces the
Efficacy of Eradication Therapy in Mice
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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 H. pylori SS1, which had been harvested directly from 48-h plate cultures into peptone trypsin broth (Organotéchnique, La Courneuve, France). 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 [12]). 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.
Three groups of mice were used in the treatment study. The mice in group 1 were used in a pilot study to determine if metronidazole resistance was induced in H. pylori after treatment with two different doses of metronidazole monotherapy. Group 2 animals were used to further characterize the acquisition of resistance after metronidazole monotherapy. The mice in group 3 were used to examine the effect of prior exposure to metronidazole on the efficacy of a metronidazole-containing H. pylori eradication regimen.Antimicrobial chemotherapy. All solutions were administered intragastrically in a final volume of 100 µl via polyethylene catheters as previously described. Seven weeks after infection, the H. pylori-colonized mice in group 1 were treated for 7 days either with peptone trypsin broth (n = 5) or 0.171 mg of metronidazole (Rhône-Poulenc Rorer, Vitry sur Seine, France) (n = 5) in a single daily dose or with peptone trypsin broth (n = 4) or 0.171 mg of metronidazole (n = 5) three times daily (TID) (see Table 1). The dose of 0.171 mg in a 30-g mouse is the body weight equivalent of a 400-mg dose in a 70-kg human. Fifteen weeks after infection, the H. pylori-colonized mice in group 2 were given either peptone trypsin broth (n = 10) or 0.171 mg of metronidazole TID for 7 days (n = 10) (see Table 1).
The H. pylori-colonized mice in group 3 were administered two treatment regimens (see Table 2). Treatment 1 was administered 15 weeks after infection and consisted of either peptone trypsin broth (n = 20) or 0.171 mg of metronidazole TID for 7 days (n = 17). Treatment 2 was administered 1 month after the completion of treatment 1 and consisted of either peptone trypsin broth (n = 19) or the mouse body weight equivalent of a recommended H. pylori eradication regimen of 20 mg of omeprazole (0.0086 mg; Astra Hassle AB, Molndal, Sweden), 250 mg of clarithromycin (0.107 mg; Abbott Laboratories, Saint-Rémy-sur-Avre, France), and 400 mg of metronidazole (0.171 mg) twice daily for 1 week (n = 18) (10).Assessment of H. pylori infection in mice. Colonization with H. pylori was assessed at least 1 month after the completion of each treatment regimen as recommended by recent guidelines (46). 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 cardia, body, and antrum. For each stomach, one fragment was immediately placed in urea-indole medium and another was 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 (12). For the performance of quantitative bacterial cultures on stomach samples, tissue fragments were homogenized in peptone trypsin broth by 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 a selective plate containing 8 µg of metronidazole/ml. To increase the sensitivity of detection of metronidazole-resistant strains, all colonies that grew on the two enumeration plates were pooled and subcultured onto plates containing 8 µg of metronidazole/ml. H. pylori colonies were identified according to standard criteria and enumerated as described above.
Serum samples were tested for H. pylori antigen-specific immunoglobulin G antibody by a previously described enzyme-linked immunosorbent assay technique (12). 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 of 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.Analysis of isolates.
Five colonies identified as
metronidazole-resistant H. pylori were
selected from each mouse for further analysis. The susceptibility to
metronidazole of each colony 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. The MIC was defined as the lowest
concentration of metronidazole 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 metronidazole if the MIC was
8 µg/ml (46). To assess if resistance to
metronidazole was stable in vitro, resistant strains
were subcultured three times on nonselective media before
redetermination of the MIC.
Intracage transmission of H. pylori. Two groups of animals were used in order to exclude the possibility of transmission of H. pylori between mice housed in the same cage. To study short-term transmission, two mice infected with H. pylori were kept in the same cage as six uninoculated mice for 9 weeks. In a further experiment, two mice infected with H. pylori were kept in the same cage as eight uninoculated mice for the period covered by the treatment studies (7 months).
Statistical analysis. Differences in the eradication rates between the groups of mice were determined by the chi-square test. A P value of <0.05 was considered significant.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Development of metronidazole resistance after
metronidazole monotherapy.
In group 1, quantitative culture of gastric tissue samples 1 month after completion
of treatment was positive for H. pylori in 18 of
19 mice (Table 1). The bacterial counts
obtained ranged from 2.0 × 103 to 1.8 × 106 CFU/g of tissue and were similar in mice treated with
peptone trypsin broth and those treated with
metronidazole. H. pylori was not
cultured from one mouse treated with metronidazole TID. The H. pylori-specific humoral response in this
mouse was of a magnitude similar to that in other infected mice,
suggesting that infection had been established and subsequently
eradicated by metronidazole in this animal. A mixed
population of metronidazole-resistant and
metronidazole-sensitive H. pylori
strains was isolated from one mouse treated with 0.171 mg of
metronidazole once daily and from two mice treated with
0.171 mg TID (Table 1). The MIC for all the resistant strains tested
from these three mice (n = 15) was 32 µg/ml (compared
to the MIC for the parental SS1 strain, which was 0.064 µg/ml).
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Prior exposure to metronidazole and the efficacy of
eradication therapy.
In group 3, none of the 10 mice inoculated
with peptone trypsin broth were infected with H. pylori
2 months after the completion of treatment 2 (Table
2). In contrast, quantitative culture of gastric tissue samples was positive for H. pylori in
all 10 SS1-inoculated mice that were treated twice with peptone trypsin
broth (Table 2), with bacterial counts of 3.1 × 104
to 1.0 × 107 CFU/g of tissue. No
metronidazole-resistant strains were isolated from
these mice. H. pylori was cultured from seven of nine
SS1-infected mice that received metronidazole
monotherapy followed by peptone trypsin broth (Table 2). Mixed
populations of metronidazole-resistant and
metronidazole-sensitive H. pylori was
isolated from six of these mice. The range of MICs for the resistant
strains tested (n = 30) was 16 to 32 µg/ml, and the
ratio of metronidazole-resistant to -sensitive
isolates was 1:100 (Table 2).
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Intracage transmission of H. pylori. Transmission from H. pylori-colonized mice to uninoculated mice was not observed. At 9 weeks and 7 months, the two infected mice were confirmed as colonized with H. pylori SS1 (by culture, biopsy urease, and serology), while the uninoculated mice remained uncolonized.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Acquired resistance of H. pylori to metronidazole and other 5-nitroimidazole drugs has been reported worldwide. There is epidemiological evidence suggesting that variation in the use of metronidazole is responsible for the uneven distribution of resistance among different populations. It has been proposed that the high level of resistance in developing countries is associated with the prior use of metronidazole to treat parasitic infections (2, 4, 11, 13). It has also been suggested that the higher prevalence of primary metronidazole resistance found in H. pylori isolated from female patients correlates with the use of metronidazole to treat bacterial vaginosis and other infections of the female genital tract (4, 11, 13, 37). However, many of these studies have been unable to demonstrate a direct association between prior use of metronidazole and the presence of resistant strains, presumably because such use often goes unrecognized by the patient (11, 13, 37). Although resistance to metronidazole in H. pylori has been shown to arise readily in vitro (18, 43), the development of resistance after metronidazole monotherapy in vivo remains poorly characterized. Given the potential implications of metronidazole resistance for the selection of optimal eradication regimens, this is an important phenomenon calling for further study.
The emergence of a single metronidazole-resistant
H. pylori strain after exposure to
metronidazole was recently reported in a euthymic mouse
model (29). However, the relevance of this model to human
infection can be questioned, particularly because only low levels of
colonization occur (24). In another study, metronidazole-resistant bacteria were isolated after
treatment of H. pylori-infected gnotobiotic
piglets (23). Although this model mimics many aspects
of human H. pylori disease, it is necessary, for
practical reasons, to use short study periods (3 weeks). In addition,
the parental H. pylori strain used to colonize piglets (26695) already possesses intermediate resistance to
metronidazole (MIC, 6.25 µg/ml; intermediate
resistance has been defined as a MIC of 4 to 8 µg/ml
[47]). It has been demonstrated that immunocompetent mice inoculated with H. pylori SS1 develop chronic
infection (16 months), with gastric bacterial loads and host
inflammatory responses similar to those found in humans (12,
24). We therefore selected the SS1 H. pylori
mouse model for the study of the emergence of acquired resistance to
metronidazole in this bacterium in vivo and for the
examination of the effect of this resistance on the efficacy of a
metronidazole-containing H. pylori
eradication regimen. All experimentation was performed on
long-term-colonized mice and, as far as was possible, in accordance
with recent guidelines for clinical trials in H. pylori
infection (46).
Using the H. pylori SS1 mouse model, we have demonstrated that H. pylori readily develops stable resistance to metronidazole in vivo after treatment with metronidazole monotherapy. In total, 73% of mice in groups 1 and 2 harbored resistant strains after receiving the TID treatment regimen. In a separate experiment, 67% of mice in group 3 that were treated with metronidazole TID followed by peptone trypsin broth developed resistant strains. The high numbers of mice colonized with resistant isolates cannot be explained by transmission of metronidazole-resistant strains between mice. We demonstrated that transmission of H. pylori SS1 does not occur between mice housed in the same cage, and similar results have recently been presented by other workers (26). These results provide direct evidence that exposure of a clonal population of H. pylori to doses of metronidazole normally used to treat parasitic and anaerobic bacterial infections does result in the emergence of resistance to this antibiotic. Moreover, the development of resistance appears to occur at a high frequency at the individual level. The fact that H. pylori survives beneath the mucous layer and in the gastric pits in both the human and murine stomachs may explain its propensity to develop resistance to metronidazole (21, 24). The antibiotic penetrates such sites poorly, exposing the bacterium to sublethal doses and encouraging the development of resistance.
The phenomenon of heteroresistance, in which susceptible and resistant bacteria may be isolated from the same patient, has previously been described for H. pylori (8, 21, 39, 45). Our study clearly demonstrates that a mixed population of sensitive and resistant bacteria may arise after exposure of a clonal, metronidazole-susceptible strain of H. pylori to either metronidazole monotherapy or a metronidazole-containing eradication regimen. Accurate determination of the proportion of resistant strains demonstrated that after metronidazole monotherapy, the ratio of metronidazole-resistant to metronidazole-sensitive isolates was 1 in 100. This ratio rose to 1 in 25 in mice treated with both metronidazole monotherapy and eradication treatment, indicating that repeated exposure to metronidazole in vivo selects for a resistant population of H. pylori in the stomach. It is recognized that current methods of susceptibility testing may underestimate the frequency of metronidazole resistance in patients coinfected with metronidazole-resistant and -sensitive H. pylori strains (8, 45). If heteroresistance after treatment with metronidazole is confirmed to occur frequently in H. pylori-infected patients, this may have important implications for the susceptibility testing of this organism.
We also observed that, despite the fact that they originated from the same metronidazole-sensitive parental strain, the degree of susceptibility to metronidazole of the emergent resistant isolates varied, with MICs ranging from 8 to 64 µg/ml. Recently, resistance to metronidazole in H. pylori was demonstrated to be associated with mutational inactivation of the rdxA gene, which encodes an oxygen-insensitive NADPH nitroreductase (14). This model will allow us to evaluate the contribution of rdxA to the development of resistance in these strains and to evaluate if other potential resistance mechanisms might exist in H. pylori.
Clinical trials of the efficacy of omeprazole, clarithromycin, and metronidazole have yielded conflicting results for the successful eradication of metronidazole-sensitive and -resistant strains. While a number of studies have demonstrated that eradication is significantly reduced for metronidazole-resistant strains (3, 19, 22, 27, 33, 35, 47), others have shown that this regimen is equally effective for both sensitive and resistant isolates (1, 25). In this study, prior exposure of H. pylori to metronidazole had a considerable negative influence on eradication; this is the first time that a direct link between prior exposure to metronidazole and the outcome of eradication therapy has been clearly demonstrated. The magnitude of this effect is likely to reflect the high frequency of resistance induced by the pretreatment of mice with metronidazole monotherapy and is consistent with the observation that the effectiveness of metronidazole-containing eradication regimens is dependent on the prevalence of metronidazole-resistant H. pylori in the population (17). In addition, secondary resistance to metronidazole, which is recognized to arise during the course of H. pylori eradication therapy (15, 20, 37, 38), was observed to arise in strains isolated from 20% of mice that received eradication therapy only.
These data establish the H. pylori SS1 mouse model as a suitable system for the study of resistance mechanisms in this organism. It will also be useful for examining factors that influence the efficacy of H. pylori eradication and for determining the optimal eradication regimens for resistant strains.
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ACKNOWLEDGMENTS |
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P. J. Jenks is supported by a Research Training Fellowship in Medical Microbiology from the Wellcome Trust, London, United Kingdom (reference no. 044330). R.L.F. acknowledges the support of the Association Charles Debré. Financial support was provided in part by Pasteur-Mérieux-Connaught (Lyon, France) and OraVax Inc. (Boston, Mass.).
We are grateful to Jani O'Rourke for helpful advice on the treatment protocols used and to Rhône-Poulenc Rorer, Astra Hassle AB, and Abbott Laboratories for the gifts of the pharmaceutical agents used in the treatment protocols.
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FOOTNOTES |
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* Corresponding author. Mailing address: Unité de Pathogénie Bactérienne des Muqueuses, Institut Pasteur, 28 Rue du Dr Roux, 75724 Paris Cedex 15, France. Phone: 33-1-40613272. Fax: 33-1-40613640. E-mail: pjenks{at}pasteur.fr.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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