Resistance of Helicobacter pylori to antibiotics and its impact on treatment options

M´egraud

Resistance of Helicobacter pylori to antibiotics and its impact on treatment options Francis M´egraud Laboratoire de Bact´eriologie, Hopital ˆ Pellegrin, Bordeaux, France

Abstract The treatment of Helicobacter pylori infection is jeopardized by resistance to the antibiotics used, which turns out to be the main risk factor for failure. Resistance is due to point mutations. For clarithromycin only two sites in the 23S rRNA sequence are concerned and can be easily detected by molecular methods, while for metronidazole several mutations on rdxA and other genes can be responsible and so do not allow such detection. The situation for the rare cases of amoxicillin resistance is not fully determined. The impact of resistance on the clinical outcome is dramatic for clarithromycin while it only decreases the success by 20% for metronidazole. ° C 2001 Harcourt Publishers Ltd

INTRODUCTION elicobacter pylori was rediscovered in 1982. Indeed, this bacterium had already been observed in the early days of microbiology but, because it could not be cultured, was not further studied and forgotten. The first culture, performed by Marshall and Warren, can be considered as the birth date of this bacterium.1 They noted already, in their first report, that H. pylori infection was associated with gastritis. This gastritis which last for decades, possibly life long, can lead to severe diseases. The most widely accepted are peptic ulcers, for which eradication of H. pylori has become the treatment of choice2 (Table 1). In addition, H. pylori infection is also the cause of a rare type of cancer: gastric MALT lymphoma. This was the first human cancer proven to be caused by a bacterium and cured by antibiotics.3 Helicobacter pylori infection is also an essential risk factor for a second, more common type of cancer, gastric carcinoma.4 However, in this case, systematic prevention by eradication of the organism is not advised at this stage. A common situation in the field of infectious diseases is also occurring for H. pylori infection, i.e. the development of resistant organisms. The first trials performed with proton pump inhibitors (PPI) and clarithromycin-based triple therapies led to very high rates of success.5 This success tends to decrease with time and especially in countries where H. pylori resistance is the highest.6 In this review, we will consider the mechanism of resistance, the techniques to detect resistance, the epidemiology of resistance and the impact on the treatment outcome of the infection.

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doi: 10.1054/drup.2001.0203, available online at http://www.idealibrary.com on

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MECHANISMS OF H. PYLORI RESISTANCE TO ANTIBIOTICS Among the different mechanisms of resistance to antibiotics at the genetic level, only one, the occurrence of a point mutation, concerns H. pylori (Table 2). These point mutations arise on the chromosome and are transmitted only to the descendants, which limits the spread of the resistance. However, horizontal transfer is theoretically possible, particularly by transformation,7 but does not seem to account for a large proportion of cases. The three antibiotics most often used to treat H. pylori infection, a macrolide (clarithromycin), a 5-nitroimidazole (metronidazole) and a β-lactam (amoxicillin), will be considered. Macrolides The macrolide target is the 23S rRNA, especially the domain V where the antibiotics bind and lead to interruption of protein synthesis. Versalovic et al. were the first to demonstrate that point mutations were associated with macrolide resistance in H. pylori.8 They showed that point mutations (adenine to guanine) in two positions, namely 2142 (A2142G) and 2143 (A2143G) (formerly designated as the 2058 and 2059 cognates in Escherichia coli, then 2143 and 2144 in H. pylori nomenclature before being correctly renamed based on the ribosomal sequence of H. pylori ) were associated with macrolide resistance.9 Later, Occhialini et al. showed that these mutations were also associated with a lack of binding of macrolides to isolated ribosomes: the amount of bound antibiotic increased proportionally with the amount of purified ribosomes from the susceptible strain but not from the resistant strain,10 (Fig. 1). Therefore a causal relationship is most likely involved and the mutations are target-associated, mutations. Originally, the two transition mutations A2142G and A2143G were found in clinical specimens. Another mutation has been described, the transversion A2142C, which rarely occurs. Debets-Ossenkop et al. were able to generate other mutations (A2143C, A2142T, A2143T) in vitro, by site-directed mutagenesis of a clarithromycin susceptible strain.11 However, these mutant strains were unstable and had reduced growth rate; similar results were reported by Wang et al.12 In addition, the mutant A2143T had an MIC of only 0.5 mg/L. It has been hypothesized that the change in nucleotide sequence in these cases induces a change in free energy and conformation within the ribosome, greater than for A2142G and A2143G, which has an impact on bacterial fitness. The mutations that are not found are most likely lethal. It is not clear why the mutant A2142C, which is apparently of the same type as the two previous ones, is found so rarely. The simultaneous mutations A2115G and G2141A described by Hulten et al. have never been found again.13 The stability of mutants A2142G and A2143G has been questioned, since resistance usually has a biological cost.14 In two studies, where the strains were subcultured 10–50 times in vitro or obtained from a given patient after several months interval, and the identity of the pre- and postisolates confirmed by Random Amplified Polymorphic DNA (RAPD), resistance was still present indicating the stability of

Resistance of H. pylori to antibiotics Table 1 Drug combinations commonly used to treat Helicobacter pylori infection (7 day treatment) 1-

Proton pump inhibitor at double dose (omeprazole, lansoprazole, pantoprazole, rabeprazole) or ranitidine bismuth citrate + clarithromycin (500 mg bd) + amoxicillin (1 g bd) This combination is the most widely prescribed. 2- Proton pump inhibitor at double dose (omeprazole, lansoprazole, pantoprazole, rabeprazole) or ranitidine bismuth citrate + clarithromycin (500 mg bd) + metronidazole (500 mg bd) 3- Proton pump inhibitor at double dose (omeprazole, lansoprazole, pantoprazole, rabeprazole) + amoxicillin (1 g bd) + metronidazole (500 mg bd) This regimen is more commonly used as second line treatment for 14 days in case of failure of treatment 1.

the mutations.13,15 In contrast, the authors of another study claimed that a reversion toward the susceptible phenotype was possible.16 Metronidazole To be active, metronidazole must be reduced inside the bacterial cells, in order to damage subcellular structures and cause lethal mutations in the DNA.17 The possibility of reduction depends on the redox potential of the intracellular environment. Any redox system with a potential lower than that of metronidazole (−415 mv) will reduce metronidazole. Such redox systems are present in anaerobes but not in aerobes. H. pylori which is a microaerobe can nevertheless usually reduce metronidazole.18 An important advance in our understanding of 5-nitroimidazole resistance comes from the work of Goodwin et al. They have shown that mutations in the gene encoding for an oxygen insensitive nitroreductase (rdxA) was responsible for this resistance.19 Another study established that 5-nitroimidazole resistance in H. pylori strain ATCC43504 was due to an insertion sequence (mini-IS605) and deletions in the rdxA gene.20 Indeed, it seems that any mechanism leading to an inactivation of rdxA, i.e. frame shift mutation, missense mutation, deletion of bases and the presence of an insertion sequence (mini-IS605), can be responsible for this resistance.21 Table 2 Genes concerned by point mutations or other genetic events leading to antibiotic resistance in Helicobacter pylori, and frequency of resistance Antibiotic group

Genes concerned

Frequency of resistance

Macrolides M´etronidazole Quinolones Rifamycins Amoxicillin

23S rRNA rdxA, frxA gyrA rpoB pbp1

0–20% 10–90% 0–10% 0–5% few cases described

Fig. 1 Macrolide binding to Helicobacter pylori ribosomes. For the wild type strain, macrolide binding is dependent on the ribosome concentration while, for the resistant strain, no binding is observed.

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Other candidates for electron donors have been suggested in previous studies using genetic or biochemical approaches: ferredoxin,22 ferrodoxin-like proteins,22−24 flavodoxin,22 NAD(P)H flavin oxidoreductase ( frxA),22,23 2-oxoglutarate oxidoreductase22,25 and pyruvate ferredoxin oxidoreductase.22,24−28 Inactivation of genes involved in some of these systems has been found to be linked to metronidazole resistance.22,23,29−31 These data support the concept that multiple enzymes within the bacterium are able to reduce metronidazole, i.e. the maintenance of an appropriate redox potential may be governed by multiple enzyme systems. Indeed, recent data have shown that point mutations in the frxA gene were involved in metronidazole resistance either in conjunction with mutations in the rdxA gene or as the only gene involved. The frxA gene from a metronidazole susceptible H. pylori strain was successfully expressed in intrinsically metronidazole resistant E. coli to render it susceptible, while the expression of a non-functional frxA gene did not change the resistant phenotype.32 βLactams Amoxicillin is the only βLactam used to treat H. pylori infection. Until recently, amoxicillin resistant H. pylori had not been found. Two types of resistance have since been described: Dore et al.33 reported the occurrence of amoxicillin resistance in 17 H. pylori strains isolated from patients in Sardinia (Italy) and in the US. These resistant isolates, grown from patients enrolled in a clinical trial on amoxicillin plus omeprazole therapy, showed pre-treatment amoxicillin resistance with MICs > 256 µg/ml and were apparently associated with a marked reduction in treatment efficacy.34 The amoxicillin resistance phenotype was unstable and lost after storage at −80◦ C but plating these strains on amoxicillin gradient plates could restore resistance. The isolates recovered had MICs and minimum bacterial concentrations (MBCs) ranging from 0.5 to 32 µg/ml and 32 to > 1024 µg/ml, respectively, and the MBC/MIC ratio for all tested strains was > 32, indicating a tolerance to amoxicillin. A report from the Netherlands35 described an H. pylori strain with stable resistance to amoxicillin (MIC of 8 µg/ml) that was isolated several times from a patient having received multiple courses of amoxicillin therapy for a respiratory tract infection. Amoxicillin resistance could be transferred to a susc °

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M´egraud ceptible strain by natural transformation at a frequency of 10−5 bacteria. The resistant transformants were stable with MIC values 400 times greater than the value of the susceptible strain and similar to the naturally resistant strain. In contrast to the report by Dore et al.34,36 amoxicillin resistance remained stable after repeated cycles of freezing and culture. Han et al.37 also described stable amoxicillin resistance (256 µg/ml) in four H. pylori clinical isolates, while unstable resistance was found in three others. No βLctamase can account for these findings, since no βLactamase homologue genes were found in the H. pylori genome and no βLactamase activity was found. Another possible target may be the penicillin-binding proteins (PBPs) which are involved in the peptidoglycan biosynthesis. Covalent binding of the βLactams to specific PBPs in susceptible organisms results in the inability of the bacterium to build a complete cell wall and leads to cell lysis. Alterations in the PBPs, by affecting the ability to bind βLactams, can confer increased resistance to these antibiotics. A specific PBP named PBP-D or PBP-4 could account for the low affinity for amoxicillin38,39 and possibly tolerance. In contrast, other studies have highlighted the role of another PBP (PBP-1A).40,41 Kusters et al.42 showed that point mutations in the PBP-1A were partially associated with stable amoxicillin resistance in their clinical isolate. Other antibiotics Point mutations are also the genetic support for resistance of H. pylori to: • Fluoroquinolones, as in other bacteria. These mutations occur in the quinolone resistance determining region (QRDR) of gyrA gene43 encoding the DNA gyrase whose main function is to relax the supercoiling of DNA to allow its replication. • Rifamycins, also as in other bacteria. The mutations occur in the rpoB gene which encodes for the subunit of the DNA-dependent RNA polymerase.44 For two other antibiotics, tetracycline and furazolidone, resistance was not supposed to occur. However, such a resistance has been reported in rare instances, but the mechanism is still unknown.

ANTIMICROBIAL SUSCEPTIBILITY TESTING OF H. PYLORI The methods used for H. pylori susceptibility testing can be divided into standard phenotypic methods based on diffusion or dilution, and genotypic methods. Phenotypic methods The dilution susceptibility testing methods can be performed in either agar or broth and yield a quantitative result. They are applicable to all antimicrobial agents that are tested as two-fold serial dilutions of various concentrations, depending on the agents tested, and the MICs obtained are expressed in µg/ml (or in mg/l). 180

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Agar dilution method Agar dilution is a reliable technique, which is usually carried out as the reference method for evaluating the accuracy of other testing methods. This method is well adapted for testing large number of strains but not for testing strains on a daily basis because it is time consuming. The National Committee for Clinical Laboratory Standards (NCCLS) has approved the agar dilution method as the method of choice for H. pylori.45 Standardization of clarithromycin susceptibility testing concerns the type of agar, growth supplement, size of inoculum, incubation atmosphere (microaeobic, anaerobic) and delay to read the plates. The breakpoints recommended are susceptible < 0.25 mg/l, intermediary 0.25– 1 mg/l and resistant >1 mg/l. Guidelines aimed at standardizing the antimicrobial susceptibility testing of H. pylori have also been drawn in Europe,46 and quality control strains deposited in the CCUG. Comparison of the NCCLS and European agar dilution methods are shown in Table 1. The agar dilution method has not been standardized for metronidazole and amoxicillin. Breakpoints of > 8 µg/ml for metronidazole, and 0.5 µg/ml for amoxicillin have been proposed but have not been officially accepted. Broth dilution method This method is not frequently used because of the difficulty in growing H. pylori in broth. However, supplementing broth with fetal calf serum has been found satisfactory.47 This method has the advantage to be more suitable for automatization. A correlation between this technique and the agar dilution method has not been made but the correlation with the E-test was excellent for both amoxicillin and clarithromycin. Breakpoint susceptibility testing This method is a simplification of one of the dilution methods previously mentioned, where only one drug concentration corresponding to the breakpoint is used. In our experience, the correlation with the agar dilution method is good, except for metronidazole. Disk diffusion method This method is the most commonly used for routine susceptibility testing, though not recommended for slow growing bacteria. However, in a recent French multicenter study, disk diffusion was found to be very accurate to detect resistance to macrolides, especially using a disk of erythromycin. A diameter >17 mm predicted resistance perfectly.48 In contrast, with regard to metronidazole resistance, conflicting results have been obtained. Epsilometer test (Etest) The Etest is a quantitative variant of the disk diffusion method. Again the correlation with the agar dilution method was excellent for all antibiotics, except metronidazole. In summary, for macrolide resistance, all of the methods can be used. However, because the disk diffusion is the simplest and cheapest, this method can be recommended, whereas an agar dilution method or Etest should be carried out when knowledge of an exact MIC is required. The difficulty arises

Resistance of H. pylori to antibiotics for metronizadole. Even when performed with the agar dilution method, resistance figures do not predict accurately the treatment outcome. The other methods are even worse, probably because a key element in the technique, i.e. the redox potential of the culture medium, is not standardized, leading to lack of reproducibility and important discrepancies compared to the agar dilution method.46,49 Additional work in the field of metronidazole resistance is definitely needed in order to obtain reliable results. Genotypic methods At this stage, these methods are only applicable to detect macrolide resistance for which there is a limited number of point mutations. Numerous techniques have been developed to detect the point mutations in positions 2142 and 2143. They are essentially PCR-based methods. First, a fragment of the 23S rRNA gene flanking the 2142 and 2143 nucleotides must be amplified. Different methods can then be applied to study the amplicons. The reference method consists of amplicon sequencing, but it cannot be performed routinely in most settings. An easy alternative method is based on the occurrence of restriction sites within the amplified fragment when one of the two most frequent mutations is present. These restriction sites are recognized by the enzymes BsaI (A2142G mutation) and BsbI (A2143G mutation), and lead to the presence of two bands instead of one on the gel. If an incomplete cleavage is observed, the presence of a mixed population is likely.8,10,50−52 This method (PCR-restriction fragment length polymorphism) has the disadvantage of not being able to detect the A2142C mutation. 30 -mismatch primers can also be used for all point mutations including the A2142C mutation.53 However, the mutations are identified by the absence of a band, which is a less preferable endpoint than a positive endpoint. Other methods, such as the PCR oligonucleotide ligation assay54 or the PCR/DNA enzyme immunoassay, include an additional step after the PCR. To perform the second method, oligonucleotide probes complementary to the DNA fragment containing the different mutations and the wild type have to be constructed. The amplified products are added to probe-coated microtiter wells. The DNA-enzyme immunoassay consists of a colorimetric hybridization in liquid phase.55 This test has been applied to detect H. pylori and its eventual clarithromycin resistance directly on biopsy specimens and has given a perfect correlation with the other methods tested.56 A similar approach has also been developed in a solid phase instead of liquid phase. This reverse hybridization line probe assay (INNO-LiPA) is already available to detect point mutations in other areas. Oligonucleotide probes corresponding to the different possible point mutations are immobilized on a nitrocellulose strip and are hybridized with the amplified products. Results were highly concordant and, as with the previous method, this assay appears to be significantly more sensitive than other methods in detecting several genotypes in the same sample.57 The preferential homoduplex formation assay (PHFA), has been applied to the direct detection of H. pylori and clarithromycin resistant mutants in gastric juice samples by Maeda

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et al.58 This assay may be more sensitive than the previous ones, since it detected more mixed populations and in half of them the resistant population was present at a concentration so low that the phenotypic method did not detect them. The authors argue that testing gastric juice gives a better picture of what happens in the whole stomach, rather than testing only biopsy specimens. An appealing method to detect H. pylori mutations stems from the development of the LightCyclerr (Roche), an apparatus designed to perform quantitative PCR.59 The method includes the use of fluorescent probes. It is very simple and quick. The only disadvantage is the need for the expensive apparatus. In addition, it has provided excellent results, and is now being applied directly to biopsy specimens.60−62 Another approach has been proposed and consists of an rRNA-based whole cell in situ hybridization using a set of fluorescent labeled oligonucleotide probes. Labelling of intact single bacteria is monitored by fluorescence microscopy. This approach allows the detection of H. pylori using a 16S rRNA probe and resistant mutants using a 23S rRNA probe simultaneously. This technique holds great promise because it avoids not only culture but also PCR. However, it has not yet been evaluated in the field.63 These genotypic methods of identification of H. pylori and antibiotic resistance are likely to be further developed in the future. Rapid detection of bacteria and resistance profiles will no doubt modify the habits of the prescribers and should contribute to a reduction of the dissemination of antimicrobial resistance.64

EPIDEMIOLOGY OF H. PYLORI RESISTANCE TO ANTIBIOTICS Current status of resistance The currently available data must take into account the technical limitations already mentioned for metronidazole resistance, and the limitations of the epidemiological method which concern most of the studies presented to date.65 The main drawback is the low number of strains usually tested which lead to very large confidence intervals, and the limited representativeness of the strains studied, sometimes including post-treatment strains and strains isolated from patients having received multiple eradication therapies. The figures that are available may come from single hospitals or better yet from multicenter clinical trials using centralized facilities. Globally, resistance to macrolides ranges from 0–20%. In the US, the pooled results from four large multicenter trials including 836 isolates showed an overall resistance rate of 8% and this level was consistent for all six regions defined.66 In Canada, the level of resistance seems to be lower (< 3%).67 A European survey was carried out in 1997–98. Twenty two centers in 17 different countries tested 1,305 isolates by the same methodology. The overall rate of primary resistance was 10% for clarithromycin. There was an increased clarithromycin resistance rate in the Southern part of Europe compared to the Northern part.68 In France in 1997, we began an active surveillance of the antimicrobial susceptibility of H. pylori. In contrast to any previous national studies, a large number of gastroenterologists c °

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M´egraud located throughout the country were asked to include one or two patients randomly. Five hundred H. pylori strains were tested for clarithromycin resistance using an agar dilution method and showed a level of primary resistance of 14.1% (95% CI [11.5–17.6]).69 A similar ongoing study shows an increase to 17%. Interestingly, the resistance rate among strains isolated from ulcer patients (2%) was significantly lower than from other patients and consistent with the result obtained for the ulcer patients included in the MACH2 study in France but has not been explained.49 The rate of secondary resistance, i.e. resistance observed after treatment failure, usually reaches 60% or more.70 This observation is a plea to avoid regimens which are not very effective, such as the combination of a proton pump inhibitor with clarithromycin. Indeed, when the percentage of acquired resistance is expressed in relation to the total number of patients included, it may reach 25% vs 2% when a triple therapy is administered.71 Resistance, once it is acquired, remains stable.13,15 For metronidazole, there is a clear-cut difference between developing countries where virtually all strains are resistant and developed countries where resistance ranges from 10–50%. In the US, the previously cited study of Weissfeld et al. indicates a resistance rate of 56%, again without apparent differences between regions. However, the authors used Etest and they suggest that this resistance was overestimated.66 In Canada, the level of resistance seems to be lower.67 The European scene also seems to be heterogeneous with a global rate of resistance of 33%.68 Our French results using agar dilution on 500 strains gave a resistance rate of 23.9% (95% CI [20.3–27.8]).69

Evolution of resistance Antimicrobial resistance of H. pylori may be the consequence of antibiotic consumption in the community. The association can be shown in hospitals, but it is not as obvious in the community. Nevertheless, patients harboring resistant bacteria have, in general, received more antibiotics than those harboring susceptible bacteria. A temporal relationship has been shown for Staphylococcus aureus and penicillin consumption, as well as for Campylobacter jejuni and fluoroquinolone consumption. In hospitals, a dose-response effect has been demonstrated. There is obviously a biological plausibility, i.e. the selection pressure. Ultimately, the definite proof of a causal association comes from the effect of an intervention, for example, the strict limitation of antibiotic use. There are, in fact, very few examples where such a link has been clearly shown in the community, one of which is the resistance of Streptococcus pyogenes to macrolides in Finland. A 50% decrease in macrolide consumption between 1988 and 1992 led to a decrease in resistance from 19 to 9% but only after a 5 year lag phase.72 With regard to H. pylori, we are currently observing an increase in macrolide resistance which seems to parallel the increased consumption of these drugs not only for H. pylori treatment but also for respiratory tract infections, although the impact of such treatment on H. pylori has never been studied. In Europe, we can hypothesize that a greater 182

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use of macrolides in countries from the South in which the policy of antimicrobial use is lax compared to countries from the North is the cause of the difference in resistance rates. Concerning metronidazole, the high resistance rate observed in developing countries has been linked to the elevated use of this inexpensive drug for parasitic infections while in developed countries it is prescribed for vaginal and dental infections. A recent survey in Belgium did not indicate an increased rate of metronidazole resistance between 1990 and 1996 which remained stable around 30%.73 If the magnitude of antimicrobial use is a major risk factor for H. pylori resistance, it is not the only one; the mechanisms of resistance, the extent of bacterial spread, the variety of prescription, and the type of susceptible hosts can affect the relationship. We anticipate that resistance will increase in the future. However, the current recommendations of the Digestive Health Initiative International Update Conference74 and the Maastricht Consensus Report,75 which was recently revised,76 are not to perform susceptibility tests for H. pylori. In fact, it may soon become cost-effective to carry out such tests77 especially in light of the latest developments which will render them quick and simple. Moreover, it has become mandatory to develop drugs with increased efficacy against resistant strains, such as nitazoxanide78 and ketolides for metronidazole and macrolide resistant strains, respectively. Ultimately, only an effective vaccine will eliminate the problem of antibiotic resistance in H. pylori. IMPACT OF H. PYLORI RESISTANCE ON TREATMENT OUTCOME Given the antibiotic combinations commonly used to treat H. pylori infection (Table 1), acquired resistance is a concern with respect to two antibiotics: clarithromycin and metronidazole. Resistance to macrolides in PPI based triple therapies In the first multicenter study performed, the ACT-10 study, the combination of omeprazole, amoxicillin, and clarithromycin was given for 10 days. There were only six clarithromycin resistant strains and only half were eradicated in contrast to 92% clarithromycin susceptible strains.71 Conversely, in the MACH2 study, the two clarithromycin resistant strains were eradicated.49 A multicenter study, carried out in southwest France, evaluated pantoprazole instead of omeprazole. None of the five strains resistant to clarithromycin were eradicated compared with 86% of susceptible strains.79 Using the omeprazole-clarithromycin-metronidazole (OCM) combination, in the MACH2 study, four of six (67%) clarithromycin resistant strains were eradicated compared with 98 of 108 (91%) clarithromycin susceptible ones.49 Two meta-analyses have been recently published on this topic. In the study of Dore et al., resistance reduced efficacy by an average of 55% (95% CI 33–78) but this analysis was not limited to triple therapies, and only 501 patients were considered.80 A similar drop in efficacy was found in the

Resistance of H. pylori to antibiotics meta-analysis of Houben et al. who differentiated between the different lengths of treatment regimens.81 An analysis of the individual data of all clinical trials undertaken in France over the last 10 years has recently been performed. More than 2,700 patients were analyzed. Unfortunately information on clarithromycin resistance was available only for 436 cases. Treatment was not successful for any of the patients harbouring a clarithromycin strain and suffering from nonulcer dyspepsia and only 20% of those with a peptic ulcer disease were treated successfully, highlighting that macrolide resistance is the primary factor for failure of H. pylori therapy.6

Resistance to metronidazole Metronidazole resistance in bismuth-based therapies In the ‘standard’ triple therapy, regardless of the antibiotic given with metronidazole, either amoxicillin or tetracyline, and regardless of the duration of treatment, significant differences in eradication rates were found between susceptible and resistant strains. In case of quadruple therapy, i.e. adding a PPI to the ‘standard’ triple therapy, given for seven days or more, metronidazole resistance can be partly overcome.82,83 Metronidazole resistance in PPI-based triple therapies The most commonly used include clarithromycin as the second antibiotic. Differences in eradication with few exceptions have been found in favour of susceptible strains compared to resistant.84 In the MACH2 study which was designed to explore the role of antibiotic resistance on the outcome, a difference of 20% was observed in the eradication rate: 95% when the strains were susceptible vs 76% when they were resistant.49 A meta-analysis including 15 arms and 791 patients found similar results: 94% eradication for susceptible strains vs 73% for resistant strains.81 More resistant strains could be eradicated (83%) if the treatment was given for two weeks instead of one week. The same applies when amoxicillin is used instead of clarithromycin in the triple therapy with an even greater difference. In the meta-analysis previously mentioned, the eradication rates were 54% and 92% for the resistant and susceptible strains, respectively, for a one-week therapy vs 72% and 88%, respectively, for a two-week therapy.81

CONCLUSIONS AND FUTURE DIRECTIONS Resistance of H. pylori to antibiotics is important to consider in the management of the infection. When a first treatment with clarithromycin has not been successful, it is mandatory not to use this antibiotic in second-line therapies, except if the strain can be proven to be susceptible by testing. Metronidazole resistance has less clinical relevance at this stage and, in addition, there are problems to determine it accurately. It can partly be overcome by extending the length of treatment beyond one week. A strict policy of antibiotic use is necessary to limit the spread of antibiotic resistance and

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surveillance programmes should be implemented at regional levels.

Received 19 April 2001; Revised and Accepted 21 May 2001 Correspondence to: Francis M´egraud, Laboratoire de Bact´eriologie, Hopital ˆ Pellegrin, Place Am´elie Raba-L´eon, 33076 Bordeaux cedex, France, Tel: 33- 5 56 79 59 10; Fax:: 33- 5 56 79 60 18; E-mail: [email protected]

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