The discovery of the key role of Helicobacter pylori

Transcription

1 GASTROENTEROLOGY 1998;115: Epidemiology and Mechanism of Antibiotic Resistance in Helicobacter pylori FRANCIS MÉGRAUD Laboratoire de Bactériologie, Hôpital Pellegrin, Université Victor Segalen, Bordeaux, France The discovery of the key role of Helicobacter pylori infection in the development of several diseases of the stomach and duodenum has been a breakthrough in gastroenterology. It has brought forth an additional area to the field of infectious diseases. The concept of treatment has therefore completely changed, and a definitive cure of these diseases can now be achieved. However, each situation has its own drawbacks, i.e., we are now faced with what is the common ground of all microorganisms: the ability to escape treatment by becoming resistant to the antibiotics used. 1 The Concept of Resistance The activity of an antibiotic on microorganisms is essentially measured by its minimal inhibitory concentration (MIC), which, although not as accurate as the minimal bactericidal concentration, is easier to measure. For a given antibiotic there is a threshold or a grey zone that separates the bacteria defined as susceptible from those defined as resistant. This threshold is determined by the concentration of antibiotic that can be achieved in the bloodstream when a therapeutic dose is administered. The problem with H. pylori is that the concentration of antibiotics should be considered in the gastric mucosa and not in the bloodstream. Furthermore, because the ph strongly influences the antimicrobial activity, this value should be taken into account. Such data are difficult, if not impossible, to obtain because the stomach is not easily accessible. It is especially difficult to follow drug kinetics in this organ over a long time period; also, the ph varies during a 24-hour period and is not the same in the different zones where the bacteria live. The mucosal concentration may also vary between the different areas of the stomach. Attempts to resolve these questions have been made but, in some instances, have led to an erroneous conclusion: it was proposed, for example, that amoxicillin had only a topical activity but was shown later that eradication can be obtained using this drug only in a parenteral treatment. 2 Because of these difficulties, clinical data obtained from carefully designed and conducted trials tend to be the solution in defining the MIC threshold (cutoff value) to separate susceptible and resistant strains. It is a pragmatic approach but has the advantage of being a global approach, because currently most of the regimens contain three drugs including two antibiotics, so it takes into account the possible synergies between the compounds. The bacteria that can tolerate high concentrations of antibiotics have developed mechanisms to escape the noxious effects of the drug. These effects can concern the different structures of the bacteria, but the most commonly used drugs interfere either with protein synthesis at the ribosome level, with cell-wall synthesis, or with DNA replication. The presence of a resistance mechanism in a strain is highly predictive of the strain s behavior. Only in rare instances is the resistance mechanism not expressed. Therefore, the detection of such a mechanism can be used to define resistant strains and will eventually lead to a genetic diagnostic approach. On a genetic basis, resistance genes can be present on plasmids that increase the risk of horizontal transmission, or on the chromosome, acquired by mutation or chromosomal rearrangement such as deletions or insertions. In H. pylori, mutations have mainly been described; however, other mechanisms may occur: transfer by transformation has been proved to be possible as well as integration of exogenous DNA. Resistance to Macrolides Macrolides are essential components of the H. pylori eradication regimen. Among them, clarithromycin has proven to be very effective because it has a low MIC for H. pylori (MIC 50, 0.03 mg/l). This MIC is less affected by a decrease in ph than that of the other compounds in this group, and the antimicrobial agent has a good diffusion in the gastric mucosa. Abbreviations used in this paper: MIC, minimal inhibitory concentration; PCR-RFLP, polymerase chain reaction restriction fragment length polymorphism; rrna, ribosomal RNA by the American Gastroenterological Association /98/$3.00

2 November 1998 ANTIBIOTIC RESISTANT H. PYLORI 1279 Resistance to macrolides in H. pylori is due to a lack of macrolide binding to the ribosome target and concerns all molecules in this family. This mechanism can be demonstrated by separating ribosomes from susceptible and resistant organisms and incubating increasing concentrations of ribosomes with radiolabeled erythromycin. The amount of antibiotic bound increases proportionally with the amount of ribosomes present in the susceptible strain, but not in the resistant strain demonstrating a lack of binding. 3 The target modification usually involves a posttranscriptional change. The so-called MLS resistance of Staphylococcus aureus, caused by methylation of adenine residues in the 23S ribosomal RNA (rrna) gene (coded by an acquired methylase), was described a long time ago but could not be documented for H. pylori. 4,5 The other known modification that can lead to resistance is a mutation, also situated in the peptidyl transferase domain of the 23S rrna, first described in Escherichia coli and subsequently in Mycoplasma pneumoniae and Mycobacterium intracellulare. Versalovic et al. 6 were the first to describe such a mutation in H. pylori in Mutations were first described in two gene positions 2142 or 2143 (formerly named 2058 and 2059, respectively, which are the cognates in E. coli, then 2143 and 2144 before to be revised as it is now). 7 More recently, mutations in positions 2116 and 2142 have also been described but seem to occur quite seldomly. 5 There are two copies of the 23S rrna gene on the H. pylori chromosome, 8,9 so one may wonder if a mutation on one copy is enough to induce resistance. Indeed, a heterozygous mutation has been found by some researchers. 5,9 It can be detected by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) of the amplified products, and this mutation has proven to be dominant. The point mutation can be a transition (A=G) or a transversion (A=C), but the transition is far more frequent. There are some data, although not unanimous, indicating that the type of mutation may influence the level of resistance. Versalovic et al. 10 studied 54 strains and found that the A2142G mutation was significantly more likely to be present in isolates with MICs of 64 mg/l than in those with the A2143G mutation (65% vs. 30%; P 0.01). The association between point mutations on the 23S rrna gene, the lack of macrolide binding to ribosomes, and high MICs of these compounds offer a new approach for diagnosing macrolide resistance. Sequencing is obviously the reference method, but other simpler approaches can also be considered. Those applied directly to strains include PCR-RFLP, because the 2142 transition creates a restriction site for the enzyme BsaI and the 2143 transition for BbsI. 3,6,11 Another approach can be made using hybridization with oligonucleotides specific to each genotype without or with prior amplification. An oligonucleotide ligase assay, 12 a DNA enzyme immunoassay, 13 and a reverse hybridization line probe assay 14 have already been developed. This last technique is the most promising because the different mutations can be detected in the same strips. Such techniques are quicker than the standard susceptibility tests like Etest (AB Biodisk, Solna, Sweden) or disk diffusion, but still require the time for culture. It may become possible to perform the test directly on a gastric specimen. The 23S rrna primers seem to be specific enough to allow a direct amplification. Once again the same kind of techniques can be used: PCR- RFLP 15 and DNA enzyme immunoassay 16 ; a result can be expected within a day. Gastric specimens could include gastric juice obtained by tubage or Entero-test (HDC Corporation, San Jose, CA), 17 rendering this method semi-invasive. Resistance to Nitroimidazoles Nitroimidazoles, especially metronidazole, were among the first drugs to be used in H. pylori eradication trials. Although their MICs are not very low (MIC 50,1 mg/l), they are not negatively influenced by a low ph; furthermore, they can concentrate in the gastric compartment. 18 This can be explained by the ph partition hypothesis known for years. 19 Metronidazole is a weak nonionized base at neutral ph (in blood and tissue) that crosses the gastric barrier and then becomes ionized because of the low ph. The consequence is that ionized metronidazole cannot cross back through the barrier and therefore accumulates in the lumen rather than reaching an equilibrium. The nitro group of metronidazole must be reduced to hydroxylamine to be active on the DNA. The exact mechanism of action is not completely understood, but it seems that the active compound breaks the DNA strand. 20 The resistance of bacteria to these compounds seems to be caused by the decreased ability to reduce the nitro group. Unfortunately, metronidazole is one of the compounds that requires one of the lowest redox potentials to be reduced ( 500 mv). A recent study has shown that a reduced nicotinamide adenine dinucleotide phosphate nitroreductase could be responsible for the reduction of the nitro group and that mutations in the corresponding gene (rdxa) would decrease the enzyme s activity. 21 It is too early to conclude that this mechanism is entirely responsible for the resistance because only a few strains have been studied. Alternatively, other enzymes intervening in the reduction such as a reduced nicotinamide adenine dinucleotide

3 1280 FRANCIS MÉGRAUD GASTROENTEROLOGY Vol. 115, No. 5 oxidase could be involved. This enzyme removes oxygen from the microenvironment, increasing the rate of drug reduction. There is clearly a difficulty in foreseeing the metronidazole resistance in vitro by employing phenotypic tests because the redox potential of the environment is not controlled; this may explain the apparent instability of this resistance. For example, it is possible to influence the result by preincubating the plates in an anaerobic atmosphere. 22 The exact redox potential level at which the test should be carried out has not been determined and probably varies with time. At present, no genotypic test is available. Phenotypic tests are used and are currently being standardized by different authority groups such as the National Committee for Clinical Laboratory Standards in the United States. In the meantime, the results are not always reliable and reproducible in individual cases; despite this major drawback, a correlation has always been found between resistance and the decreased efficacy of the regimen in clinical trials, indicating a real clinical significance of metronidazole resistance. Resistance to Amoxicillin Amoxicillin is the only -lactam used to treat H. pylori infection. This is due to its very low MIC on this bacterium, usually 0.03 mg/l. Interestingly, resistance is virtually absent for this compound. This was also the case during many years for Streptococcus pneumoniae; subsequently a decreased susceptibility was observed, which was relative to a decreased affinity to the penicillinbinding proteins, enzymes that participate in cell wall synthesis. Indeed, some H. pylori strains have been described that repeatedly showed an MIC of 0.25 or 0.5 mg/l, i.e., up to 100 times the MIC of the most susceptible strains. Recently, Dore et al. 23 found that one of four penicillin-binding proteins, normally present in susceptible strains, was missing in isolates with higher MICs. Currently, this phenomenon seems to be rare, and its clinical significance has not been documented. It nevertheless indicates the need for a surveillance. Resistance to Fluoroquinolones Fluoroquinolones are not commonly used in H. pylori eradication treatments. However, when they have been used either alone or in a combination of antibiotics, resistance has been documented. Fluoroquinolones inhibit the DNA gyrase and topoisomerase IV enzymes that transform the supercoiling of DNA to allow its replication. Resistance in H. pylori has been studied extensively by Moore et al., 24 who described point mutations associated with higher MICs. The gyra gene was cloned and sequenced. Sequence analysis from ciprofloxacin-resistant mutants revealed four classes of mutations with substitutions at amino acids 87, 88, and 91 and a double substitution at amino acids 91 and 97. The mutations concerned the quinolone resistance determining region in 10 of 11 ciprofloxacin-resistant mutants. Moore et al. were able to transform susceptible strains using the amplified fragment from resistant strains as donor DNA. 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 that concern most of the studies presented to date. The main drawback is the low number of strains tested in most studies, which led to very large CIs, and the limited representativity of the strains studied, sometimes including posttreatment strains and strains isolated from patients undergoing multiple eradication therapies. The available figures may come from single hospitals or, better yet, from multicenter clinical trials using centralized facilities. Globally, resistance to macrolides ranges from 0% to 15%. In the United States, the pooled results from four large multicenter trials including 836 isolates have shown an overall resistance rate of 8%; this level was consistent for all six regions defined. 25 In Canada, the level of resistance seems to be lower ( 3%). 26 In Europe, there is a north-to-south gradient. In Mediterranean countries such as France, Italy, and Spain, the level of resistance is consistently between 10% and 15%, whereas in Scandinavian countries and in the Netherlands it is less than 3%. 27 Countries like the United Kingdom and Germany are found in an intermediary position. In 1997, an active surveillance of the antimicrobial susceptibility of H. pylori was begun in France. In contrast to any previous national studies, a large number of gastroenterologists located throughout the country were asked to include 1 or 2 patients randomly. Five hundred H. pylori strains were tested for clarithromycin resistance using an agar dilution method and gave a level of primary resistance of 14.1% (95% confidence interval, ). 28 Interestingly, the resistance rate among strains isolated from patients with ulcers (2%) was significantly lower than from other patients, consistent with results obtained for ulcer patients included in the MACH 2 study in France.

4 November 1998 ANTIBIOTIC RESISTANT H. PYLORI 1281 The rate of secondary resistance, resistance observed after treatment failure, may reach 60% or more. 29 This observation is a plea to avoid regimens that are not very effective such as the combination of a proton pump inhibitor with clarithromycin because, when the percentage of acquired resistance is expressed in relationship to the total number of patients included, it may reach 25% vs. 2% when a triple therapy is administered. 29 Resistance, once acquired, remains stable. 4 For metronidazole, there is a clear-cut difference between developing countries where virtually all strains are resistant and developed countries where resistance ranges between 10% and 50%. In the United States, the previously cited study of Weissfeld et al. 25 indicates a resistance rate of 56%, again without apparent differences between regions. However, they used the Etest and suggest that this resistance was overestimated. In Canada, rates of 15% 30% have been cited. The European scene also seems to be heterogeneous. Our French results using agar dilution on 500 strains gave a resistance rate of 23.9% (95% confidence interval, ). 28 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 is not as obvious in the community. Nevertheless, patients with resistant bacteria have, in general, received more antibiotics than those with susceptible bacteria. A temporal relationship has been shown for S. aureus and penicillin consumption as well as for Campylobacter jejuni and fluoroquinolone consumption. In hospitals, a doseresponse effect has been found. There is obviously a biological plausability that is the selection pressure. Ultimately, the definite proof of causal association comes from the effect of an intervention, i.e., the strict limitation of antibiotic use. Such a link has been clearly shown in very few instances, one of which is the resistance of Streptococcus pyogenes to macrolides in Finland. A 50% decrease in macrolide consumption between 1988 and 1992 has led to a decrease in resistance from 19% to 9%, but only after a 5-year lag phase. 30 We are currently observing an increase in macrolide resistance that 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 use of macrolides in countries from the south, in which the policy of antimicrobial use is laxist, compared with countries from the north is the cause of the difference in resistance rates. The high resistance rate to metronidazole observed in developing countries has been linked to the elevated use of this inexpensive drug for parasitic infections, whereas in developed countries it may be prescribed for vaginal or 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%. 31 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, extent of bacterial spread, variety of prescription, and type of susceptible hosts can affect the relationship. We anticipate that resistance will increase. The current recommendations of the Digestive Health Initiative International Update Conference 32 and the Maastricht Consensus Report, 33 which are not to perform susceptibility tests, may have to be revised. It may soon become cost-effective to carry out such tests, especially in light of the latest developments, rendering them quick and simple. It has become mandatory to develop drugs with increased efficacy against resistant strains, such as nitazoxanide and ketolides for metronidazole- and macrolide-resistant strains, respectively. Ultimately, only an effective vaccine will eliminate the problem of antibiotic resistance in H. pylori. References 1. Mégraud F. Resistance of Helicobacter pylori to antibiotics. Aliment Pharmacol Ther 1997;11(suppl): Adameck RJ, Wegener M, Labenz J, Freitag M, Opferkuch W, Ruhl GH. Medium-term results of an oral and intravenous omeprazole/ amoxicillin Helicobacter pylori eradication therapy. Am J Gastroenterol 1994;89: Occhialini A, Urdaci M, Doucet-Populaire F, Bébéar CM, Lamouliatte H, Mégraud F. Macrolide resistance in Helicobacter pylori: rapid detection of point mutations and assays of macrolide binding to ribosomes. Antimicrob Agents Chemother 1997;41: Debets-Ossenkopp YJ, Sparrius M, Kusters JG, Kolkman JJ, Vandenbroucke-Grauls CMJE. Mechanism of clarithromycin resistance in clinical isolates of Helicobacter pylori. FEMS Microbiol Lett 1996;142: Hultén K, Gibreel A, Sköld O, Engstrand L. Macrolide resistance in Helicobacter pylori: mechanism and stability in strains from clarithromycin-treated patients. Antimicrob Agents Chemother 1997;41: Versalovic J, Shortridge D, Kliber K, Griffy V, Beyer J, Flamm RK, Tanaka SK, Graham DY, Go MF. Mutations in 23S rrna are associated with clarithromycin resistance in Helicobacter pylori. Antimicrob Agents Chemother 1996;40: Taylor DE, Zhongming GE, Purych D, Lo T, Hiratsuka K. Cloning and sequences analysis of two copies of a 23S rrna gene from Helicobacter pylori and association of clarithromycin resistance with 23S rrna mutations. Antimicrob Agents Chemother 1997;41:

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The effect of changes in the consumption of macrolide antibiotics on erythromycin resistance in group A streptococci in Finland. N Engl J Med 1997;337: de Koster E, Cozzoli A, Vandenborre C, Jonas C, Denis P, Otero J, Deltenre M. Helicobacter pylori resistance to macrolides increases, to imidazoles remains stable (abstr). Gastroenterology 1997;112:A The report of the Digestive Health Initiative international Update Conference on Helicobacter pylori. Gastroenterology 1997; 113(suppl):S4 S Current European concepts in the management of Helicobacter pylor infection. The Maastricht Consensus Report. Gut 1997;41: Received May 11, Accepted August 3, Address requests for reprints to: Francis Mégraud, M.D., Laboratoire de Bactériologie, Hôpital Pellegrin, Place Amélie Raba Léon, Bordeaux Cedex, France. francis.megraud@chuaquitaine.fr; fax: (33)