Journal of Applied Microbiology 2001, 90, 134S±144S

Antibiotic resistance of Helicobacter pylori J.G. Kusters1 and E.J. Kuipers2 1

Department of Medical Microbiology, and 2Department of Gastroenterology, Vrije Universiteit, Amsterdam, the Netherlands

1. 2. 3. 4. 5.

Summary, 134S History, 134S Whom to treat, 135S Treatment, 135S Antimicrobial resistance, 136S 5.1 Resistance to clarithromycin (and related macrolides), 136S 5.2 Resistance to metronidazole (and related nitroimidazoles), 137S

1. SUMMARY Peptic ulcer disease due to infection with Helicobacter pylori is an extremely common chronic and disabling disease, which in the past was attributed to factors such as stress, genetics and personality. Reports of an association with ¯agellated bacilli had already appeared in the late 19th century, but this association was considered to be due to bacterial overgrowth in the presence of delayed gastric emptying and the signi®cance of this observation was not realized. For decades, patients with ulcer disease were treated with rest, various diets, and ®nally, often with surgery. The recognition that chronic ulcer disease can result from infection with H. pylori turned it into a curable and preventable infectious disorder. Only a few antibiotics can be used successfully for eradication of H. pylori; these are metronidazole, tetracycline, amoxycillin, clarithromycin and azithromycin. Other drugs with some in vitro antimicrobial effect are bismuth salts and proton pump inhibitors. Successful eradication of H. pylori infection requires a combination of two or three antibiotics and an antacid drug. The increased prevalence of antibiotic-resistant H. pylori strains has serious implications as, apart from patient compliance, antimicrobial resistance is the most important factor determining the outcome of antibiotic treatment. Resistance against clarithromycin and metronidazole is of

Correspondence to: J.G. Kusters, Dijkzigt Hospital,Dr Molewaterplein 40,3015 GD Rotterdam, the Netherlands (Tel.: + 31 10 4635946; fax + 31 10 4634682; e-mail: [email protected]).  Present address: Department of Gastroenterology and Hepatology, Dijkzigt Hospital, Rotterdam, The Netherlands.

5.3

Resistance to amoxycillin (and related beta-lactams), 138S 5.4 Resistance to tetracycline, 139S 5.5 Resistance to less commonly used antibiotics, 139S 5.6 Resistance to bismuth compounds and antisecretory agents, 139S 6. Transfer of antibiotic resistance, 139S 7. Conclusions, 140S 8. References, 140S

particular clinical importance as these two drugs are used in almost all standard H. pylori eradication regimens. Our current understanding of the mechanisms of antibiotic resistance for these two drugs now allows for rapid testing of resistance and provides clues on how to reduce the spread of resistance. Spontaneous resistance to rifampicin, tetracycline, amoxycillin, streptomycin, trova¯oxacin and cipro¯oxacin has also been reported. The underlying mechanisms for resistance against these drugs have been characterized in some detail and will be discussed. 2. HISTORY In 1892, the Italian pathologist Bizzozero described for the ®rst time the presence of spiral-shaped microorganisms associated with vacuoles in the cells of the gastric mucosa. 1 (Bizzozero 1892). Around the same time, a few other clinicians also reported a relation between the presence of spiral-shaped bacteria in gastric juice and upper gastrointestinal disorders (Pel 1899). In 1938, Doenges found, during an autopsy study, spirochetes in 43% of the stomachs of 242 subjects (Doenges 1938). It was known that peptic ulcer disease responded well to bismuth therapy and doses up to 25 g daily were frequently prescribed. However, research on this subject was hampered by the dif®culty in obtaining fresh clinical specimens of human gastric tissue, and by the fact that the observed gastric bacilli could not be cultured in the lab. As a result, these ®ndings were soon forgotten. This changed with the arrival of endoscopic procedures, and almost one century after the ®rst description by Bizzozero, Warren and Marshall (Warren and Marshall 1983, Marshall and Warren 1984) were the ®rst to ã 2001 The Society for Applied Microbiology

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culture these curved bacteria from gastric biopsy specimens obtained from patients with upper gastrointestinal complaints. Dr Barry Marshall then proved that these bacteria were the cause of upper GI disease by infecting himself and developing gastritis (Marshall et al. 1985). This experiment thus ful®lled Koch's postulates with respect to the association between H. pylori and upper gastrointestinal disease. Marshall and Warren therefore started to study ways to eradicate this bacterium in patients with peptic ulcer disease and soon came back to bismuth therapy (Marshall et al. 1987). As a result of their discovery, peptic ulcer disease has changed from a chronic, relapsing disease of uncertain cause to a curable infectious disease. 3. WH OM TO T R E A T Indications for H. pylori treatment are a topic of repeated discussions and consensus meetings (NIH Consensus Conference 1994; Berg 1996; Cotton 1994; Zoorob 1996). In general, indications are becoming more liberal over time. It remains clear and widely accepted that patients who mainly bene®t from H. pylori eradication are those with clinical and symptomatic disease associated with H. pylori (Walsh and Peterson 1985). This includes in particular patients with duodenal or gastric ulcer disease. In the persistent presence of H. pylori, ulcer disease is a chronic recurring disorder (van der Hulst et al. 1997). Even during acid-suppressive maintenance therapy, 20±30% of the patients suffer from a recurrent ulcer within the ®rst year. This proportion is reduced to only a few percent after successful H. pylori eradication (Xia et al. 1997). Eradication treatment has therefore become the therapy of choice for infected patients suffering from ulcerative disease (NIH Consensus Conference 1994). Other disorders that bene®t from H. pylori eradication include patients with primary gastric B-cell lymphomas originating from mucosa-associated lymphoid tissue (MALT lymphoma) and individuals suffering from hypertrophic protein-losing gastritis (Walsh and Peterson 1995). Both are rare disorders, which have been shown to regress partially or completely after eradication of H. pylori. Other indications for H. pylori eradication are less clear (Walsh and Peterson 1995; Talley et al. 1998). Almost all H. pylori-infected individuals have histological signs of chronic, active gastritis (Price 1988). Although eradication treatment does result in the disappearance of this gastritis, it is not common to treat `gastritis only' patients, as they do not display any symptoms or complications. A small portion of the infected subjects suffers from dyspepsia, but it is unclear to what extent these dyspeptic symptoms result from the H. pylori infection; in most cases eradication of H. pylori in these nonulcer dyspepsia patients does not lead to complete loss of dyspeptic symptoms (Talley 1994).

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Dyspepsia without endoscopical abnormalities is therefore generally not considered to be an indication for H. pylori eradication therapy. A future indication for H. pylori treatment, which is now widely being discussed, could be the prevention of atrophic gastritis and gastric cancer (Cats et al. 1998). 4. TREATMENT Once an infection with H. pylori has established, it will last for life unless treated. In vitro, H. pylori is extremely susceptible to the majority of antibiotics, however, most antibiotics are unable to successfully eradicate this bacterium from infected subjects (Graham 1998). This is ®rst explained by the inability of drugs to achieve appropriate levels in the gastric mucus layer. For antimicrobial agents to be effective, they must accumulate in the gastric mucus layer where Helicobacter resides, either by penetrating the mucus layer from the gastric lumen directly after ingestion, or by secretion across the gastric mucosa. Achievement of therapeutic antibiotic concentrations at the site of infection is adversely affected by the poor secretion of most antibiotics by the cells of the gastric mucosa and their slow diffusion into the gastric mucus layer (McNulty et al. 1988; van Zanten et al. 1992). In addition, the low pH of the mucus layer has an adverse effect on the activity and stability of most antibiotics (Hardy et al. 1988; Grayson et al. 1989; Malanoski et al. 1993; Debets-Ossenkopp et al. 1995). Furthermore, the slow growth rate of H. pylori, and the acidic milieu, which negatively in¯uences the permeability of the bacterial cell membrane, both result in an increase of the minimal inhibitory concentration (MIC) of most antibiotics (Todt and McGroarty 1992). The inability of most antibiotics to reach therapeutic concentrations at the site of infection not only negatively affects treatment outcome, but also facilitates the induction of antimicrobial resistance. Patients need to be properly informed of the possible sideeffects of treatment as these may provoke noncompliance, which signi®cantly affects the ef®cacy of treatment (Graham et al. 1995). Side-effects depend on the treatment regimen, and are more frequent in patients treated with metronidazole or clarithromycin than in patients treated with amoxycillin, and during triple therapy (compared to mono/dual therapy). The frequency of patients with side-effects varies from approximately 10±40% (Penston and McColl 1997), but only very few patients (1±4%) have to stop therapy because of severe side-effects (Deltenre et al. 1998). The development of antimicrobial resistance is also an important factor complicating successful eradication of H. pylori infection (Graham et al. 1992; Deltenre et al. 1998). Due to the above requirements, only a few antibiotics can be used to eradicate H. pylori. The most commonly used of these are tetracycline, amoxycillin, metronidazole and clarithromycin.

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Unfortunately none of them is active enough to use as a mono-therapy, and the successful eradication of H. pylori infections requires a combination of two or three antibiotics and an antacid drug (van der Hulst et al. 1996). The combination of colloidal bismuth subcitrate with two antibiotics (classical triple therapy) was the ®rst therapeutic regimen that widely came into use (Chiba et al. 1992). Nowadays, this regimen is largely replaced by the better tolerated and less complicated combination of a proton pump inhibitor (PPI) and two or three of the abovementioned antibiotics (van der Hulst et al. 1996; Houben et al. 1999a). The latest improvement comes with the introduction of ranitidine bismuth citrate (a salt of the H2-receptor antagonist ranitidine and a bismuth compound). Combination of ranitidine bismuth citrate with clarithromycin has resulted in a simple and effective H. pylori eradication regime (Williamson et al. 1998; Houben et al. 1999a). It has recently been claimed that, in addition to its bene®cial effects, H. pylori eradication may also have negative effects: more than 10% of duodenal ulcer patients who were successfully treated for H. pylori subsequently developed signs of gastro-oesophageal re¯ux disease (Labenz and Malfertheiner 1997; Labenz et al. 1997; SaccaÁ et al. 1997; Werdmuller and Loffeld 1997). Although there is evidence of a correlation between the presumed protective effect of H. pylori and genetic traits of the infecting strain (Vicari et al. 1998), it is at present unclear on what mechanisms this observed protection is based. Suggested mechanisms include the positive effect of gastritis healing on acid production, a disappearance of the H. pylori urease buffering of acid, and an effect of H. pylori eradication on the lower oesophageal sphincter. Whatever the mechanism is, these ®ndings are important as they further stress that H. pylori infection may have bene®cial effects (Blaser 1999a; 1999b). Hence, H. pylori eradication therapy should not be considered as a universal remedy for upper abdominal symptoms in general. 5. ANTIMICROBIAL RESISTANCE The accurate determination of the minimal inhibitory concentration (MIC) of an antimicrobial drug is important when assessing the susceptibility of strains. The methods used to determine the susceptibility of H. pylori to a given drug are not standardized. E-test, agar dilution and disc diffusion tests are commonly used, but consistent results are not always found when these methods are compared (Midolo et al. 1997; AlarcoÂn et al. 1998a). In addition, factors such as the medium used, the inoculation size, and incubation conditions all affect the outcome. In spite of problems with the lack of a standard method to determine the MIC, it is clear that the widespread use of antibiotics has

resulted in a signi®cant increase in the prevalence of antibiotic-resistant H. pylori strains (LoÂpez-Brea et al. 1997; van der Wouden et al. 1997). Spontaneous resistance to rifampicin, tetracycline, amoxycillin, streptomycin, trova¯oxacin, cipro¯oxacin, clarithromycin and metronidazole have all been reported. The underlying mechanisms for several of these drugs have been characterized in some detail and will be discussed below. Although the clinical signi®cance of in vitro-determined resistance levels of H. pylori has long been disputed, various recent meta-analyses have shown beyond any doubt that resistance to metronidazole or clarithromycin signi®cantly reduces the ef®cacy of clarithromycin- or metronidazole-containing regimens for which the infecting H. pylori is shown to be resistant (Houben et al. 1999b; van der Wouden et al. 1999; Dore et al. 2000). These two drugs are used in almost all current H. pylori eradication regimens, hence it is safe to claim that resistance against clarithromycin and metronidazole is of clinical importance. In vitro, the spontaneous development of resistance to these two antibiotics occurs at a frequency of l0)5 to l0)9, which indicates that resistance most probably results from single point mutations. This was subsequently con®rmed when the molecular basis for these resistances was uncovered. Although there are regional differences in the prevalence of antibiotic resistance (with resistance generally increasing as one approaches the equator), there has recently been a global increase of the antibiotic resistance in H. pylori. Resistance rates are highest for metronidazole, followed by clarithromycin. There are only a few reports on resistance to tetracycline and on resistance or tolerance to amoxycillin. Apart from patient compliance, resistance of the infecting H. pylori strain to the antibiotics used is currently thought to be the most important factor determining the outcome of antibiotic treatment (Graham 1998). Given the large numbers of subjects colonized with H. pylori, the considerable proportion that develop disease, the expanding indications for H. pylori treatment and the relative dif®culty of curing this infection, it is not surprising that a much effort is spent on understanding the mechanisms of antibiotic resistance. Understanding the mechanism may allow for rapid testing for resistance and provide clues on how to slow down its spread. 5.1. Resistance to clarithromycin (and related macrolides) The majority of the H. pylori strains are still very sensitive to clarithromycin, but resistance (MIC > 2 g l)1) is rapidly developing and striking regional differences exist. Even within a small area such as western Europe, resistance to clarithromycin ranges from approximately 1á5% in countries such as the Netherlands and Sweden, to > 15% in

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France and Italy. Clarithromycin belongs to the macrolides, a group of antibiotics that all bind to the bacterial ribosome and thus block the synthesis of proteins (Goldman et al. 1994). The antibacterial spectrum of clarithromycin is similar to that of erythromycin, but clarithromycin is better absorbed, more acid-stable, and hence more effective against H. pylori (Peters and Clissold 1992). Clarithromycin resistance is associated with mutations in the 23S ribosomal RNA genes resulting in A to G substitutions in one of two adjacent adenosine residues, but occasionally an A to C mutation is also observed (Debets-Ossenkopp et al. 1996; Stone et al. 1996). These mutations decrease the af®nity of the macrolide drug for the ribosome and thus result in an increased MIC (Occhialini et al. 1997). The A2142 to G substitution generally results in a higher MIC (> 64 g l)1) than the A2143 to G, or A to C substitution (MIC < 64 g l)1) (Versalovic et al. 1996; Debets-Ossenkopp et al. 1998). It was recently shown for some of these mutations that there is a clear negative effect on the growth rate of the bacterium (Debets-Ossenkopp et al. 1998; Wang et al. 1999) and this may promote the reversal to the sensitive wildtype 23S ribosomal gene sequence that is observed when a resistant strain is passaged several times on drug-free medium (Xia et al. 1996; Debets-Ossenkopp et al. 1998). As expected from the resistance mechanism, susceptibility and resistance to clarithromycin usually coincides with those for the related antibiotics azithromycin, erythromycin, clindamycin and streptogramin type-B (Taylor et al. 1997; Debets-Ossenkopp et al. 1998; GarcõÂa-Arata et al. 1999) rendering these antibiotics useless for the treatment of patients infected with clarithromycin-resistant isolates. As clarithromycin resistance seems to be based uniquely on the mutation of the two nucleotides mentioned above, it opened the possibility of rapid polymerase chain reaction (PCR)-based detection methods. Recently, various methods based on detection of the speci®c mutations in PCR-ampli®ed products have been proposed (Stone et al. 1997; van Doorn et al. 1999). The results of this look very promising as they can easily be automated and can be applied directly to biopsy specimens in combination with PCR-based detection of other relevant H. pylori-speci®c markers. 5.2. Resistance to metronidazole (and related nitroimidazoles) Metronidazole was introduced in 1959 for the treatment of gynaecological infections caused by the parasite Trichomonas vaginalis it soon became apparent that the drug was also active against anaerobic and some microaerophilic bacteria such as H. pylori, and metronidazole rapidly became a popular drug for the treatment of infections with anaerobic microorganisms (Ingham et al. 1980). At that time, the

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majority of strains of H. pylori were highly sensitive to metronidazole, but resistance rapidly developed and is currently believed to be > 30% worldwide (Glupczunski 1992). Strains are considered susceptible to metronidazole if the MIC is greater than 8 g l)1. However, studies have shown that treatment of patients infected with strains with intermediate resistance to metronidazole (MIC 2±8 g l)1) is also less successful, but the exact extent of this intermediate resistance on eradication ef®cacy is not clearly de®ned. Metronidazole is actively secreted into the gastric juice and its antimicrobial activity is only marginally affected by a decrease in pH (van Zanten et al. 1992; DebetsOssenkopp et al. 1995). The drug itself is not toxic, but upon entering the bacterium, metronidazole is reduced to an active anion radical (Edwards 1993). This forms the active compound, which acts by causing lethal damage to vital molecules such as DNA, RNA, proteins and fatty acids. An extremely low redox potential is required to allow conversion of the drug into the active form, and the cells of the host and most aerobic bacteria lack such a low redox potential. There has been much speculation on the exact mechanism for resistance to metronidazole in H. pylori. Suggested mechanisms for which evidence has been provided include reduced uptake and/or increased ef¯ux of metronidazole from the bacterium (Lacey et al. 1993; Moore et al. 1995a), inactivation of metronidazole through `futile cycling' (Cederbrant et al. 1992), increased repair of radical-induced damage (Chang et al. 1997; Smith and Edwards 1995; Jorgensen et al. 1998), and mutations in the metronidazolereducing enzymes (Hoffman et al. 1996; Goodwin et al. 1998; Kaihovaara et al. 1998). In contrast with anaerobic bacteria, where transport of metronidazole into the cell is thought to occur via passive diffusion, evidence suggests the presence of an active metronidazole transporter in H. pylori. However, in vitro experiments have shown no differences in the activity of the transporter between metronidazolesusceptible and -resistant bacteria (Moore et al. 1995a). In vitro, metronidazole resistance is completely lost upon exposure to anaerobic conditions and also in cultures of high bacterial cell density (Cederbrant et al. 1992; Smith and Edwards 1995; van Zwet et al. 1995). It has been suggested that this reduced sensitivity to metronidazole results from a loss or decrease in the ability to remove oxygen from the intracellular milieu, such that the low redox potential required for metronidazole activation cannot be maintained (Smith and Edwards 1997). If correct, this would stress that decreased ability to achieve a low redox potential is indeed an important factor in resistance (Smith and Edwards 1995). There have however, been serious critiques on this as it assumes that resistance in H. pylori is based on the same mechanisms present in anaerobic microrganisms. H. pylori, however, is not an anaerobic bacterium but a microaerophilic

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one, hence molecular oxygen is likely to be present in its cytoplasm where it can compete with metronidazole for electrons. The process of re-oxidation of metronidazole radicals by molecular oxygen is called `futile cycling' and results in the reformation of the parent molecule, and superoxide (Perez-Reyes et al. 1980; Cederbrant et al. 1992). Superoxide is a highly reactive molecule and as a result, in¯icts damage to bacterial proteins, RNA and DNA. H. pylori, however, contains a highly ef®cient reactiveoxygen scavenger system, consisting of large amounts of superoxide dismutase and catalase, enabling the neutralization of superoxide (Mori et al. 1997). Smith and Edwards' initial work led to the hypothesis that in H. pylori the one-electron reduction of metronidazole produced enough toxic radicals (both metronidazole radicals and superoxide) to overwhelm the oxygen scavenger system and thus in¯ict lethal damage to the cell. The recent research into the mechanism of metronidazole resistance in H. pylori, however, has shed new light on this subject. In a very elegant study, Goodwin et al. recently identi®ed a molecular mechanism responsible for the high-level metronidazole resistance observed in H. pylori (Goodwin et al. 1998). In contrast to anaerobic bacteria, H. pylori contains a NADPH nitroreductase (encoded by the rdxA gene) that is able to reduce metronidazole by a two-electron transfer step into a toxic metabolite that cannot be retransformed to its parent molecule under in¯uence of molecular oxygen. Goodwin et al. showed that only resistant clinical isolates contained null mutations in the rdxA gene (i.e. mutations that result in production of an inactive enzyme). In addition,they showed that disruption of rdxA in a sensitive strain resulted in resistance to metronidazole, while introduction of an intact copy in a resistant strain resulted in sensitivity (Goodwin et al. 1998). Several others have subsequently con®rmed these ®ndings. One such study used the ®rst H. pylori strain ever cultured (Debets-Ossenkopp et al. 1999a). Interestingly, this strain (NCTC11637) was found to be metronidazoleresistant. In 1983, Marshall and Warren isolated this strain from a female patient, and it is likely that this patient had received metronidazole for the treatment of a gynaecological infection, thereby selecting for a metronidazoleresistant H. pylori isolate. It was found also that in this strain the rdxA gene was disrupted. In addition, it was con®rmed that no mutations were found in any other genes previously suggested to be associated with resistance (Debets-Ossenkopp et al. 1999a). Additional con®rmation for the role of rdxA in metronidazole resistance comes from a controlled treatment study in a mouse model which revealed that where treatment failed due to the development of metronidazole resistance, 25 out of 27 metronidazole resistant isolates from these mice contained null

mutations in the rdxA gene (Jenks et al. 1999). In metronidazole-resistant clinical isolates, the same group found mutations in the rdxA gene in 12 of 13 cases (Tankovic et al. 2000). These studies show that high-level resistance to metronidazole (> 32 g l)1) is associated with mutations in the rdxA gene in the majority of, but not in all, cases. The other mechanisms that are responsible for resistance are still unknown, but also in these instances a role of RdxA has been postulated; mutations in genes regulating the expression of the rdxA gene or in the promoter sequence of the rdxA gene (van der Wouden et al. 2000). 5.3. Resistance to amoxycillin (and related beta-lactams) H. pylori is sensitive to amoxycillin (MIC < 1 g l)1), both in vitro and in vivo. Amoxycillin acts by interfering with the synthesis of the bacterial cell wall, resulting in the lysis of replicating bacteria (as do related penicillin derivatives). Amoxycillin is stable at neutral pH, but its antimicrobial activity rapidly decreases under increasingly acidic circumstances (Grayson et al. 1989). In contrast to the closely related drug ampicillin, amoxycillin is actively secreted from the blood into the gastric juice (van Zanten et al. 1992), hence, intravenous amoxycillin can eradicate H. pylori infections (Adamek et al. 1993). Until recently, spontaneous amoxycillin resistance in clinical isolates of H. pylori had not been described, neither could such resistance be induced by repeated in vitro passage with sub-MIC concentrations of these antimicrobial agents. This is strange, as resistance to beta-lactams is very common in most other bacteria as a result of their extensive use in the community for more than 30 years. In most other bacteria, resistance is based on the production of beta-lactamases, but no such activity has been reported in H. pylori. A less common mechanism of bacterial resistance to beta-lactams is based on the modi®cation of penicillin-binding proteins (PBPs) that are associated with a decreased af®nity of these PBPs for beta-lactam antibiotics. It has recently been observed that where resistance against beta-lactams in Streptococcus pneumoniae and Neisseria gonorrhoeae is based on mutations in PBPs that were initially only associated with relative low-level resistance, as mutations accumulated over time, the MIC increased accordingly (Maiden 1998). Dore et al. recently isolated several amoxycillin-tolerant H. pylori strains (i.e. strains inhibited but not killed at high antibiotic concentrations) from patients in Italy and in the US (Dore et al. 1998; Dore et al. 1999a). The basis of this tolerance seems to lie in the altered expression of a PBP, however, this phenomenon is dif®cult to maintain in subcultures and tended to disappear following storage of the strains at low temperature (Dore et al. 1999b; 1999c).

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Our laboratory also identi®ed and characterized an amoxycillin-resistant strain (van Zwet et al. 1998). In contrast with the strains described by Dore et al., this strain does not lose its resistance upon repeated subculture in the absence of amoxycillin, or on storage of the strains at low temperature. Preliminary data indicate that the resistance is based on a single point mutation in a PBP. If so, resistance against amoxycillin will probably increase rapidly in the near future. 5.4. Resistance to tetracycline Almost all H. pylori strains (> 99%) are sensitive to tetracycline (MIC < 4 g l)1), both in vivo and in vitro. Tetracycline acts by inhibiting bacterial protein synthesis, and its activity is barely affected by low pH. Although there have been some reports of spontaneous tetracycline-resistant clinical isolates (Midolo et al. 1996; Piccolomini et al. 1997), the mechanism responsible for this resistance is unknown. Our own unpublished data indicate that, when H. pylori is subjected to serial passage with increasing concentrations of tetracycline, resistant bacteria with an MIC exceeding 32 g l)1 can easily be obtained. However, these bacteria rapidly lose their resistance when passaged once again on tetracycline-free media. This indicates that these bacteria are tolerant rather than resistant to tetracycline. In contrast with this, we recently obtained a strain with a low level of resistance to tetracycline (MIC 8 g l)1). The resistance of this strain is stable upon repeated passage on tetracycline-free media and using the puri®ed chromosomal DNA of this resistant strain as source, resistance could easily be transmitted to a sensitive strain through natural transformation. 5.5. Resistance to less commonly used antibiotics Cipro¯oxacin and most related ¯uoroquinolones display a signi®cant decrease of their activity at low pH, rendering them less effective for the treatment of H. pylori infections. Cipro¯oxacin acts by inhibiting the bacterial DNA gyrase, which results in inhibition of bacterial replication. Resistance of H. pylori to cipro¯oxacin depends on the development of a mutation in the gyrA gene that encodes the DNA gyrase A subunit. The majority of the observed mutations occur at a single position (Asp-91) within this subunit and are associated with resistance to 8 g l)1 cipro¯oxacin (Moore et al. 1995b). Trova¯oxacin is a novel ¯uoroquinolone that is not yet approved for treatment of H. pylori infections. It is a promising candidate for treatment because its activity is barely affected by low pH. On the downside, it has been associated with severe side-effects, and in spite of the fact that trova¯oxacin has not yet been widely introduced to the

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market, there is already a high level of resistance to this antibiotic (Rozynek et al. 1997; Debets-Ossenkopp et al. 1999b). Most likely, the observed resistance is the result of exposure to related quinolones used, for instance, in the treatment of respiratory infections and severe hospital infections. Because of this, the feasibility of using trova¯oxin in the treatment of H. pylori infections needs further investigation. Both rifampicin and streptomycin are not commonly used in the treatment of H. pylori infections. Because bacteria that are resistant to rifampicin or streptomycin can easily be acquired in the laboratory through simple in vitro selection of spontaneous mutants, these antibiotics have been used as convenient markers for successful transformation. By analogy with other bacteria, resistance to rifampicin and streptomycin are believed to be the result of a mutation in the b-subunit of the RNA polymerase and the S12 ribosomal protein of H. pylori, respectively. However, no formal con®rmation of this has been presented. 5.6. Resistance to bismuth compounds and antisecretory agents Bismuth is a topical antimicrobial drug that disrupts the integrity of the bacterial cell wall; it effectively lyses H. pylori. Bismuth compounds are widely used in combination therapies because they act by a mechanism that differs from that of antibiotics and, hence, have a complementary effect. Until now, no resistance to bismuth compounds has been described for H. pylori. Proton-pump inhibitors, such as omeprazole and lansoprazole, have some direct action against H. pylori in vitro, the effect mediated through the activated, sulphenamide form of these compounds (AlarcoÂn et al. 1998b). In vivo, this activation takes place selectively within the vesicles of the parietal cell where the compound is subsequently and irreversibly bound to the proton pump. Therefore, it is not surprising that when used alone, these drugs are unable to eradicate H. pylori. Nevertheless, the addition of these antisecretory agents to a combination of antibiotics signi®cantly increases the ef®cacy of H. pylori eradication regimens (Lind et al. 1999; Bayerdorffer 2000). This is thought to be due to the higher ef®cacy of most antibiotics under less acidic circumstances. 6 . T R A N S F E R O F A N TI B I O T I C RESISTANCE In spite of the fact that clinical isolates of H. pylori often contain small cryptic plasmids, thus far all antibiotic resistance mechanisms in H. pylori seem to be chromosomally mediated. This is in contrast to antibiotic resistance

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found in other bacteria where resistance genes are frequently found on extrachromosomal elements such as plasmids, or located on mobile elements such as transposons. There are indications that most antibiotic resistances are acquired de novo, but transfer from resistant to sensitive bacteria probably also occurs. Under laboratory conditions, natural transformation and transformation by electroporation has been used by several groups as a means of transferring antibiotic resistance markers to other H. pylori strains (Nedenskov-Sorensen et al. 1990; Haas et al. 1993; Segal and Tompkins 1993; Tsuda et al. 1993; Wang et al. 1993; Segal 1995; Taylor 1996). There is also experimental evidence for the occurrence of conjugation (Kuipers et al. 1998), and also bacteriophages have been isolated from H. pylori (Schmid et al. 1990; Heintschel von Heinegg et al. 1993), hence it is likely that transduction will also occur. It is, however, unknown whether the exchange of genetic material through natural transformation, transduction or conjugation plays a signi®cant role in the acquisition of antibiotic resistance of H. pylori. The observation that antibiotic resistance is located on the chromosome of H. pylori rather than on plasmids, combined with the lack of amoxycillin-resistant H. pylori, despite the fact that such resistance is easily acquired by other bacteria, makes extensive exchange of DNA with other bacterial species unlikely, but it cannot be excluded as there is ample evidence for frequent genetic exchange (Achtman et al. 1999; Covacci et al. 1997). 7. CONCLUSIONS Ultimately, antimicrobial resistance results in treatment failure. On the other hand, treatment failure is often the result of the induction of antimicrobial resistance. The development of multidrug-resistant organisms endangers the ef®cacy of our current treatment regimens. The observed increase in antimicrobial resistance calls for the development of new active compounds for the treatment of H. pylori infections. Knowledge of the entire genomic sequence of H. pylori may allow the rapid development of novel drugs that speci®cally target vital functions of H. pylori, but until we have those, we should try to stop the rapid spread and induction of resistance. To this purpose, monotherapy should never be used and susceptibility testing should be performed whenever drugs are prescribed for which a high local resistance has been reported. In the light of H. pylori-associated disease and its prevalence, it is remarkable that most countries do not have regional surveillance programmes that monitor the evolution of H. pylori resistance, in order to allow timely adaptation of the treatment regimens. Perhaps the latter is explained by the lack of a standardized susceptibility

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