Susceptibility of Various Strains of Helicobacter pylori to Selected Agents

F. Siavoshi Ph.D*., A.H. Pourkhajeh MD**, Sh. Merat MD**, H. Asl-Soleimani MD**, E. Heydarian*, M. Khatibian MD**, R. Malekzadeh MD* **

*Department of Microbiology, Tehran University, ** Digestive Disease Research Center, Tehran University of Medical Sciences

  • Abstract

    Background-It is well known that eradicating Helicobacter pylori (HP) is an essential step in curing peptic ulcer disease. Many regimens are currently available but none of them can achieve a 100% eradication rate. Furthermore, the eradication rate among Iranian patients appears to be even lower.
    Objective-We designed a study to determine the antimicrobial susceptibility of HP isolated from Iranian patients to the most commonly used antibiotics for HP eradication regimens.
    Methods-Isolates of HP were obtained from patients with dyspepsia during upper GI endoscopy. The samples were assessed for susceptibility to nitroimidazoles and clarithromycin (188 samples) and furazolidone (140) using the agar dilution method. Sixty-eight of the isolates were tested for susceptibility to amoxicillin and tetracycline using the disc diffusion procedure.
    Results- From 186 isolates, 69 (37.5%) were resistant to nitroimidazoles, and 27 (14.5%) to clarithromycin. Among the 140 isolates tested for susceptibility to furazolidone, 7 (5%) showed resistance. All 68 isolates tested with the disc diffusion procedure were susceptible to tetracycline but 5 (7%) exhibited resistance to amoxicillin.
    Conclusion- Resistance of HP to metronidazole and tinidazole is high among Iranian patients, with tetracycline and amoxicillin remaining the most efficient drugs. Furthermore, HP isolates from Iranian patients were found to be highly susceptible to furazolidone. This drug is readily available at low cost in Iran and may be a good substitute for metronidazole in HP eradicating regimens in this country.

    •

    Keywords · Helicobacter pylori · resistance · antibiotic · furazolidone · metronidazole · Iran

    Introduction

    Therapy has aimed at eradicating Helicobacter pylori (HP),which is the major etiologic agent in peptic ulcer disease (PUD). Unfortunately, none of the currently available regimens are able to achieve a 100% eradication rate, which is even less effective in Iran.1-3 It is presumed that failure to achieve an eradication rate as high as that observed in some other countries could be related to either patient compliance or bacterial resistance. The one-year reinfection rate in Iran is about 16.4% which points to the sub-optimal treatment regimens.2

    The antimicrobial most commonly used in HP eradication regimens include nitroimidazoles (such as metronidazole or tinidazole), clarithromycin, amoxicillin, tetracycline, and bismuth. Some of these (e.g. metronidazole and amoxicillin) are readily available in Iran and other developing countries and are less judiciously used in treating various infectious diseases, leading to conse-quently increased likelihood of resistance to these agents. Some other antibiotics like clarithromycin and tinidazole are not readily available and the pre-treatment exposure of Iranians to these agents is virtually zero.

    There are many reports concerning the emergence of resistant HP strains, especially to metronidazole.4-7 But to the best of our knowledge, there is no information on this pattern of resistance in Iran.

    The aim of the present study is to evaluate bacterial resistance among HP isolates obtained from Iranian patients. Based on our results, we will also suggest a regimen, which may be the most effective in eradicating HP in Iran and probably other developing countries.

    Materials and Methods

    Isolates of HP were obtained from Iranian patients with dyspepsia during upper GI endoscopy. None of the patients had received antimicrobial therapy for at least one week before endoscopy. Biopsies from antrum and corpus were cultured on brucella agar supplemented with 5-7% sheep blood, amphotericin B (6 mg/l) vancomycin incubation(10 mg/l) , trimethoprim (5 mg/l), and polymixin B (2500 IU/l). After 3-5 days of microaerobic at 37ºC, the cultures were tested for antimicrobial susceptibility using agar dilution5 and disc diffusion methods,8 bacterial suspensions were prepared in phosphate-buffered saline (pH 7.4, 0.01 M) with a density equivalent to a No. 1 McFarland standard. Egg yolk agar was prepared by adding egg yolk and 2,3,5-triphenyltetrazolium chloride to brucella agar as previously described.5,9,10

    Double dilutions of antimicrobials were added to the egg yolk agar in final concentrations ranging 0.5-64 µg/ml for nitroimidazoles, 0.125-64 µg/ml for clarithromycin, and 0.31-4 µg/ml for fura-zolidone. The plates were incubated at 37ºC for 24 hours to ensure sterility and were used within one week of preparation.

    The isolates were assessed for susceptibility to nitroimidazoles and clarithromycin (188 isolates), and furazolidone (140). Plates were spot-inoculated with 10 µl of bacterial suspension and incubated under microaerobic conditions for 3-5 days. Bacterial cultures related to each dilution of antimicrobials were examined and minimum inhibitory concentrations (MICs) were determined. The disc diffusion procedure was performed with 68 isolates by depositing 25 mg/ml amoxicillin, and 30 mg/ml tetracycline discs on the streak-inoculated egg yolk agar. Plates were incubated as mentioned earlier, and the diameters of the inhibition zones were recorded for each antibiotic.

    Results

    Resistant strains were defined as those that grew on the egg yolk agar medium containing more than 8 g/ml nitroimidazoles,11,12 2 µg/ml clarithromycin,13 and 0.5 µg/ml furazolidone, and those with growth inhibition zone diameter of equal or less than 13 mm for amoxicillin and 14 mm for tetracycline.

    From the 186 isolates, 69 (37.5%) were resistant to nitroimidazoles (Tables 1 and 2), and 27 (14.5%) to clarithromycin (Table 3). Among the 140 isolates tested for susceptibility to furazolidone, 7 (5%) were resistant (Table 4). All 68 isolates tested were susceptible to tetracycline with growth inhibition zone diameters of 20-60 mm. However 5 isolates exhibited resistance to amoxicillin (7%) with growth inhibition zone diameters of 10-12 mm. Inhibition zone diameters for the remaining 63 isolates were 18-60 mm.

    Discussion

    Reinfection and failure of HP eradication is a major problem in the treatment of PUD. The reported failure rates vary with different regimens and in different countries.

    Metronidazole-resistant strains occur in all populations and at different percentages app-roaching 70% in some areas.14,15 In our study, 37.5% of HP isolates were resistant to metronidazole. Metronidazole-based regimens are reported to have eradication rates above 90% in patients carrying metronidazole-sensitive strains.16 In Iran, the eradication rate for such regimens is about 55%17 which can be explained by the 37.5% resistance of HP to metronidazole observed in our study.

    Metronidazole is a frequently used drug in Iran, and therefore it is not unexpected to find such a high rate of resistance. However, the rate of resistance to tinidazole (which is not available in Iran) is similarly as high (38%). This can only be explained by cross-resistance with metronidazole.

    Substituting clarithromycin for metronidazole in traditional triple therapy has been reported to achieve eradication rates as high as 93%.18 This is in agreement with reports of 1-10% resistance of HP strains to macrolides.13 In our study, only 14.5% of the isolates were resistant to clarithro-mycin making this antibiotic a viable option for replacing metronidazole in HP eradication regimens. Unfortunately, clarithromycin is not currently available in Iran. Furthermore, its high cost and the likelihood of emergence of resistant strains make it a less attractive choice both in Iran and in other developing countries.19

    The prevalence of HP is about 90% in Iran,20 whereas in the Caucasian population of the United States and other developed countries the prevalence increases with age from approximately 10% at 20 years of age to about 50% at 60 years of age.21 This high prevalence of HP in Iran further emphasizes the importance of having an effective and low cost regimen to eradicate HP.

    Furazolidone is a low cost, highly efficient and well-tolerated antibiotic which has been used in HP eradicating regimens (in combination with metronidazole or as a substitute for it).22-25 In our study, 95% of the isolates were susceptible to furazolidone which is in accordance with the 81.6% eradication rate reported in Iranian patients treated with furazolidone-based regimens.22 A similar eradication rate of 80-86% has been reported from Colombia26 and even higher rates from other countries.25,27

    All isolates tested for tetracycline and 92.6% of those tested for amoxicillin were susceptible to the respective antibiotic. This high susceptibility has also been reported by others,28,29 indicating their importance in HP eradicating regimens.

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