a Department of Microbiology and d Gastroenterology, Miyagi University, Miyagi pref. Japan; c Department of Pediatrics, Tohoku University School of Medicine; b Department of Respiratory Oncology, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan
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Abstract |
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Introduction |
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Materials and methods |
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A total of 52 patients underwent endoscopy and gastric biopsy because of gastrointestinal symptoms at Tohoku University Hospital and Social Insurance Sendai Hospital. Among these patients, 27 were paediatric patients aged 516 years. Diagnosis included chronic gastritis (n = 15), gastric ulcer (n = 25) and duodenal ulcer (n = 12). Fifty-two H. pylori strains were isolated from gastric biopsy samples. All patients had no previous history of treatment for H. pylori. Informed consent was obtained from all patients or their parents before inclusion in the study.
MIC determinations
Antimicrobial susceptibility was tested under microaerobic conditions (5% CO2, 5% O2, 90% N2) for 72 h on Mueller Hinton agar (Eiken, Tokyo, Japan) supplemented with 5% defibrinated sheep blood (Sigma Chemical Co., St Louis, MO, USA) at 37°C. MICs of amoxicillin, clarithromycin, metronidazole and rifampicin were determined by the Etest (AB Biodisk, Solna, Sweden). Briefly, colonies were suspended in 1 mL of saline to achieve turbidity equivalent to that of a no. 2.0 McFarland opacity standard. Mueller Hinton agar plates supplemented with 5% defibrinated sheep blood were inoculated by confluent swabbing of the surface with the adjusted inoculum suspension. Etest strips were applied to the surface of the agar plates. H. pylori were considered to be resistant to rifampicin, amoxicillin, clarithromycin and metronidazole when the MICs were greater than 16, 0.5, 0.5 and 8 mg/L, respectively.68 H. pylori NCTC 49503 was included as control organism.
In vitro inducement of drug resistance studies
Forty H. pylori strains susceptible to each antibiotic were tested for in vitro inducement of drug resistance. The strains were incubated and transferred 10 times after being exposed to x MIC of each antibiotic by the agar dilution method. Briefly, a saline suspension equivalent to a no. 2.0 McFarland standard was prepared from a 72 h subculture from MuellerHinton agar supplemented with 5% defibrinated sheep blood. The inoculum was replicated directly on to the antibiotic-containing agar dilution plates. Agar dilution MIC determination was performed using two-fold dilutions (range, 16<0.063 mg/L) of each antibiotic in blood agar plates. The plates were incubated at 37°C under microaerobic conditions. Antibiotics tested were as follows: amoxicillin (Takeda Chemical Industries Ltd, Osaka, Japan); clarithromycin (Dainabot Co. Ltd, Osaka, Japan); metronidazole (Shionogi & Co. Ltd, Osaka, Japan); and rifampicin (Daiichi Pharmaceutical Co. Ltd, Tokyo, Japan).
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Results |
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MICs of each antibiotic for H. pylori are presented in the Table. Eleven (41%) (MIC range 616 mg/L) of 27 strains in children and one (4%) (MIC, 16 mg/L) of 25 strains in adults, were clarithromycin resistant. There was a significant difference in resistance rate between children and adults (P < 0.01). All strains studied were susceptible to amoxicillin and metronidazole. The MIC of rifampicin ranged from <0.016 to 0.25 mg/L in children and from 0.125 to 0.75 mg/L in adults; the MIC90 of rifampicin was 0.19 mg/L in children and 0.125 mg/L in adults. No rifampicin resistance was detectable by the Etest among the 52 strains.
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For metronidazole, the MIC90 rose gradually during in vitro drug resistance inducement procedures and acquired resistance after the ninth transfer (Figure). These strains retained susceptibility to amoxicillin, clarithromycin and rifampicin during the inducement, although the MIC90 of amoxicillin rose.
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Discussion |
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The incidence of clarithromycin-resistant strains has increased in recent years. Rifampicin has retained its activity against clarithromycin-resistant isolates of H. pylori, and does not appear to induce drug resistance during prolonged exposure. These results suggest that rifampicin may be useful as second-line therapy in patients with eradication failure. One advantage of rifampicin is that the frequency of its clinical use is extremely low, allowing little opportunity for the development of resistance. Furthermore, we have reported that the prevalence of H. pylori infection is low in patients with tuberculosis and/or atypical tuberculosis who were treated with rifampicin.10 Considering the data presented here and the results from in vitro inducement of resistance experiments, we believe that H. pylori strains would continue to be susceptible to rifampicin during therapy. However, further work would be required to establish efficacy.
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Notes |
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References |
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2
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9 . Furesz, S., Scotti, R., Pallanza, R. & Mapelli, E. (1967). Rifampicin: A new rifamycin: Absorption, distribution, and elimination in man. Arzneimittel Forschung 7, 5347.
10 . Fujimura, S., Kawamura, T., Asou, N., Takahashi, H. & Watanabe, A. (2000). Helicobacter pylori infection rate in patients treated with rifampicin: Eradication effect of rifampicin on Helicobacter pylori. Japanese Journal of Chemotherapy (in Japanese with English abstract) 48, 83942.
Received 29 August 2001; returned 9 November 2001; revised 22 November 2001; accepted 7 December 2001