In vitro activity of rifampicin against Helicobacter pylori isolated from children and adults

Shigeru Fujimuraa,b,*, Seiichi Katoc, Takeshi Kawamurad and Akira Watanabeb

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antibiotic resistance in Helicobacter pylori has been a growing problem with current treatment regimens. However, second-line therapy for cases with eradication failure remains to be established. The aim of this study was to evaluate (in vitro) the potential use of rifampicin for H. pylori eradication. The susceptibility of 52 H. pylori strains isolated from children and adults to amoxicillin, clarithromycin, metronidazole and rifampicin was determined. Forty H. pylori isolates susceptible to these antibiotics were investigated for in vitro inducement of resistance. All isolates were susceptible to rifampicin. None of the H. pylori isolates tested acquired rifampicin resistance during prolonged exposure in in vitro inducement experiments. It is suggested that rifampicin has stable activity against H. pylori.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Helicobacter pylori colonization is an important factor in the pathogenesis of chronic gastritis, peptic ulcers and gastric cancer. A combination of proton pump inhibitor and two antibiotics (new triple therapy) has been widely preferred as the standard regimen for eradicating H. pylori. At present, amoxicillin, clarithromycin and metronidazole are the antibiotics used most frequently in eradication regimens.1 Adult studies have demonstrated that this new triple therapy achieves high eradication rates of between 80% and 90%.2 On the other hand, H. pylori resistance to clarithromycin and metronidazole has been found worldwide, and is thought to be the major factor in eradication failure.3 In strains isolated from patients with eradication failure, acquired resistance to the antibiotic used, especially clarithromycin, is frequently detected.3 Efficient second-line therapy has not yet been established. Clarithromycin, a new macrolide, exhibits excellent antimicrobial activity against respiratory pathogens and consequently has commonly been used in both adult and paediatric populations; however, H. pylori shows cross-resistance among macrolides.4 In contrast, in many countries, rifampicin is used almost exclusively for tuberculosis or atypical tuberculosis. There are fewer rifamycins in clinical use generally and there is only one preliminary study on the susceptibility of H. pylori to rifabutin.5 In the present study, we investigated the in vitro susceptibility of H. pylori to rifampicin with the view that rifampicin is reserved as second-line therapy for the eradication of H. pylori. Inducement of resistance to each antibiotic (in vitro) was also determined.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients and bacterial strains

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 5–16 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.6–8 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 1/2 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 Mueller–Hinton 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).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
MICs

MICs of each antibiotic for H. pylori are presented in the TableGo. Eleven (41%) (MIC range 6–16 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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Table. MICs (mg/L) of antibiotics for 52 clinical isolates of H. pylori
 
In vitro inducement of drug resistance

For metronidazole, the MIC90 rose gradually during in vitro drug resistance inducement procedures and acquired resistance after the ninth transfer (FigureGo). These strains retained susceptibility to amoxicillin, clarithromycin and rifampicin during the inducement, although the MIC90 of amoxicillin rose.



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Figure. Comparison of in vitro inducement of amoxicillin, clarithromycin, metronidazole and rifampicin resistance. Symbols denote amoxicillin (open triangles), clarithromycin (filled triangles), metronidazole (open circles) and rifampicin (filled circles). These susceptible strains did not acquire resistance to rifampicin, clarithromycin or amoxicillin after 10 transfers and being exposed to 1/2 x MIC of each antibiotic.

 

    Discussion
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 Materials and methods
 Results
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In this study, the percentage of resistance to clarithromycin was significantly higher for children (41%) than for adults (4%) (P < 0.01). In children, macrolides including clarithromycin have frequently been used in the treatment of various diseases such as respiratory tract infections in clinical practice. This may be the reason for the high rate of clarithromycin resistance in H. pylori observed in children. Although the MIC breakpoint of rifampicin for H. pylori has not yet been established, Heep et al.6 reported that the MIC of rifampicin for resistant mutants of H. pylori ranged from 32 to 64 mg/L. This study demonstrated that the MIC of rifampicin for H. pylori remains low. No resistance to rifampicin was observed in any H. pylori strains studied. The concentration of rifampicin in gastric mucosa is 1.2 mg/L 12 h after administration of 450 mg of the drug.9 Because the MIC range of rifampicin was <0.016– 0.75 mg/L in this study, it is assumed that H. pylori would be susceptible to the usual dose of rifampicin in clinical practice. Furthermore, rifampicin is stable in acid and its side effects are infrequent. It can therefore be expected to exhibit a high degree of clinical efficacy.

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.


    Notes
 
* Correspondence address. Department of Microbiology, Miyagi University, 1 Gakuen, Taiwa-cho, Miyagi pref. 981-3298 Japan. Tel/Fax: +81-22-377-8289; E-mail: hujimura{at}mail.sp.myu.ac.jp Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Lind, T., Megraud, F., Unge, P., Bayerdorffer, E., O'Morain, C., Spiller, R. et al. (1999). The MACH 2 study: role of omeprazole in eradication of Helicobacter pylori with 1 week triple therapies. Gastroenterology 116, 248–53.[ISI][Medline]

2 . European Helicobacter pylori Study Group. (1997). Current European concepts in the management of Helicobacter pylori infection: the Maastricht Consensus Report. Gut 41, 8–13.[Abstract/Free Full Text]

3 . Adamek, R. J., Suerbaum, S., Pfaffenbach, B. & Opferkuch, W. (1998). Primary and acquired Helicobacter pylori resistance to clarithromycin, metronidazole and amoxicillin: Influence on treatment outcome. American Journal of Gastroenterology 93, 386–9.[ISI][Medline]

4 . Glupczynski, Y. & Burette, M. (1999). Drug therapy for Helicobacter pylori infection: Problems and pitfalls. American Journal of Gastroenterology 85, 1545–51.

5 . Pilotto, A., Franceschi, M., Rassu, M., Furlan, F. & Scagnelli, M. (2000). In vitro activity of rifabutin against strains of Helicobacter pylori resistant to metronidazole and clarithromycin. American Journal of Gastroenterology 95, 833–4.[ISI][Medline]

6 . Heep, M., Beck, D., Bayerdorffer, E. & Lehn, N. (1999). Rifampin and rifabutin resistance mechanism in Helicobacter pylori. Antimicrobial Agents and Chemotherapy 43, 1497–9.[Abstract/Free Full Text]

7 . National Committee for Clinical Laboratory Standards. (2000). Approved Standard M100-S10. MIC Testing Supplemental Tables. NCCLS, Villanova, PA.

8 . Glupczynski, Y., Megraud, F., Andersen, L. P. & Lopez-Brea, M. (1999). Antibiotic susceptibility of H.pylori in Europe in 1998: results of the third multicentre study. Gut 45, Suppl. 3, A3.[ISI]

9 . Furesz, S., Scotti, R., Pallanza, R. & Mapelli, E. (1967). Rifampicin: A new rifamycin: Absorption, distribution, and elimination in man. Arzneimittel Forschung 7, 534–7.

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, 839–42.

Received 29 August 2001; returned 9 November 2001; revised 22 November 2001; accepted 7 December 2001