Resistance of Helicobacter pylori isolated in Israel to metronidazole, clarithromycin, tetracycline, amoxicillin and cefixime

Zmira Samra1,2,*, Haim Shmuely2,3, Yaron Niv2,3, Gabriel Dinari4, Douglas J. Passaro5, Alex Geler3, Eyal Gal3, Michal Fishman3, Jacklin Bachor1 and Jacob Yahav2

1Microbiology Laboratories, 2Helicobacter pylori Research Institute and 3Department of Gastroenterology, Rabin Medical Center, Beilinson Campus, Petah Tiqva; 4Institute of Pediatric Gastroenterology and Nutrition, Schneider Children’s Medical Center of Israel, Petah Tiqva, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 5Division of Epidemiology and Biostatistics, University of Illinois, Chicago, IL, USA

Received 13 November 2001; returned 21 January 2002; revised 18 February 2002; accepted 27 February 2002.


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The resistance of Helicobacter pylori isolated in Israel to metronidazole, clarithromycin, tetracycline, amoxicillin and cefixime was tested in 138 isolates, including 28 from treatment failures. No resistance to tetracycline was detected. Resistance to amoxicillin was found in one isolate (MIC = 1.5 mg/L) from an untreated patient, and resistance to cefixime in two isolates from each group (P = 0.18). Resistance to metronidazole and clarithromycin was much higher in the isolates from treated than from untreated patients: 60.7% and 38.2% for metronidazole (MIC >= 8 mg/L) (P = 0.03); 46.4% and 8.2% for clarithromycin (MIC >= 2 mg/L) (P < 0.001). Therapeutic outcome would benefit from susceptibility testing, especially after treatment failure.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Helicobacter pylori infection has been linked to peptic ulcer disease and gastric cancer.1 In Israel, the prevalence of H. pylori infection is c. 60%, and the annual incidence of gastric cancer is about 15 per 100 000 population.2 Treatment of H. pylori infection usually consists of a proton-pump inhibitor (PPI) in combination with two antibiotics; the cure rate ranges from 80 to 90%.3 The antibiotics most commonly used are clarithromycin, metronidazole, amoxicillin and tetracycline. Resistance to metronidazole or clarithromycin is high and is expected after treatment failure with therapeutic combinations containing one of these compounds.4 There are few data on antibiotic resistance of H. pylori in Israel.5

The aim of this study was to assess the resistance of H. pylori isolates from untreated and treated patients in Israel to five antimicrobial agents. Cefixime was included because previous investigations have found it to possess anti-H. pylori activity both in vivo and in vitro.6


    Materials and methods
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 Materials and methods
 Results
 Discussion
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Bacterial strains

Between January 2000 and June 2001, 138 clinical isolates of H. pylori were isolated from antral biopsy specimens of 138 dyspeptic adults (71 females, 67 males; median age 55 years), one isolate from each patient, at the Rabin Medical Center, a major tertiary hospital in central Israel. Twenty-eight patients had been treated previously for H. pylori infection with a PPI and two or more antimicrobial agents: 15 with amoxicillin, clarithromycin and metronidazole; seven with clarithromycin and metronidazole, three with amoxicillin and clarithromycin; two with clarithromycin, metronidazole and tetracycline; and one with amoxicillin and metronidazole.

The specimens were inoculated directly on to Columbia blood agar (Difco, Detroit, MI, USA) supplemented with yeast extract (5 g/L), laked lysed horse blood (7%), vancomycin (3 mg/L), colistin sulphate (7.5 mg/L), nystatin (12 500 IU/L) and co-trimoxazole (5 mg/L). Cultures were incubated for 72 h at 37°C under microaerophilic conditions. H. pylori isolates were identified by colony morphology, characteristic spiral morphology on Gram staining, and positive findings for catalase, urease and oxidase tests.

MIC determination

The MICs of amoxicillin, clarithromycin, metronidazole, tetracycline and cefixime were determined by Etest (AB Biodisk, Solna, Sweden). Suspensions to a density of a 3 McFarland standard were prepared in Columbia broth (Difco) from 72 h blood agar subculture and spread (100 µL) on Petri plates (90 mm diameter) containing Mueller–Hinton agar (BBL, Becton Dickinson Microbiology Systems, Cockeysville, MD, USA) supplemented with yeast extract (5 g/L), horse serum (12%) and nicotinamide adenine dinucleotide (25 mg/L). Etest strips were placed on the plates as soon as the inocula were absorbed into the agar. The plates were incubated at 37°C under microaerophilic conditions. MIC values were determined after 72 h incubation. Resistance was defined as follows: amoxicillin, MIC >= 1.5 mg/L; tetracycline >= 4 mg/L; clarithromycin >= 2 mg/L; metronidazole >= 8 mg/L7 and cefixime >= 2 mg/L. H. pylori strain ATCC 43526 was used for quality control of the selective medium and ATCC 43504 for the quality control of the susceptibility tests.

Prevalence of resistance was calculated between treated and untreated patients for each antibiotic with the Mantel–Haenszel {chi}2 test or Fisher’s exact test. Cornfield or exact 95% confidence intervals (CIs) were calculated as appropriate.


    Results
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The distribution of the MICs of the five antimicrobial agents is given in Table 1. All H. pylori isolates were sensitive to tetracycline, with an MIC of <=0.38 mg/L. Resistance to amoxicillin was detected in only one isolate (MIC = 1.5 mg/L) recovered from an untreated patient. This isolate was found to be sensitive to all the other antimicrobial agents tested. MICs of clarithromycin and metronidazole of >=256 mg/L were observed, respectively, in 5.4% and 30% of isolates from untreated patients, and 25% and 42.6% of isolates from treated patients.


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Table 1.  MICs for H. pylori isolates from 110 untreated patients and 28 treated patients
 
The prevalence of H. pylori resistance to all five antibiotics tested is given in Table 2. Resistance to cefixime was found in two isolates (1.9%) from untreated patients, both sensitive to all the other antibiotics, and in two isolates (7.2%) from treated patients, of whom one was also resistant to clarithromycin and the other to both clarithromycin and metronidazole. Of the isolates recovered from untreated patients, 8.2% and 38.2% were resistant to clarithromycin and metronidazole, respectively. Corresponding rates for these agents for the isolates from treated patients were 46.4% (P < 0.001) and 60.7% (P = 0.03). Dual resistance was found in 5.5% of untreated patients (6/110) and 32.1% of treated patients (9/28) (P < 0.001). In eight of the nine isolates from the treated patients, dual resistance was to metronidazole and clarithromycin.


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Table 2.  Prevalence of resistance in H. pylori isolates from untreated and treated patients
 
In the treated group, all the clarithromycin- or metronidazole-resistant isolates were from patients treated previously with clarithromycin or metronidazole, respectively. No statistically significant association was found between gender and resistance for any of the antibiotics studied (data not shown).


    Discussion
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This study shows that the prevalence of metronidazole and clarithromycin resistance is significantly higher in H. pylori isolates recovered from treated patients than in isolates from untreated patients.

The clinical significance of the resistance level of H. pylori on in vitro studies has long been disputed. Nevertheless, recent meta-analyses have provided conclusive evidence that H. pylori resistance to metronidazole or clarithromycin reduces the efficacy of regimens containing these agents.8 As antimicrobial resistance is the most important determinant after patient compliance of outcome of treatment,9 and metronidazole and clarithromycin are used in almost all regimens, the increasing resistance of H. pylori to these agents is of clinical importance. In our study, all clarithromycin- or metronidazole-resistant isolates from the treated group were recovered from patients treated previously with clarithromycin or metronidazole, respectively.

No resistance to tetracycline was found, and the sole strain that was resistant to amoxicillin was recovered from an untreated patient (MIC = 1.5 mg/L). Tetracycline possessed the most significant anti-H. pylori activity in vitro, and it may be used as an alternative agent in the presence of clarithromycin and/or metronidazole resistance in adult patients.

Cefixime was found to be highly active against clarithromycin-resistant strains of H. pylori. It had similar activity to tetracycline against both clarithromycin-sensitive and clarithromycin-resistant H. pylori isolates from untreated and treated patients.

Dual resistance to metronidazole and clarithromycin was common (28%) in isolates from treated patients. Previous studies have shown that for patients infected by clarithromycin-resistant strains, the use of treatment regimens containing both metronidazole and clarithromycin dramatically decreases the H. pylori eradication rate.10 Thus, in countries where metronidazole resistance is common, physicians might well refrain from using a combination of metronidazole and clarithromycin in order to prevent the emergence of more resistant strains.

In conclusion, the high prevalence of both single and dual metronidazole and clarithromycin resistance in isolates recovered from treated patients warrants special attention. Tetracycline and cefixime are potential alternative agents for the treatment of clarithromycin- and metronidazole-resistant H. pylori infection. Further clinical studies are needed to confirm these data in vivo.


    Acknowledgements
 
We thank Professor I. Ofek for helpful discussions and critical review of the manuscript. We also thank Charlotte Sachs and Gloria Ginzach of the Editorial Board, Rabin Medical Center, Beilinson Campus, for their editorial assistance.


    Footnotes
 
* Correspondence address. Department of Clinical Microbiology, Rabin Medical Center, Beilinson Campus, Petah Tiqva 49100, Israel. Tel: +972-3-937-6725; Fax: +972-3-921-8466; E-mail: zsamra{at}clalit.org.il Back


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1 . Parsonnet, J. (1998). Helicobacter pylori: The size of the problem. Gut 1, Suppl. I, S6–9.

2 . Central Cancer Registry. (1995). Annual Report. Ministry of Health, Jerusalem, Israel.

3 . Walsh, J. H. & Peterson, W. L. (1995). The treatment of Helicobacter pylori infection in the management of peptic ulcer disease. New England Journal of Medicine 333, 984–91.[Free Full Text]

4 . Hass, C. E., Nix, D. E. & Schentag, J. J. (1990). In vitro selection of resistant Helicobacter pylori. Antimicrobial Agents and Chemotherapy 34, 1637–41.[ISI][Medline]

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6 . Ikeda, F., Yokota, Y., Mine, Y. & Tatsuta, M. (1990). Activity of cefixime against Helicobacter pylori and affinities for the penicillin-binding proteins. Antimicrobial Agents and Chemotherapy 34, 2426–8.[ISI][Medline]

7 . King, A. (2001). Recommendations for susceptibility tests on fastidious organisms and those requiring special handling. Journal of Antimicrobial Chemotherapy 48, 77–80.[Abstract/Free Full Text]

8 . Van der Wouden, E. J., Thijs, J. C., van Zwet, A. A., Sluiter, W. J. & Kleibeuker, J. H. (1999). The influence of in vitro metronidazole resistance on the efficacy of nitroimidazole-containing anti-Helicobacter pylori regimens: a meta-analysis. American Journal of Gastroenterology 94, 1751–9.[ISI][Medline]

9 . Graham, D. Y. (1998). Antibiotic resistance in Helicobacter pylori: implications for therapy. Gastroenterology 115, 1272–7.[ISI][Medline]

10 . Miyaji, H., Azuma, T., Ito, S., Suta, H., Ito, Y., Yamazaki, Y. et al. (1997). Susceptibility of Helicobacter pylori isolates to metronidazole, clarithromycin and amoxicillin in vitro and in clinical treatment in Japan. Alimentary Pharmacology and Therapeutics 11, 1131–6.[ISI][Medline]