JAC Advance Access originally published online on November 3, 2006
Journal of Antimicrobial Chemotherapy 2007 59(1):160-161; doi:10.1093/jac/dkl449
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Correspondence |
Antimicrobial effectiveness of ketoconazole against metronidazole-resistant Helicobacter pylori isolates from Iranian dyspeptic patients
1 Microbiology Department, Faculty of Sciences, University of Tehran Tehran, Iran 2 Digestive Diseases Research Centre, Tehran University of Medical Sciences Tehran, Iran
*Corresponding author. Tel: +98-2161112460; Fax: +98-2166405141; E-mail: siavoshi{at}khayam.ut.ac.ir
Keywords: antimycotics , oral yeasts , dual activity
Sir,
The rate of Helicobacter pylori infection in Iran reaches up to 85% and a considerable proportion of infected individuals develop gastric diseases.1 Furthermore, the eradication rate of H. pylori infection is low, mainly due to a considerable number (37%) of metronidazole-resistant strains.2 Accordingly, investigators have been trying to substitute this antibiotic. Since fatty acids, namely cholesteryl glucosides, have been found in the cell membrane of Helicobacter species, investigators have speculated that imidazole antimycotics such as ketoconazole might interfere with the biosynthesis of these fatty acids from cholesterol.3 The dual activity of ketoconazole against both H. pylori and fungi might be valuable because oral yeasts have been proposed as potent reservoirs as well as protective vehicles for transmission of H. pylori to the human gastrointestinal tract.4 Thus design of chemotherapeutic regimens containing ketoconazole will benefit patients by eradicating H. pylori as well as reducing the number of yeast microflora harbouring H. pylori.
In this study the antibacterial activity of ketoconazole against metronidazole-resistant and -susceptible strains of H. pylori was assessed by agar dilution method (ADM) and disc diffusion method (DDM).
Fifty H. pylori isolates were obtained (2004–2005) from biopsy cultures of 50 patients who were referred to endoscopy units at the Digestive Diseases Research Centre of Tehran University of Medical Sciences, Tehran, Iran. Patients were grouped as those with oesophageal reflux (18), gastritis (17), ulcer (13) and gastric cancer (2). Biopsies were cultured on selective brucella agar (Merck) containing blood under microaerobic conditions. Bacterial isolates were identified as H. pylori on the basis of Gram's stain, showing Gram-negative spiral forms, and positive urease, oxidase and catalase tests. The resistance of 50 H. pylori strains to metronidazole (Sigma) was assessed by ADM according to CLSI (formerly NCCLS) guidelines. Metronidazole in ethanol was added to Mueller–Hinton agar (Merck) plates containing blood, to reach final dilutions of 32, 16, 8, 4 and 2 mg/L. Aliquots (5 µL) of bacterial suspensions with turbidities equivalent to that of a no. 2 McFarland standard were spot-inoculated on the surface of agar. Plates were examined for growth or inhibition after 3 days of appropriate incubation. The MIC of metronidazole was determined as >8 mg/L. The antibacterial effectiveness of ketoconazole (Sigma) against 50 H. pylori isolates was assessed, using ADM (15 isolates) and DDM (35 isolates). In ADM, ketoconazole in DMSO was added to Mueller–Hinton blood agar, with final dilutions of 32, 16, 8, 4 and 2 mg/L. Among fifteen isolates, five were resistant to metronidazole. Aliquots (5 µL) of bacterial suspensions with turbidities equivalent to that of a no. 2 McFarland standard were spot-inoculated on the surface of agar plates. Plates were examined after 3 days of microaerobic incubation and MICs were determined. Similar bacterial suspensions of Escherichia coli were spot-inoculated on Mueller–Hinton agar plates with serial dilutions of ketoconazole (128–4 mg/L). In DDM, serial dilutions of ketoconazole (32, 16, 8, 4 and 2 mg/L) were prepared in DMSO. Among 35 recruited isolates, eleven were metronidazole-resistant. Aliquots (100 µL) of bacterial suspensions were surface-inoculated on Mueller–Hinton blood agar. Each ketoconazole dilution (10 µL) was introduced into paper discs on the surface of the agar. After microaerobic incubation, growth inhibition zones were measured. Strains with inhibition zone diameters (IZDs) of 17–21 and >21 mm were considered as susceptible and highly susceptible, respectively.
Out of 50 isolates, 16 (32%) were resistant to metronidazole. Susceptibility to ketoconazole for 14/15 (93.3%) isolates recruited in ADM was determined at MIC 8 mg/L, although 8/15 (53.3%) were also inhibited at MIC 
4 mg/L and one (6.6%) was inhibited at an MIC of 16 mg/L. Five out of 15 isolates that were resistant to metronidazole showed high susceptibility to ketoconazole. Control E. coli exhibited growth on plates containing 128 mg/L ketoconazole. In DDM, the means of IZDs for 35 H. pylori isolates at different dilutions of ketoconazole were determined (Table 1). Susceptibility of bacterial isolates was determined according to IZDs at 8 mg/L. Nineteen isolates (54.1%) were susceptible and 16 isolates (45.9%) were highly susceptible to ketoconazole. Among 11 metronidazole-resistant strains, 7 (63.6%) were susceptible and 4 (36.4%) were highly susceptible to ketoconazole. H. pylori isolates from patients with gastritis, gastric ulcer or cancer were similarly susceptible to ketoconazole.
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All the 50 isolates were inhibited by ketoconazole, although E. coli was highly resistant. The MIC of ketoconazole was determined as 8 mg/L in both ADM and DDM. Similarly, inhibitory concentrations of the antimycotic miconazole (MIC 2–32 mg/L) against H. pylori have been reported.5
Ketoconazole can be considered as a substitute for metronidazole. Regarding the safety of ketoconazole, it is proposed that the dose required to inhibit mammalian cells is much higher than that required for fungi6 or bacteria.5 Since the intracellular existence of H. pylori in yeast plays an important role in the persistence of H. pylori in the human oral cavity,4 administration of ketoconazole not only leads to eradication of H. pylori, but might also reduce the chance of recurrence of bacterial infection by affecting colonization of yeasts in the gastrointestinal tract.
Transparency declarations
None to declare.
Acknowledgements
Part of this study was presented at the European Helicobacter Study Group Workshop, Stockholm, Sweden, 2003. This study was performed as part of an MSc project, funded by the Research Council of the University of Tehran.
References
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2 Siavoshi F, Pourkhajeh AH, Merat S, et al. (2000) Susceptibility of various strains of Helicobacter pylori to selected agents. Arch Iranian Med 3:60–3.
3
Haque M, Hirai Y, Yokota K, et al. (1996) Lipid profile of Helicobacter spp: presence of cholesteryl glucoside as a characteristic feature. J Bacteriol 178:2065–70.
4 Siavoshi F, Salmanian AH, Akbari F, et al. (2005) Detection of Helicobacter pylori-specific genes in the oral yeast. Helicobacter 10:318–22.[CrossRef][ISI][Medline]
5 von Recklinghausen G, Di Maio C, Ansorg R. (1993) Activity of antibiotics and azole antimycotics against Helicobacter pylori. Zentralbl Bakteriol 280:279–85.[ISI][Medline]
6
Hitchcock C, Dickinson K, Brown SB, et al. (1990) Interaction of azole antifungal antibiotics with cytochrome P-450-dependent 14
-sterol demethylase purified from Candida albicans. J Biochem 266:475–80.
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