a Department of Microbiology, Hospital Universitario de la Princesa, Diego de León 62, 28006 Madrid; b Gastroenterology Unit, Hospital del Niño Jesus, Madrid, Spain
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Abstract |
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Introduction |
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Currently, treatment with antibiotics is widely recommended for several of these diseases, such as peptic ulcer or MALT lymphoma.3 Amoxycillin, tetracycline, metronidazole and macrolides (mainly clarithromycin) are used most frequently, combined with proton pump inhibitors or bismuth salts, for the treatment of H. pylori infections, although tetracycline is not used in children. However, side effects, poor compliance and resistance to the antibiotics used are common causes of treatment failure. Several authors have found a correlation between infection with resistant H. pylori clinical isolates and a lower eradication rate compared with susceptible H. pylori-infected patients.4
Some studies report that strains obtained from children show higher percentages of resistance to clarithromycin than strains from adults in the same area, although similar data are not found for metronidazole.5
The purpose of this study was to determine the evolution of clarithromycin, metronidazole and amoxycillin resistance in Spanish H. pylori clinical isolates obtained from paediatric patients during a 9 year period.
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Materials and methods |
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A total of 246 strains of H. pylori were included: 45 from 19911993, 81 from 19941996, and 120 from the 19971999 period. All the strains were tested against clarithromycin and amoxycillin; against metronidazole, 42 strains were tested from the 19911993 period, 79 from the 19941996 period and 118 from the 19971999 period. Patients aged 318 years old living in the Madrid area were included in the study.
H. pylori clinical isolates were obtained from diagnostic gastric biopsies, cultured on selective and non-selective plates, which were incubated at 37°C in a microaerophilic atmosphere for 710 days. Strains were identified according to colony morphology, Gram's stain and positive reaction with urease, catalase and oxidase tests. They were then stored at 80°C in trypticase soy broth containing 20% glycerol. H. pylori NCTC 11638 was used as control in the susceptibility tests.
MIC determination
Clarithromycin was obtained from Abbott Laboratories (Madrid, Spain) and metronidazole and amoxycillin from SigmaAldrich (Madrid, Spain). Antibiotics were dissolved following manufacturers' recommendations, to a standard concentration of active drug.
MICs were determined by an agar dilution technique using MuellerHinton agar plus 7% lysed horse blood. Plates contained two-fold dilutions of each antibiotic from 128 to 0.008 mg/L. Isolates were grown for 48 h in brainheart infusion broth plus 10% fetal calf serum, and 106 cfu applied to plates using a Steer replicator. Plates were incubated in 5% CO2 at 37°C for 35 days and the MIC recorded as the lowest concentration of the antibiotic inhibiting visible growth. Resistance was defined as: clarithromycin, MIC 1 mg/L (criteria recently recommended by the NCCLS6); metronidazole, MIC
8 mg/L; and amoxycillin, MIC
2 mg/L.
Statistical analysis
The confidence intervals of the prevalence rates were calculated and a 2 linear trend was used to compare the increase of resistance throughout the study period.
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Results |
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Discussion |
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Several authors have used different in vitro methods to detect susceptibility, including disc diffusion, Etest, agar dilution and agar breakpoint, although discrepancies between methods have been reported.7 Recently, agar dilution has been recommended by the NCCLS8 and should be used to detect in vitro susceptibility or resistance to antibiotics in H. pylori. The method used by us was similar to that recommended by the NCCLS, although the incubation atmosphere was different: we used a CO2 incubator instead of a jar with a gas generating system, because we found no discrepancies when we compared both methods in a previous study and it is more practical for our laboratory. The NCCLS have recently published breakpoints for clarithromycin,6 but these are lacking for metronidazole and amoxycillin.
The overall percentage of resistance to clarithromycin was 21.1%; this was rare before 1994 and had increased to 28.3% during 19971999. This is high compared with other data obtained in a different area.9 Metronidazole resistance has also increased over the 9 years and now exceeds clarithromycin resistance.5 Our patients attended for the first time with H. pylori infection at the Gastroenterology Unit. Thus, antibiotic resistance can be considered primary.
Very few data exist for resistance in strains isolated from children; most authors have studied strains from adults. The prevalence of clarithromycin resistance is frequently lower than metronidazole resistance, being usually <10%, although a 50% rate was reported in Peru.7 Resistance seems to be lower in countries where macrolides are not widely used. In European countries the resistance rate ranged from 2.3 to 10%.4,7,10 Even where macrolide resistance is rare, metronidazole resistance can be as high as 90%.9
In contrast to clarithromycin and metronidazole, no amoxycillin resistance was observed. This is despite the wide use of this antibiotic, both alone and combined with clavulanic acid, to treat H. pylori infections or respiratory tract infections in children and adults. However, resistance has already appeared in some countires and although the prevalence is still low, it could increase.
As the prevalence of resistance to clarithromycin, metronidazole and amoxycillin varies according to the country or even the population within a country, local data are necessary in order to choose the best treatment for H. pylori.
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Acknowledgments |
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Notes |
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References |
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2 . Drumm, B., Koletzko, S. & Oderda, G. (2000). H. pylori infection in children: a consensus statement. European Pediatric Task Force on H. pylori. Journal of Pediatric Gastroenterology and Nutrition 30, 20713.[Medline]
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European Helicobacter pylori Study Group. (1997). Current European concepts in the management of Helicobacter pylori infection. The Maastricht Consensus Report. Gut 41, 813.
4 . Megraud, F. (1997). Resistance of Helicobacter pylori to antibiotics. Alimentary Pharmacology and Therapeutics 11, Suppl. 1, 4353.[ISI][Medline]
5 . Glupczynski, Y., Megraud, F., Andersen, L. P. & López-Brea, M. (1999). Antibiotic susceptibility of H. pylori in Europe in 1998: results of the third multicentre study. Gut 45, Suppl. III, A3.[ISI]
6 . National Committee for Clinical Laboratory Sandards. (2000). Performance Standards for Antimicrobial Susceptibility Testing: Tenth Informational Supplement (Aerobic Dilution) M100-S10 (M7). NCCLS, Wayne, PA.
7 . Alarcón, T., Domingo, D. & López-Brea, M. (1999). Antibiotic resistance problems with Helicobacter pylori. International Journal of Antimicrobial Agents 12, 1926.[ISI][Medline]
8 . National Committee for Clinical Laboratory Standards. (1999). Performance Standards for Antimicrobial Susceptibility Testing: Ninth Informational Supplement M100-S9. NCCLS, Wayne, PA.
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Mukhopadhyay, A. K., Kersulyte, D., Jeong, J.-Y., Datta, S., Ito, Y., Chowdhury, A. et al. (2000). Distinctiveness of genotypes of Helicobacter pylori in Calcutta, India. Journal of Bacteriology 182, 321927.
10 . López-Brea, M., Domingo, D., Sánchez, I. & Alarcón, T. (1997). Evolution of resistance to metronidazole and clarithromycin in Helicobacter pylori clinical isolates from Spain. Journal of Antimicrobial Chemotherapy 40, 27981.[Abstract]
Received 5 October 2000; returned 15 February 2001; revised 21 March 2001; accepted 21 May 2001