A 9 year study of clarithromycin and metronidazole resistance in Helicobacter pylori from Spanish children

Manuel López-Breaa,*, Maria Josefa Martínezb, Diego Domingoa and Teresa Alarcóna

a Department of Microbiology, Hospital Universitario de la Princesa, Diego de León 62, 28006 Madrid; b Gastroenterology Unit, Hospital del Niño Jesus, Madrid, Spain


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The evolution of clarithromycin, metronidazole and amoxycillin resistance in 246 Spanish Helicobacter pylori clinical isolates, obtained from paediatric patients during a 9 year period, was determined by an agar dilution technique. Clarithromycin resistance (MIC 1 mg/L) was 2.27% (IC95 0.05–12.02) in 1991–1993, 20.98% (IC95 12.72–31.46) in 1994–1996 and 28.33% (IC95 20.48–37.28) in 1997–1999 (P < 0.01). Metronidazole resistance (MIC 8 mg/L) was 7.14% (IC95 1.49–19.48) in 1991–1993, 20.25% (IC95 12.04–30.79) in 1994–1996 and 43.90% (IC95 32.95–55.30) in 1997–1999 (P < 0.01). Amoxycillin resistance was not found (all strains showed MICs < 2 mg/L).


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Helicobacter pylori is a Gram-negative microaerophilic bacillus found in the human gastric mucosa and is associated with various digestive diseases, such as peptic ulcer, gastritis and mucosa-associated lymphoid tissue (MALT) lymphoma, and is considered a risk factor for the development of gastric cancer.1 H. pylori infection is most frequently acquired during childhood, causing various digestive diseases both in childhood and adulthood. Symptoms such as vomiting, epigastric or recurrent abdominal pain have been associated with H. pylori infection in children.2

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Microorganisms

A total of 246 strains of H. pylori were included: 45 from 1991–1993, 81 from 1994–1996, and 120 from the 1997–1999 period. All the strains were tested against clarithromycin and amoxycillin; against metronidazole, 42 strains were tested from the 1991–1993 period, 79 from the 1994–1996 period and 118 from the 1997–1999 period. Patients aged 3–18 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 7–10 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 Sigma–Aldrich (Madrid, Spain). Antibiotics were dissolved following manufacturers' recommendations, to a standard concentration of active drug.

MICs were determined by an agar dilution technique using Mueller–Hinton 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 brain–heart 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 3–5 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 {chi}2 linear trend was used to compare the increase of resistance throughout the study period.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
When considering all isolates of H. pylori from children over the 9 year period, the overall clarithromycin resistance was 21.13% (IC95 16.20–26.77). Metronidazole resistance was 23.01% (IC95 17.83–28.87). All the strains had MICs of amoxycillin >2 mg/L. The TableGo shows the percentage of resistance to clarithromycin and to metronidazole in the three 3 year study periods. A statistically significant difference was found for both clarithromycin and metronidazole when the linear trend of increase in resistance was studied. The cumulative percentage of strains inhibited at the different antibiotic concentration is shown in the FigureGo.


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Table. Number and percentage of clarithromycin- and metronidazole-resistant H. pylori strains during the study period
 


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Figure. Cumulative percentage of strains inhibited at the different MICs for the three antibiotics studied. Symbols: {diamondsuit}, amoxycillin; {blacksquare}, clarithromycin; {blacktriangleup}, metronidazole.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Currently, clarithromycin and metronidazole are considered as basic antibiotics in any H. pylori eradication regimen.4 However, resistance to both of these agents could appear during therapy, and primary resistance due to previous use of macrolides may be seen for other infections, such as respiratory infections, or nitroimidazoles for treatment of diseases, such as giardiasis, amoebiasis and vaginal infections.

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 1997–1999. 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.


    Acknowledgments
 
We are grateful to Brenda Ashley for her English lan-guage assistance and to Juan Carlos Sanz for statistical analysis and critical reading of manuscript. This work was supported in part by Fondo de Investigaciones Sanitarias de la Seguridad Social FIS 93/0114, FIS 95/0222 and FIS 99/0025. T.A. is a post-doctoral fellow of the Consejeria de Educación de la Comunidad Autónoma de Madrid.


    Notes
 
* Corresponding author. Tel/Fax: +34-913-090047; E-mail: mlbrea{at}microb.net Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Dunn, B. E., Cohen, H. & Blaser, M. J. (1997). Helicobacter pylori. Clinical Microbiology Reviews 10, 720–41.[Abstract]

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, 207–13.[Medline]

3 . 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]

4 . Megraud, F. (1997). Resistance of Helicobacter pylori to antibiotics. Alimentary Pharmacology and Therapeutics 11, Suppl. 1, 43–53.[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, 19–26.[ISI][Medline]

8 . National Committee for Clinical Laboratory Standards. (1999). Performance Standards for Antimicrobial Susceptibility Testing: Ninth Informational Supplement M100-S9. NCCLS, Wayne, PA.

9 . 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, 3219–27.[Abstract/Free Full Text]

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, 279–81.[Abstract]

Received 5 October 2000; returned 15 February 2001; revised 21 March 2001; accepted 21 May 2001