Clarithromycin resistance stability in Helicobacter pylori: influence of the MIC and type of mutation in the 23S rRNA

Teresa Alarcón*, Diego Domingo, Nuria Prieto and Manuel López-Brea

Department of Microbiology, Hospital Universitario de la Princesa, Diego de León 62, 28006 Madrid, Spain


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Thirty clarithromycin-resistant Helicobacter pylori strains (MIC range 8–64 mg/L) were subcultured in a drug-free medium and the MIC was determined every five passages to detect in vitro stability of resistance. Three out of the 30 (10%) lost their resistance after 10, 13 or 18 subcultures (MIC decrease from 8 to 0.008, from 16 to 0.064 and from 32 to 0.016 mg/L). The effect of four macrolides at subinhibitory concentrations on the development of resistance was studied in H. pylori NCTC 11638 and TIGR 26695. A change in the MIC was observed only when NCTC11638 was exposed to 0.5 x MIC of erythromycin for 20 days.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Helicobacter pylori is a Gram-negative microaerophilic rod found in the human gastric mucosa and is associated with a variety of digestive diseases, such as peptic ulcer, gastritis and MALT lymphoma, and is considered a risk factor in the development of gastric cancer.1 Amoxycillin, tetracycline, metronidazole and macrolides (mainly clarithromycin) are the most frequently used antimicrobials, combined with proton pump inhibitors or bismuth salts, for the treatment of H. pylori infections.2 However, side effects, poor compliance and resistance to the antibiotics used are common causes of treatment failure.3,4

The prevalence of clarithromycin resistance varies with geographical location but it is generally less than 10%. In Spain, 3.5% of H. pylori are clarithromycin resistant, whilst in other European countries the percentage has ranged from 0 to 10%.3,4 The mechanism of resistance to clarithromycin described in H. pylori is a transition mutation in the peptidyl transferase region of the 23S rRNA at Escherichia coli-equivalent base A2058 or A2059.5 Point mutations in which an adenine residue was replaced by a guanine were found by Versalovic et al.5 in H. pylori clinical isolates and confirmed by other authors who also found A to C mutation.69

Xia et al.10 found that nine of the 20 (45%) clarithromycin-resistant H. pylori clinical isolates tested reverted to sensitive after two to five subcultures on drug-free agar. However, Hulten et al.8 could not reproduce those results; the seven clarithromycin-resistant H. pylori strains remained stably resistant after 50 subcultures in vitro.

The aim of this study was to determine the influence of the MIC and the type of mutation in the 23S rRNA on the loss of in vitro clarithromycin resistance after several subcultures in the laboratory in 30 clarithromycin-resistant H. pylori clinical isolates. The effect of macrolide subinhibitory concentrations in the development of resistance to these antibiotics was also studied.


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

Thirty clarithromycin-resistant H. pylori strains were obtained from gastric biopsy samples cultured on selective and non-selective plates. Plates were incubated at 37°C in a microaerobic atmosphere for 7–10 days for primary culture and every 2–3 days for subculture. Strains were identified according to colony morphology, Gram's stain and positive reaction with urease, catalase and oxidase tests, and stored at –80°C in trypticase soy broth containing 20% glycerol until use. TIGR 26695 and NCTC 11638 were used to study the effect of subinhibitory concentrations of macrolides on the development of resistance. The latter strain was also used as a control in the susceptibility tests.

MIC determination

MIC was determined by an agar dilution method. Serial dilutions of the antibiotic ranging from 0.008 to 128 mg/L were prepared in Mueller–Hinton agar supplemented with 10% horse blood. Isolates were grown for 48 h in BHI plus 10% fetal calf serum, a suspension of 109 cfu/mL applied with a Steers replicator and plates were incubated for 3–5 days. Resistance was considered as a clarithromycin MIC >=4 mg/L.

Evaluation of stability

The 30 clarithromycin-resistant H. pylori clinical isolates were subcultured in drug-free Columbia agar plus 7% blood every 2–3 days 10–20 times and the MIC determined every five passages to detect in vitro stability.

Genotypic determination of resistance

H. pylori DNA was extracted by the CTAB reagent method. A2142G and A2143G mutations were determined by the PCR–RFLP method.5 A 3'-mismatched PCR was used to detect A2142C as described previously.9

Effect of subinhibitory concentrations

NCTC 11638 and TIGR 26695 strains were subcultured every 2 days in blood medium containing one of the three macrolides (erythromycin, azithromycin or clarithromycin) at 1/2 x or 1/8 x MIC, for up to 2, 4 or 6 days. Moreover, NCC 11638 was subcultured at 1/2 x MIC for up to 10 and 20 days. Resistance after exposure to subinhibitory concentrations was determined by studying MICs the three macrolides and confirmed by PCR–RFLP to detect A2142G and A2143G mutations in the 23S rRNA gene.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The clarithromycin MICs ranged from 8 to 64 mg/L in the 30 clarithromycin-resistant H. pylori strains selected for this study (Table IGo). Seven of the 30 (23.3%) resistant strains carried the A2142G mutation, three (10%) carried the A2142C mutation and 20 (66.6%) the A2143G mutation. Three of the 30 (10%) clarithromycin-resistant strains tested lost their resistance after 10, 13 or 18 subcultures (MIC changed from 8 to 0.008, from 16 to 0.064 and from 32 to 0.016 mg/L). Resistance was unstable in one of 17 strains with MIC 8 mg/L, one of six with MIC 16 mg/L and one of four with MIC 32 mg/L. Resistance was unstable in two of seven strains with the A2142G mutation and in one of 20 strains with the A2143G mutation.


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Table I. Initial and final MICs (mg/L) after the maximum number of subcultures and type of rRNA mutation in the 30 clarithromycin-resistant Helicobacter pylori strains studied
 
When the effect of subinhibitory concentrations was studied, no significant changes in the macrolide MIC values were observed at either 1/2 x or 1/8 x MIC for up to 10 days. The results are shown in Table IIGo. A change in the MIC was observed only when NCTC 11638 was exposed to 0.25 mg/L (1/2 x MIC) of erythromycin for 20 days. A genomic study of the colonies showed an A2143G mutation in this strain. This effect was not observed with clarithromycin or azithromycin.


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Table II. Effect on the MIC (mg/mL) of erythromycin (E), azithromycin (A) and clarithromycin (C) after exposure of Helicobacter pylori to different subinhibitory concentrations of macrolides for a varying number of days
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Infection by clarithromycin-resistant strains is correlated with lower eradication rates, although in some patients eradication is achieved, in spite of them being infected by clarithromycin-resistant strains. The presence of unstable resistance in vivo could explain the efficacy of the antibiotic in such cases.

According to our data, clarithromycin resistance was unstable in three of the 30 (10%) H. pylori strains. When other authors studied stability they found different results. Whilst Xia et al.10 found that 45% of strains (9 out of 20) showed unstable resistance after two to five subcultures, Hulten et al.8 found that in all the seven strains tested resistance was stable after as many as 50 subcultures. Debets-Ossenkopp et al.,6 studying 20 clinical isolates, found that after 21 passages in clarithromycin-free medium all strains remain resistant.6 In our study, we needed more than the two to five subcultures described by Xia et al., but we found that resistance was not stable after 10, 13 or 18 subcultures.

We found that the MIC for most of our strains was 8 mg/L (17 strains) compared with 16 mg/L for six strains, 32 mg/L for four strains and 64 mg/L for three strains. We observed that only one of the 8 mg/L group lost resistance (5.8%) compared with one of six in the 16 mg/L group (16.6%) or one of four with an MIC of 32 mg/L (25%). In contrast, Xia et al.10 observed that isolates for which the MICs were <=32 mg/L were more likely to revert to susceptible (6/9), than those for which they were >32 mg/L (3/11).

Most of our clarithromycin-resistant strains had an A2143G mutation (20 strains), seven had A2142G and three had A2142C. In two of the seven strains with the A2142G mutation, resistance was unstable (28.5%) compared with only one of 20 with A2143G mutation (5%) or none out of three with A2142C. Hulten et al.8 studied four strains with the A2142G mutation and two with A2143G and they found no loss of resistance. In the study performed by Debets-Ossenkopp et al.,6 13 strains with A2142G and seven with A2143G lost no resistance.

When NCTC 11638 and TIGR 26695 were exposed to subinhibitory concentrations of macrolides no changes were observed after 10 days' exposure to clarithromycin, erythromycin or azithromycin. However, cross-resistance to the three macrolides was obtained after 20 days' exposure to erythromycin but not to clarithromycin or azithromycin.

The reversibility of clarithromycin resistance could have at least two clinical consequences: (i) clarithromycin may eradicate H. pylori, in spite of apparent resistance in vitro due to the instability of the resistance; (ii) the clarithromycin resistance rate in vitro may be underestimated due to the subcultures performed from the first isolation to the moment of performing the susceptibility testing. However, the clinical importance of these data is at the moment unknown and further studies are needed.

In summary, 10% of the clarithromycin-resistant strains studied here lost their resistance after 10–18 subcultures in vitro. A very long exposure of NCTC 11638 to a subinhibitory concentration of erythromycin led to the development of resistance to all macrolides in vitro. However, this effect was not observed with other macrolides and for shorter periods of exposure.


    Acknowledgments
 
We wish to thank Brenda Ashley for English language assistance. This study was funded in part by a grant from Abbott Laboratories S.A.


    Notes
 
* Corresponding author. Tel: +34-91-520-23-17; Fax: +34-91-309-00-47; E-mail: talarcon{at}helicobacterspain.com 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 . Unge, P. (1998). Antimicrobial treatment of Helicobacter pylori infection—a pooled efficacy analysis of eradication therapies. European Journal of Surgery 582, Suppl., 16–26.

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

4 . Alarcón, T., Domingo, D. & López-Brea, M. (1999). Antibiotic resistance problems with Helicobacter pylori. International Journal of Antimicrobial Agents 12, 19–26.[ISI][Medline]

5 . Versalovic, J., Shortridge, D., Kibler, K., Griffy, M. V., Beyer, J., Flamm, R. K. et al. (1996). Mutations in 23S rRNA are associated with clarithromycin resistance in Helicobacter pylori. Antimicrobial Agents and Chemotherapy 40, 477–80.[Abstract]

6 . Debets-Ossenkopp, Y. J., Brinkman, A. B., Kuipers, E. J., Vandenbroucke-Grauls, C. M. J. R. & Kusters, J. G. (1998). Explaining the bias in the 23S rRNA gene mutations associated with clarithromycin resistance in clinical isolates of Helicobacter pylori. Antimicrobial Agents and Chemotherapy 42, 2749–51.[Abstract/Free Full Text]

7 . Domingo, D., Alarcon, T., Sanz, J. C., Sánchez, I. & López-Brea, M. (1998). High frequency of mutations at position 2144 of the 23S rRNA gene in clarithromycin-resistant Helicobacter pylori strains isolated in Spain. Journal of Antimicrobial Chemotherapy 41, 573–4.[Free Full Text]

8 . Hulten, K., Gibreel, A., Skold, O. & Engstrand, L. (1997). Macrolide resistance in Helicobacter pylori: mechanism and stability in strains from clarithromycin-treated patients. Antimicrobial Agents and Chemotherapy 41, 2550–3.[Abstract]

9 . Alarcón, T., Domingo, D., Prieto, N. & López-Brea, M. (2000). PCR using 3'-mismatched primers to detect A2142C mutation in 23S rRNA conferring resistance to clarithromycin in Helicobacter pylori clinical isolates. Journal of Clinical Microbiology 38, 923–5.[Abstract/Free Full Text]

10 . Xia, H.-X., Buckley, M., Keane, C. T. & O'Morain, C. A. (1996). Clarithromycin resistance in Helicobacter pylori: prevalence in untreated dyspeptic patients and stability in vitro. Journal of Antimicrobial Chemotherapy 37, 473–81.[Abstract]

Received 8 November 1999; returned 23 February 2000; revised 3 April 2000; accepted 30 May 2000