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Journal of Clinical Microbiology, July 2001, p. 2648-2651, Vol. 39, No. 7
Departments of Medical
Microbiology1 and
Gastroenterology,3 Academic Medical
Center, University of Amsterdam, Amsterdam, and Delft
Diagnostic Laboratory, Delft,2 The Netherlands
Received 3 January 2001/Returned for modification 28 March
2001/Accepted 20 April 2001
Of the Helicobacter pylori populations from 976 patients, six contained clarithromycin-resistant as well as
-susceptible colonies. In each heterogeneous H. pylori
population, resistant H. pylori colonies harbored identical
23S ribosomal DNA (rDNA) mutations associated with clarithromycin
resistance, while the susceptible H. pylori colonies all
had wild-type 23S rDNA. The resistant and susceptible colonies of each
of the heterogeneous H. pylori populations had identical
randomly amplified polymorphic DNA-PCR genotypes. In conclusion,
evaluation of antimicrobial susceptibility can be misinterpreted if
only a single colony from the primary H. pylori population
is used to test for clarithromycin susceptibility.
Helicobacter pylori
infection has been established as an etiologic factor in nonautoimmune
gastritis, peptic ulcer disease (PUD), gastric carcinoma, and lymphoma
(1, 3, 10). Since curing H. pylori infection
prevents peptic ulcer recurrence, the eradication of the organism has
become the cornerstone in the treatment of PUD (15).
Clarithromycin (CLR) in combination with metronidazole (MTZ) is often
used in H. pylori eradication regimens (4).
H. pylori resistance to these drugs substantially reduces the success rate of CLR-plus-MTZ eradication regimens (4). The 23S rRNA gene is present in two copies in the H. pylori
genome (12, 13). In H. pylori, seven different
point mutations (A2115 It has been reported that the H. pylori population in a
patient can be heterogeneous with respect to MTZ susceptibility
(20). Infection by a mixed population of CLR-susceptible
and CLR-resistant H. pylori has been reported (6, 7,
8, 16). However, in most of these studies (6, 7,
16), discrimination between mixed H. pylori infection
and infection with H. pylori heterozygous for 23S rRNA was
not established. The aim of this study was to evaluate the relevance of
heterogeneity in susceptibility to CLR in H. pylori
populations from patients with gastritis or PUD prior to anti-H.
pylori treatment. In this study, a distinction was made between
mixed H. pylori infection and infection with H. pylori heterozygous for 23S rRNA.
H. pylori isolates were cultured from gastric biopsy
specimens from 976 patients from the Amsterdam area who were referred for upper gastrointestinal tract endoscopies in 1997 and 1998 because
of dyspeptic symptoms (14). Briefly, each specimen was smeared on Columbia agar (Oxoid CM 331; Unipath Ltd., Basingstoke, England) plates containing 7% (vol/vol) horse blood. Colonies that
exhibited the characteristic morphology were identified as H. pylori if they were urease, catalase, and oxidase positive. The
cultures of the antrum and corpus were collected separately with swabs,
which were subsequently shaken in 8% glycerol-peptone. These bacterial
suspensions were stored at Among the 976 H. pylori populations cultured, 51 (5.2%)
were resistant to CLR. Close examination of the E-test plates revealed that six (12%) of these 51 H. pylori populations were
heterogeneous regarding CLR susceptibility. In these H. pylori populations, the majority of bacteria were susceptible to
CLR, but Helicobacter colonies were also growing within the
zones of growth inhibition of the bacterial lawn. For each
heterogeneous H. pylori population, the MICs of CLR for two
resistant H. pylori colonies and for two susceptible
H. pylori colonies were reassessed by E-test, and the DNAs
of the colonies were subjected to the 23S ribosomal DNA (rDNA) LiPA
(Fig. 1; Table 1). In all heterogeneous
H. pylori populations, the MICs of CLR for the two
CLR-resistant colonies were high, and the colonies harbored identical
23S rDNA mutations in both copies of rRNA operons, associated with CLR
resistance. The MICs for all susceptible H. pylori colonies
were low, and the colonies had wild-type 23S rDNA. RAPD-PCR profiles of
the resistant and susceptible H. pylori colonies of each of
the heterogeneous H. pylori populations were identical,
indicating identical genotypes (Fig. 2).
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.7.2648-2651.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Clarithromycin-Susceptible and -Resistant
Helicobacter pylori Isolates with Identical Randomly
Amplified Polymorphic DNA-PCR Genotypes Cultured from Single
Gastric Biopsy Specimens Prior to Antibiotic Therapy
ABSTRACT
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G, G2141A, A2142G, A2142C, A2143G,
A2143C, and A2142T) in the peptidyltransferase region of the V
domain of the 23S rRNA gene have been found to be associated with
resistance to CLR (5, 9, 11, 12, 18). In a recent study,
it was found that among CLR resistant H. pylori, the
predominant mutations were A2143G and A2142G, while virtually all
CLR-susceptible H. pylori had no mutation in their 23S rRNAs
(17).
70°C. CLR susceptibilities were
determined by the E-test (AB Biodisk, Solna, Sweden) according to the
instructions of the manufacturer (20). Colonies growing within the zone of growth inhibition of the bacterial lawn were subcultured on blood agar for 3 days before assessment of the CLR MIC
was performed. Isolates were considered resistant to CLR if the MIC was
2 mg of CLR/liter (7). Mutations in the 23S rRNA gene
were assessed by a PCR-based reverse hybridization onto a line probe
assay (INNO-LiPA) (16). This LiPA allows the simultaneous detection of all of the aforementioned point mutations in the 23S rRNA
gene associated with CLR resistance. In addition, the genotype of each
H. pylori strain was assessed by randomly amplified polymorphic DNA (RAPD)-PCR using four different primers
(14). For analysis, the four profiles were combined.
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FIG. 1.
LiPA results for clarithromycin-resistant H. pylori isolates from six patients. The results from one of the
clarithromycin-resistant H. pylori colonies from the
heterogeneous H. pylori population of each of the six
patients are presented here. WT, wild type.
TABLE 1.
Comparison of 23S rDNA LiPA results with E-test results
for two CLR-resistant and two CLR-susceptible colonies of heterogeneous
H. pylori populations
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FIG. 2.
RAPD profiles of CLR-resistant and -susceptible H. pylori isolates from single biopsy specimens. Two CLR-susceptible
(S) and two CLR-resistant (R) colonies from the heterogeneous
H. pylori populations of six patients were assessed by
RAPD-PCR. m, 100-bp molecular size markers.
Our results are in accordance with the results from recent studies by Maeda and coworkers (6), Matsuoka and colleagues (8), and van Doom and collaborators (16) reporting mixed infections with CLR-susceptible and CLR-resistant H. pylori. However, genotyping was not performed in those studies. In addition, in another study, the susceptibility of H. pylori to CLR was assessed by molecular biological techniques directly on biopsy specimens from H. pylori-infected patients (7). Seventeen percent of the H. pylori-positive biopsy specimens yielded 23S rDNA PCR products that hybridyzed with both the wild-type probe and one of the mutant probes. The results were explained by either mixed infection with resistant and susceptible H. pylori or infection by H. pylori heterozygous for the 23S rRNA gene. Our findings favor the first explanation.
Only a limited number of different 23S rDNA point mutations were found
among the heterogeneous H. pylori populations. The CLR-resistant H. pylori isolates of four of the six
heterogeneous H. pylori populations had the A2142G point
mutation, one had the A2143G mutation, and another had the A2142C
mutation in their 23S rRNA genes. Possibly, in an environment without
CLR, the disadvantage of these point mutations in the 23S rRNA gene in
H. pylori is insignificant, resulting in a lack of negative selection of these 23S rRNA mutants. This is supported by the results
of in vitro experiments (2, 19). In these experiments, it
was found that the growth rates of H. pylori isolates
with the A2142G, A2142C, or A2143G mutation did not
differ from that of the wild type, but H. pylori isolates
with other 23S rDNA mutations grew more slowly (2). In
addition, Wang and coworkers showed identical growth rates of wild-type
H. pylori and H. pylori with the A2142G or
A2143G 23S rDNA mutation (19). From the individual
growth rates and the patterns of competitive growth, it was concluded
that the order of preference of competitive accumulation is
A2142G > A 2143G >>> A2142C > A2143C (A2143T). The prevalence of the A2142G, A2143G, and
A2142C mutations among the heterogeneous H. pylori
populations in our study is consistent with this order.
In conclusion, the results show coexistence of CLR-resistant and -susceptible H. pylori isolates with identical genotypes in patients prior to treatment. If only a single colony from the primary H. pylori populations is used to test for CLR susceptibility, the results can be misinterpreted. Assessment of 23S rRNA mutations in H. pylori directly from biopsy specimens by molecular biological techniques, such as the LiPA, has the advantage that infection with a mixed H. pylori population is easily detected. In addition, knowledge of the type of 23S rRNA mutation may be important since CLR MICs are associated with the type of 23S rRNA mutation in H. pylori (17).
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands. Phone: 31-20-5664862. Fax: 31-20-6979271. E-mail: a.vanderende{at}amc.uva.nl.
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REFERENCES |
|---|
|
Top
Abstract
Text
References
|
|---|
| 1. |
Blaser, M. J.,
G. I. Perez-Perez, and H. Kleanthous.
1995.
Infection with Helicobacter pylori strains possessing cagA associated with an increased risk of developing adenocarcinoma of the stomach.
Cancer Res.
55:2111-2115 |
| 2. |
Debets-Ossenkopp, Y. J.,
A. B. Brinkman,
E. J. Kuipers,
C. M. J. E. Vandenbroucke-Grauls, and J. G. Kusters.
1998.
Explaining the bias in the 23S rMA gene mutations associated with clarithromycin resistance in clinical isolates of Helicobacter pylori.
Antimicrob. Agents Chemother.
42:2749-2751 |
| 3. | Foreman, D., and the Eurogast Study Group. 1993. An international association between Helicobacter pylori infection and gastric cancer. Lancet 341:359-362. |
| 4. |
Houben, M. H.,
D. van der Beek,
E. F. Hensen,
A. J. Craen,
E. A. J. Rauws, and G. N. J. Tytgat.
1999.
A systematic review of Helicobacter pylori eradication therapy the impact of resistance on eradication rates.
Aliment. Pharmacol. Ther.
13:1047-1055[CrossRef][Medline].
|
| 5. | Hultén, K., A. Gibreel, O. Skold, and L. Engstrand. 1997. Macrolide resistance in Helicobacter pylori: mechanism and stability in strains from clarithromycin-treated patients. Antimicrob. Agents Chemother. 41:2550-2553[Abstract]. |
| 6. |
Maeda, S.,
H. Yoshida,
H. Matsunaga,
K. Ogura,
O. Kawamata,
Y. Shiratori, and M. Omata.
2000.
Detection of clarithromycin-resistant Helicobacter pylori strains by a preferential homoduplex formation assay.
J. Clin. Microbiol.
38:210-214 |
| 7. |
Marais, A.,
L. Monteiro,
A. Occhialini,
M. Pina,
H. Lamouliatte, and F. Megraud.
1999.
Direct detection of Helicobacter pylori resistance to macrolides by a polymerase chain reaction/DNA enzyme immunoassay in gastric biopsy specimens.
Gut
44:463-467 |
| 8. |
Matsuoka, M.,
Y. Yoshida,
K. Hayakawa,
S. Fukuchi, and K. Sugano.
1999.
Simultaneous colonisation of Helicobacter pylori with and without mutations in the 23S rRNA gene in patients with no history of clarithromycin exposure.
Gut
45:503-507 |
| 9. | Occhialini, A., M. Urdaci, F. Doucet-Populaire, C. M. Bebear, H. Lamouliatte, and F. Megraud. 1997. Macrolide resistance in Helicobacter pylori: rapid detection of point mutations and assays of macrolide binding to ribosomes. Antimicrob. Agents Chemother. 41:2724-2728[Abstract]. |
| 10. |
Parsonnet, J.,
S. Hansen,
L. Rodriguez,
A. B. Gelb,
R. A. Warnke,
E. Jellum,
N. Orentreich,
J. H. Vogelman, and G. D. Friedman.
1994.
Helicobacter pylori infection and gastric lymphoma.
N. Engl. J. Med.
330:1267-1271 |
| 11. | Stone, G. G., D. Shortridge, R. K. Flamm, J. Versalovic, J. Beyer, K. Idler, L. Zulawinski, and S. K. Tanaka. 1996. Identification of a 23S rRNA gene mutation in clarithromycin-resistant Helicobacter pylori. Helicobacter 1:227-228[Medline]. |
| 12. | Taylor, D. E, Z. Ge, D. Purych, T. Lo, and K. Hiratsuka. 1997. Cloning and sequence analysis of two copies of a 23S rRNA gene from Helicobacter pylori and association of clarithromycin resistance with 23S rRNA mutations. Antimicrob. Agents Chemother. 41:2621-2628[Abstract]. |
| 13. | Tomb, J.-F., O. White, A. R. Kervage, R. A. Clayton, G. G. Sutton, R. D. Fleischmann, K. A. Ketchum, H. P. Klenk, S. Gill, B. A. Dougherty, K. Nelson, J. Quackenbush, L. Zhou, E. F. Kirkness, S. Peterson, B. Loftus, D. Richardson, R. Dodson, H. G. Khalak, A. Glodek, K. Mckenney, L. M. Fitzgerald, N. Lee, M. D. Adams, E. K. Hickey, D. E. Berg, J. D. Gocayne, T. R. Utterback, J. D. Peterson, J. M. Kelley, M. D. Cotton, J. M. Weidman, C. Fujii, C. Bowman, L. Watthey, E. Wallin, W. S. Hayes, M. Borodovsky, P. D. Karp, H. O. Smith, C. M. Fraser, and J. C. Venter. 1997. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388:539-547[CrossRef][Medline]. |
| 14. | van der Ende, A., E. A. J. Rauws, M. Feller, C. J. Mulder, G. N. J. Tytgat, and J. Dankert. 1996. Heterogeneous Helicobacter pylori isolates from members of a family with a history of peptic ulcer disease. Gastroenterology 111:638-647[CrossRef][Medline]. |
| 15. | van der Hulst, R. W. M., E. A. J. Rauws, B. Köycü, J. J. Keller, J. G. P. Tyssen, M. Bruno, and G. N. J. Tytgat. 1997. Prevention of ulcer recurrence after successful eradication of Helicobacter pylori infection: a prospective long term follow-up study. Gastroenterology 113:1082-1086[CrossRef][Medline]. |
| 16. |
van Doorn, L.-J.,
Y. J. Debets-Ossenkopp,
A. Marais,
R. Sanna,
F. Mégraud,
J. G. Kusters, and W. G. V. Quint.
1999.
Rapid detection, by PCR and reverse hybridization, of mutations in the Helicobacter pylori 23S rRNA gene, associated with macrolide resistance.
Antimicrob. Agents Chemother.
43:1779-1782 |
| 17. |
van Doorn, L.-J.,
Y. Glupczynski,
J. G. Kusters,
F. Mégraud,
P. Midolo,
N. Maggi-Solcà,
D. M. M. Queiroz,
N. Nouhan,
E. Stet, and W. G. V. Quint.
2001.
Accurate prediction of macrolide resistance in Helicobacter pylori by a PCR line probe assay for detection of mutations in the 23S rRNA gene: multicenter validation study.
Antimicrob. Agents Chemother.
45:1500-1504 |
| 18. |
Versalovic, J.,
M. S. Osato,
K. Spakovsky,
M. P. Dore,
R. Reddy,
G. G. Stone,
D. Shortridge,
R. K. Flamm,
S. K. Tanaka, and D. Y. Graham.
1996.
Point mutations in the 23S rRNA gene of Helicobacter pylori associated with different levels of clarithromycin resistance.
J. Antimicrob. Chemother.
40:283-286 |
| 19. |
Wang, G.,
M. S. Rahman,
M. Z. Humayun, and D. E. Taylor.
1999.
Multiplex sequence analysis demonstrates the competitive growth advantage of the A-to-G mutants of clarithromycin-resistant Helicobacter pylori.
Antimicrob. Agents Chemother.
43:683-685 |
| 20. | Weel, J. F., R. W. M. van der Hulst, Y. Gerrits, G. N. J. Tytgat, A. van der Ende, and J. Dankert. 1996. Heterogeneity in susceptibility to metronidazole among Helicobacter pylori isolates from patients with gastritis or peptic ulcer disease. J. Clin. Microbiol. 34:2158-2162[Abstract]. |
Journal of Clinical Microbiology, July 2001, p. 2648-2651, Vol. 39, No. 7
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.7.2648-2651.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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