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Antimicrobial Agents and Chemotherapy, May 2001, p. 1500-1504, Vol. 45, No. 5
Delft Diagnostic Laboratory, Delft, The
Netherlands1; Cliniques Universitaire
UCL de Mont-Godinne, Yvoir, Belgium2;
Department of Medical Microbiology, Free University, Amsterdam,
The Netherlands3; Laboratoire de
Bactériologie, Hôpital Pellegrin, Bordeaux,
France4; Monash Medical Center, Clayton,
Australia5; Istituto Cantonale
Batteriosierologico, Lugano, Switzerland6; and
Laboratory for Research in Bacteriology, Faculty of
Medicine/UFMG, Belo Horizonte, Brazil7
Received 18 September 2000/Returned for modification 4 January
2001/Accepted 9 February 2001
Helicobacter pylori strains from 299 patients were
tested in six laboratories in different countries. Macrolide
susceptibility of the strains was determined by agar dilution (17.4%)
or the epsilometer test (82.6%). Mutations in the 23S ribosomal DNA
(rDNA) that are associated with macrolide resistance were analyzed by PCR and reverse hybridization (PCR-line probe assay [LiPA]). This method identifies A2115G, G2141A, A2142G, A2142C, A2142T, A2143G, and
A2143C mutations in the 23S rDNA. vacA s-region (s1a, s1b, s1c, and s2) and m-region (m1, m2a, and m2b) genotypes and
cagA status were also determined using another PCR-LiPA
system. Of the 299 strains investigated by MIC testing, 130 (43.5%)
were resistant and 169 (56.5%) were susceptible to clarithromycin. Of
the 130 resistant strains, 127 (97.7%) contained 23S rDNA mutations, whereas 167 (98.8%) of the 169 susceptible strains contained wild-type sequences. The predominant mutations were A2143G (45.2%) and A2142G (33.3%). Twenty-eight (19.8%) strains contained multiple 23S rDNA mutations. Only five resistant strains contained the A2142C mutation (three of these in combination with the A2142G mutation), and the
A2115G, G2141A, A2142T, and A2143C mutations were not found. MICs of
clarithromycin for the A2142G mutant strains were significantly higher
than MICs for the A2143G strains. Although there was no significant
association between 23S rDNA mutations and the vacA and
cagA status, clarithromycin-susceptible strains more often contained mixed vacA genotypes, indicating the presence of
multiple H. pylori strains. In conclusion, our data
confirmed the very strong association between 23S rDNA mutations and
macrolide resistance and showed that the PCR-LiPA permits accurate and
reliable diagnosis of macrolide resistance in H. pylori.
Helicobacter pylori is a
gram-negative bacterium that colonizes the human stomach. Persistent
infection with H. pylori is associated with chronic
gastritis and peptic ulcer disease and may eventually result in the
development of atrophic gastritis, mucosa-associated lymphoid tissue
lymphoma, and gastric cancer (1, 7).
Infection with H. pylori can be effectively treated by a
combination of a proton pump inhibitor and/or an
H2-receptor antagonist with multiple antibiotics.
Metronidazole, amoxicillin, clarithromycin, and tetracycline are
frequently included in triple or quadruple regiments (4,
21). Resistance to antimicrobial agents has a significant
impact on the efficacy of anti-Helicobacter treatment. Resistance to metronidazole is observed in 10 to 50% of the
cases in developed countries but can be as high as 90% in developing countries (15). The prevalence of macrolide-resistant
strains varies among countries and ranges from less than 2% of the
strains in The Netherlands and Norway (16, 27) to more
than 10% in France and some other countries (2, 5, 15,
18). Moreover, the prevalence of resistant strains appears to be
increasing (5, 8, 12, 20). Resistance to clarithromycin
decreases the effectiveness of antibiotic therapy by an average of 55%
(6, 10).
The major cause of macrolide resistance in H. pylori is the
lack of binding of the macrolides to the 23S rRNA components of the
bacterial ribosome due to modification of the target site by
methylation or point mutations in the peptidyltransferase region of
domain V of the 23S rRNA (30). H. pylori
contains two copies of the 23S ribosomal DNA (rDNA) gene, and five
distinct point mutations have been reported that are associated with
macrolide resistance in natural H. pylori strains, i.e.,
G2115 Since macrolide resistance is clinically important, and because the
prevalence of resistant strains is increasing, there is a clear need
for rapid and accurate diagnostic methods to determine macrolide
resistance. The conventional method to determine the antibiotic
resistance of H. pylori is based on the analysis of cultured
strains by agar diffusion or dilution or the epsilometer (E-test)
(9, 15, 17). These methods are tedious and strongly dependent on the experimental conditions and subjective interpretation and hence are not always reproducible (15). DNA-based
diagnostic methods may offer a rapid and reliable alternative approach
for macrolide susceptibility testing. Furthermore, molecular assays can
be applied directly on gastric biopsy specimens, without the need for
in vitro culture of the bacterium (26). Therefore, we have
developed a PCR-based reverse hybridization assay that permits
simultaneous identification of the different mutations in the 23S rDNA
(22).
The aims of the present study were to (i) assess the performance of the
PCR-line probe assay (LiPA) method in a multinational study in six
different laboratories, (ii) compare the results of the PCR-LiPA with
results obtained by microbiological susceptibility testing, (iii)
determine the prevalence of the specific 23S rDNA mutations in
clarithromycin-resistant strains in different parts of the world, and
(iv) investigate whether 23S rDNA mutations are related to the
virulence-associated vacA and cagA genotypes.
Multinational study.
Six laboratories (in Australia,
Belgium, Brazil, France, The Netherlands, and Switzerland) participated
in this multicenter study. Each laboratory used its own method to
culture H. pylori and tested for clarithromycin
susceptibility by agar dilution or E-test. Each laboratory was asked to
select from its strain collection approximately 50 nonduplicate and
well-documented clarithromycin-susceptible and clarithromycin-resistant
strains (approximately 25 of each) to be tested by PCR-LiPA. Most
strains were obtained from patients after unsuccessful eradication
therapy. Approval of ethics committees was obtained where necessary.
All reagents and instructions used for PCR amplification of the 23S
rDNA gene and reverse hybridization analysis of the PCR products by
LiPA were provided by the organizing laboratory. 23S rDNA LiPA results
were interpreted in each laboratory, and DNA from all strains was sent
to the organizing laboratory, where the vacA and
cagA genotypes were determined.
H. pylori cultures and isolation of DNA.
Gastric
biopsy specimens were obtained from patients by upper endoscopy. From
these specimens, H. pylori was cultured under microaerobic
conditions. Genomic DNA was isolated from cultured strains by
proteinase K treatment. Bacteria were harvested from plates and
resuspended in phosphate-buffered saline. After centrifugation, the
bacterial cell pellet was resuspended in 500 µl of a solution containing 10 mM Tris HCl (pH 8.0), 5 mM EDTA, 0.1% sodium dodecyl sulfate, and 0.1 of proteinase K per ml and incubated for at least 2 h at 55°C. Proteinase K was inactivated by incubation at
95°C for 10 min. The lysate was centrifuged at 10,000 × g, and the supernatant was transferred to a new tube.
Macrolide susceptibility testing.
Susceptibility to
macrolides was assessed by MIC testing using either agar dilution or
E-test in the participating laboratories (9, 16). Each
laboratory used its own MIC cutoff value to classify strains as
clarithromycin susceptible or resistant.
PCR-LiPA for 23S rDNA mutations.
Specific mutations in the
23S rDNA were determined by PCR-LiPA as described earlier
(22). Briefly, part of the 23S rDNA was amplified by PCR,
using biotinylated primers. PCR products were denatured and hybridized
to specific oligonucleotide probes, which were immobilized on a
nitrocellulose strip in parallel lines. This method specifically
identifies A2115G, G2141A, A2142G, A2142C, A2142T, A2143G, and A2143C
mutations in the 23S rDNA. Hybridization was performed under highly
stringent conditions, to ensure complete specificity as described
previously (22). Hybrids were detected by alkaline
phosphatase-conjugated streptavidin and a substrate, resulting in a
purple precipitate at the specific probe line. Hybridization results
were interpreted visually.
PCR-LiPA for vacA and cagA
genotyping.
vacA (s and m region) and cagA
genotypes were determined by PCR-LiPA as described earlier
(23-25).
Statistical analyses.
Statistical analyses were performed
with SPSS for Windows, version 8, using the chi-square and Wilcoxon
rank sum tests.
A total of 299 strains were studied in six different laboratories.
Since the samples were tested under code, it was not possible to obtain
demographic data or clinical data for the patients. First, macrolide
susceptibility was tested by agar dilution (n = 52;
17.4%) or E-test (n = 247; 82.6%), and MICs were
determined. Overall, among this group of selected strains, 169 (56.5%)
were susceptible and 130 (43.5%) were resistant to clarithromycin.
The 23S rDNA was studied by PCR-LiPA, and the results are shown in
Table 1. Since there were no significant
differences among the laboratories, results from all strains were
combined. Of the 299 strains, 170 (56.9%) contained wild-type 23S rDNA
sequences, whereas mutations were detected in 129 (43.1%). As shown in
Table 2, the predominant mutations among
the 129 mutant strains were A2143G (44.1%) and A2142G (32.6%). The
A2142C mutation was found as a single 23S rDNA genotype in two
resistant strains from Belgium. In three other strains (one from France
and two from Switzerland), the A2142C and A2142G mutations were both
present. None of the strains contained the A2115G, G2141A, A2142T, or
A2143C mutation. Uniform 23S rDNA genotypes were found in 271 (90.6%)
of the strains, whereas 28 (9.4%) strains contained combinations of
different 23S rDNA genotypes, suggesting either infection with multiple H. pylori strains or the presence of different 23S rDNA
alleles in a single strain (Table 3).
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.5.1500-1504.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
A, G2141A, A2142G, A2142C, and A2143G (11,
19, 29).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Susceptibility of H. pylori strains to
clarithromycin and detection of 23S rDNA mutations by PCR-LiPA in six
laboratories
TABLE 2.
Rate of detection of specific 23S rDNA mutations among
129 strains containing non-wild-type 23S rDNA sequences from six
laboratories
TABLE 3.
Characteristics of H. pylori cultures
containing multiple 23S rDNA genotypes
There was a very strong association between the presence of 23S rDNA mutations and macrolide resistance. Overall, 167 (98.8%; 95% confidence interval, 95.8 to 99.7%) of the 169 clarithromycin-susceptible strains contained wild-type sequences, whereas 127 (96.7%; 95% confidence interval, 93.4 to 99.2%) of the 130 clarithromycin-resistant strains contained mutant 23S rDNA sequences. One of the two susceptible strains containing 23S mutations showed a mixture of the wild-type and A2143G mutant sequences.
The MICs for strains with different 23S rDNA mutations were compared (data not shown). MICs for strains containing the A2142G mutation were significantly higher than those for strains containing the A2143G mutation (Wilcoxon rank sum test, P < 0.001).
The vacA (s and m region) and cagA genotypes
could be determined for 296 (99.0%) of the strains, and the results
are shown in Table 4. There was a strong
association between the vacA s1 genotype and the presence of
cagA (chi-square test, P < 0.001). Of the
28 strains showing combinations of 23S rDNA genotypes, only 1 (3.6%)
showed multiple vacA genotypes. Although there were no
significant associations between the vacA and
cagA genotypes and the status of the 23S rDNA, a few
trends were observed. First, among susceptible strains, a higher
frequency of multiple vacA s and m genotypes was observed.
Second, the subset of 99 strains containing the A2142G
(n = 42) and A2143G (n = 57) mutations
was analyzed. Among these, the A2143G strains more often contained multiple vacA s (chi-square test, P = 0.04)
or vacA m (chi-square test, P = 0.08) genotypes
than did the A2142G strains. Finally, the A2143G strains more often
contained vacA s1- and cagA-positive genotypes,
whereas the A2142G strains were often of the vacA s2- and
cagA-negative genotype (chi-square test, P = 0.007 and P = 0.04, respectively).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Rapid and accurate detection of antibiotic resistance plays an increasingly important role in the management of H. pylori-infected patients. Due to the low growth rate of the bacterium, conventional susceptibility testing methods, such as agar dilution and E-test, require culture of the bacterium and are time-consuming. In contrast, molecular tools can be directly applied to gastric biopsy specimens, thereby omitting the necessity for culture (26). Since molecular methods are not dependent on bacterial density, viability, and growth rate, the results are more reliable and reproducible than results of growth-based susceptibility assays.
In the present study, the results of conventional clarithromycin susceptibility testing were compared with the results of molecular analysis by PCR-LiPA in six laboratories around the world. The strains were initially tested by agar dilution or E-test; these methods are known to be equally reliable for clarithromycin susceptibility testing (16).
The strong association between resistance to macrolides and specific mutations in the 23S rDNA was confirmed. The positive and negative predictive values of the PCR-LiPA for detection of macrolide resistance were higher than 97%, indicating the high degree of accuracy of the assay.
The PCR-LiPA method permits detection of seven distinct 23S rDNA mutations. Of these, the A2143G and A2142G mutations were the most prevalent and accounted for 98% of the macrolide-resistant strains containing single 23S rDNA genotypes. The A2142C mutation was found in only five strains.
Multiple 23S rDNA alleles were found in more than 20% of the mutant strains. Other studies also have found multiple 23S rDNA alleles (13, 14). Since H. pylori contains two copies of the 23S rDNA, detection of multiple 23S rDNA sequences may reflect the presence of different mutations in either copy of the gene in a single strain or may indicate the presence of multiple H. pylori strains. However, only 1 of the 28 strains that contained multiple 23S rDNA genotypes also showed multiple vacA genotypes. This may suggest that the majority of the strains containing multiple 23S rDNA genotypes were not mixed strains but were single strains containing two different mutant copies of the 23S rDNA gene, although the discriminatory power of vacA genotyping is obviously limited. None of the strains contained either of the A2115G and G2141A mutations, which were described by Hultén et al. (11). Also, other studies failed to identify strains with these mutations (16) among at least several hundreds of strains, implying that these particular mutants are extremely rare.
PCR-LiPA also permits specific detection of the A2142T and A2143C mutations. Strains containing these mutations have been artificially constructed and were clarithromycin resistant (3). Since both mutations appeared to be stable upon passage of H. pylori strains, specific probes were included in the LiPA. However, natural strains containing these mutations have not been isolated from patients so far and were also not found in the present study, suggesting that they are extremely rare or nonexistent.
All strains with discrepant results between PCR-LiPA and clarithromycin resistance testing were retested by agar dilution or E-test and PCR-LiPA. Of the initial 15 cases with discrepant results, 10 could be resolved because retesting showed a different MIC, which now was in agreement with the PCR-LiPA result (data not shown). Repeated LiPA confirmed the first results in all cases except one. In a single strain for which the MIC was >256 µg/ml, only wild-type sequences were found first, whereas retesting showed the presence of both wild-type and A2142G mutant sequences. These results show that the reproducibility of the LiPA is very high and also indicate the lack of reproducibility of conventional MIC testing.
A significant association was found between the specific 23S rDNA mutation and the MIC. The MICs for strains with the A2142G mutation were higher than those for strains with the A2143G mutation, confirming earlier observations (28). Also, the prevalences of these mutants appear to be different among strains of H. pylori obtained before treatment and after treatment with regimens containing clarithromycin (Y. Glupczynski et al., unpublished observations).
The A2142G and A2143G mutations were most predominant among the resistant strains. There was no significant difference among strains from different geographic origins, indicating that strains with these mutations account for the great majority of resistant strains worldwide.
Overall, 23S rDNA genotyping and susceptibility testing results for five strains remained discrepant. Two strains were clarithromycin susceptible but contained mutant 23S rDNA sequences. These findings may be due to inaccurate MIC testing, although repeated testing showed the same results. Conversely, three strains were clarithromycin resistant, but only wild-type 23S rDNA sequences were found. Again, repeated MIC testing and PCR-LiPA yielded identical results. In these strains, other mechanisms which are not related to 23S rDNA mutations may play a role (15).
Hypothetically, virulent strains of H. pylori will more often result in peptic ulcer disease, and patients infected by such strains will be treated. Consequently, the prevalence of macrolide-resistant strains may be epidemiologically related to the virulence of the bacterium. Considering the entire group of strains in this study, there was no significant association between the virulence-associated vacA and cagA genotypes of the H. pylori strains and the 23S rDNA status. However, among susceptible strains, a considerably higher frequency of multiple vacA s and m genotypes was observed. Clarithromycin-resistant strains are mainly obtained from patients who have been treated with antibiotics, which will often result in selection of a single resistant strain (with a single vacA genotype). In contrast, clarithromycin-susceptible strains are mainly obtained from patients who have not been treated and have not been subjected to the antibiotic selection pressure. Therefore, multiple strains will be less prevalent among clarithromycin-resistant strains.
In a subgroup of 99 strains (containing either the A2142G or the A2143G mutation), there were significant differences between A2143G strains and A2142G strains with respect to the frequency of vacA and cagA genotypes. This may indicate that there is a weak clonal relationship between virulence-associated and 23S rRNA genes, although the mechanism remains unclear. This weak relationship is a further indication that the mechanism mentioned above may influence the selection of resistant and maybe more-virulent strains from a mixed infection present before the onset of therapy, probably resulting in a weak clonal relationship between virulence-associated and 23S rRNA genes. However, a bias in the selection of the strains in the present study population cannot be excluded, as they all represent strains obtained from patients who underwent gastroscopy for upper abdominal complaints. Therefore, this selection bias might result in the overrepresentation of certain virulence-associated genotypes in the isolates tested.
In conclusion, macrolide resistance of H. pylori can be effectively determined by molecular analysis of 23S rDNA mutations. PCR-LiPA offers a reliable molecular tool for rapid and accurate identification of macrolide-resistant H. pylori strains. The use of such tools, especially when used directly with gastric biopsy specimens, may facilitate the choice of effective antibiotic therapies for H. pylori infection and prevent further increase of the prevalence of antibiotic resistance.
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ACKNOWLEDGMENTS |
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All authors from the six participating centers contributed equally to this work.
We thank Frank Hulstaert and Hans Pottel for statistical assistance and helpful discussions.
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FOOTNOTES |
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* Corresponding author. Mailing address: Delft Diagnostic Laboratory, R. de Graafweg 7, 2625 AD, Delft, The Netherlands. Phone: 31-15-2604581. Fax: 31-15-2604550. E-mail: L.J.van.Doorn{at}ddl.nl.
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Abstract
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Discussion
References
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Antimicrobial Agents and Chemotherapy, May 2001, p. 1500-1504, Vol. 45, No. 5
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.5.1500-1504.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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