ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 2005, p. 3166–3170 0066-4804/05/$08.00⫹0 doi:10.1128/AAC.49.8.3166–3170.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 49, No. 8
Real-Time PCR Screening for 16S rRNA Mutations Associated with Resistance to Tetracycline in Helicobacter pylori Erik Glocker,1 Marco Berning,1 Monique M. Gerrits,2 Johannes G. Kusters,2 and Manfred Kist1* National Reference Center for Helicobacter pylori, Department of Microbiology and Hygiene, Institute of Medical Microbiology and Hygiene, University Hospital Freiburg, Hermann-Herder-Str. 11, 79104 Freiburg, Germany,1 and Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center, Rotterdam, The Netherlands2 Received 17 December 2004/Returned for modification 3 February 2005/Accepted 11 April 2005
The effectiveness of recommended first-line therapies for Helicobacter pylori infections is decreasing due to the occurrence of resistance to metronidazole and/or clarithromycin. Quadruple therapies, which include tetracycline and a bismuth salt, are useful alternative regimens. However, resistance to tetracycline, mainly caused by mutations in the 16S rRNA genes (rrnA and rrnB) affecting nucleotides 926 to 928, are already emerging and can impair the efficacies of such second-line regimens. Here, we describe a novel real-time PCR for the detection of 16S rRNA gene mutations associated with tetracycline resistance. Our PCR method was able to distinguish between wild-type strains and resistant strains exhibiting single-, double, or triple-base-pair mutations. The method was applicable both to DNA extracted from pure cultures and to DNA extracted from fresh or frozen H. pylori-infected gastric biopsy samples. We therefore conclude that this real-time PCR is an excellent method for determination of H. pylori tetracycline resistance even when live bacteria are no longer available.
(15, 16) or ciprofloxacin (7) in H. pylori. The major advantage of this technique over classical microbiological resistance testing is not in its enhanced speed and standardization but is mostly in the option to test for resistance in clinical specimens that no longer contain live bacteria. In this study, a real-time PCR for the detection of 16S rRNA gene mutations associated with tetracycline resistance was developed and tested with seven unrelated tetracycline-resistant (Tetr) strains from The Netherlands (n ⫽ 1), South America (n ⫽ 5), and Canada (n ⫽ 1); eight mutants with artificially created 16S rRNA mutations; and 150 tetracycline-sensitive (Tets) H. pylori clinical isolates and their corresponding gastric biopsy samples.
Helicobacter pylori infection is chronic in nature, causes gastritis, and increases the risk of development of peptic ulcer disease, mucosa-associated lymphoid tissue lymphoma, and gastric cancer (for reviews, see references 13, 18, and 20). Increasing rates of resistance to the first-line antibiotic drugs (e.g., clarithromycin) are compromising the eradication of H. pylori and result in therapy failures (8). Thus, alternative treatments that include tetracyclines are recommended (1, 2, 14). Tetracyclines are bacteriostatic drugs which exert their antimicrobial effects by affecting the 30S subunit of the ribosome and block the binding of aminoacyl-tRNA, resulting in impaired protein biosynthesis (3, 22). The resistance of H. pylori to tetracyclines is reported to be caused by mutations in the 16S rRNA. H. pylori isolates exhibiting AGA926-9283 TTC triple-base-pair mutations (4, 5, 22) show MICs higher than 4 mg/liter and probably represent the clinically most relevant strains, whereas single- or double-base-pair mutations were, rather, associated with MICs between 1 mg/liter and 4 mg/liter (3, 5). The susceptibility of H. pylori to tetracycline is routinely examined by agar diffusion (Etest) or agar dilution tests, which are accepted to be the “gold standards” (10). These methods are slow and time-consuming, and they sometimes fail due to a lack of growth of the infecting H. pylori strain or due to overgrowth with contaminating bacteria. It has already been shown that real-time PCR assays are elegant methods for prediction of resistance to clarithromycin
MATERIALS AND METHODS Gastric tissue specimens. Culture positive gastric tissue samples were derived from 150 patients with gastritis or peptic ulcer disease. After the tissue specimens were cultured for H. pylori and the antimicrobial susceptibility of the isolates recovered were tested, DNA was isolated from the gastric tissue specimens by using a QIAmp DNA mini kit (QIAGEN, Hilden, Germany). The DNA samples were stored at ⫺20°C until use. Before the DNA preparations were subjected to real-time PCR, amplification of the vacA signal sequence was performed by a specific conventional PCR, as described earlier (19), to demonstrate the presence of H. pylori DNA. Ten H. pylori-negative samples (confirmed by a negative urease test, negative culture, and negative vacA PCR results) were used as negative controls. Bacterial strains, culture conditions, and determination of susceptibility to tetracycline by Etest. All H. pylori strains used in this study were cultured under microaerophilic conditions at 37°C for 48 h and identified as H. pylori by the use of standard criteria (11, 19). The Etest method (AB Biodisk, Sweden) was used to determine the MIC of tetracycline for all H. pylori strains. The method was performed by a previously published protocol (10). Strains were classified as resistant to tetracycline when the MIC was ⱖ1 mg/liter and as sensitive when the MIC was ⬍1 mg/liter. Canadian H. pylori isolate KC617 was kindly provided by S. van Zanten and G. Cooper-Lesins from the Queen Elizabeth II Health Sciences Centre, Halifax, Nova Scotia, Canada.
* Corresponding author. Mailing address: Nationales Referenzzentrum fu ¨r Helicobacter pylori, Abteilung Medizinische Mikrobiologie und Hygiene, Institut fu ¨r Medizinische Mikrobiologie und Hygiene, Universita¨tsklinikum Freiburg, Hermann-Herder-Strae 11, D-79104 Freiburg, Germany. Phone: 49-761-203-6590. Fax: 49-761-203-6562. E-mail:
[email protected]. 3166
VOL. 49, 2005
SCREENING FOR TETRACYCLINE RESISTANCE IN H. PYLORI
DNA extraction, 16S rRNA gene amplification, and sequencing. The DNA of the H. pylori isolates was extracted by using a QIAmp DNA mini kit (QIAGEN). Amplification of a 120-bp fragment of the 16S rRNA genes (GenBank accession no. AF512997) was performed by using primers 16S-880fw (5⬘-ATAGACGGG GACCCGCACAAG-3⬘) and 16S-999rv (5⬘-TGGCAAGCCAGACACTCCA-3⬘) (all primers were delivered from Hermann GmbH, Germany). PCR amplicons were examined by applying 10 l on a 1.2% agarose gel (Peqlab, Germany) and were then purified by using the QIAquick PCR purification kit (QIAGEN). DNA preparations of five Brazilian Tetr strains (BZ002, BZ197, BZ261, BZ288, and BZ291) isolated from unrelated patients were previously characterized as carrying the Tetr-inducing AGA926-9283TTC triple-base-pair mutation and were kindly provided by M. Ribeiro and J. Pedrazzoli, Jr., from the University Medical School, Sao Francisco, Brazil (17). The purified PCR products were sequenced with an ABI PRISM BigDye Terminator cycle sequencing ready reaction kit (Applied Biosystems, United Kingdom) by using the PCR primers as sequencing primers. Sequencing was accomplished with an ABI 310 DNA sequencer (Applied Biosystems). 16S rRNA gene real-time PCR. For the detection of 16S rRNA gene mutations, a pair of hybridization probes (TIB Molbiol, Germany) consisting of an anchor and a mutation probe was designed. Anchor probe 16S-Anc (5⬘-TCT AGC GGA TTC TCT CAA TGT CAA GCC TAG-3⬘; 3⬘ labeled with fluorescein) covers nucleotides 975 to 946, and mutation probe 16S-AGA-Sensor (5⬘-AAG GTT CTT CGT GTA TCT TCG-3⬘; 5⬘ labeled with LightCycler Red640 and 3⬘ phosphorylated) covers nucleotides 943 to 923. An additional probe, 16S-TTC-Sensor (5⬘-AAG GTT CTT CGT GTA GAA TCG-3⬘; 5⬘ labeled with LightCycler Red705 and 3⬘ phosphorylated), that matched the sequence of the TTC mutant and that covered the same nucleotides as probe 16S-AGA-Sensor was used in combination with probe 16S-Anc for a more accurate discrimination of the various mutants. Real-time PCR was performed with bacterial DNA extracted from isolates and with DNA preparations from the gastric tissue samples as well. The method included the amplification of the 16S rRNA gene fragment of H. pylori by use of the primers mentioned above and the simultaneous detection of the PCR product with the hybridization probes. Real-time PCRs were accomplished in 20-l volumes in glass capillaries (Roche Diagnostics, Germany) by using a LightCycler instrument (Roche Diagnostics, Germany). Twenty microliters of PCR mixture contained 10 l of QuantiTect hybridization master mix (QIAGEN), 0.4 l of primers 16S-880fw and 16S-999rv (25 M each), 2 l of the anchor probe and 2 l of the mutation probe (2 M each), 3.2 l of H2O, and 2 l of template DNA (10 ng/l). The cycling conditions consisted of an initial activation at 95°C for 15 min and 10 cycles of denaturation at 95°C for 10 s and annealing at 56°C for 10 s, with an elongation step at 72°C for 10 s. Then, the annealing temperature was decreased stepwise by 1°C per cycle to a final annealing temperature of 51°C, followed by a further 35 cycles (50 cycles in total). After amplification, the samples were denatured at 95°C for 0 s and cooled down to 30°C, where they were held at that temperature for 30 s. Then, samples were slowly heated to 85°C at a ramping rate of 0.1°C/s with continuous acquisition of the decline in fluorescence. Melting curves were plotted automatically and analyzed with the LightCycler software. In order to evaluate the detection limit of the 16S real-time PCR assay, DNA of the H. pylori strain 26695 was isolated as described above and quantified by spectrophotometry at 260 nm. Afterwards, serial 1:10 dilutions were prepared, resulting in bacterial DNA concentrations ranging from 2 ng/l to 2 fg/l, and then the bacterial DNA was applied as the template DNA to the real-time PCR mixture. The specificities of the PCR primers and the hybridization probes were investigated by applying the DNA of various bacterial species, including Escherichia coli (clinical isolates), Campylobacter fetus (clinical isolates), Campylobacter coli (clinical isolates), Campylobacter jejuni (clinical isolates), Lactobacillus spp. (clinical isolates), Helicobacter mustelae (ATCC 43772), Helicobacter pullorum (NCTC 12827), and Helicobacter felis (our own strain collection). The sensitivity of the PCR assay for the detection of the tetracycline-resistant genotype among the tetracycline-sensitive wild-type isolates was investigated by applying DNA mixtures of the TTC mutant and the AGA wild type to the real-time PCR mixture. The DNAs were mixed at ratios (mutant to wild type) of 1:1, 1:2, 1:5, 1:10, and 1:20 and then used as the template DNA. Nucleotide sequence accession number. The 120-bp fragment of the 16S rRNA genes has been submitted to GenBank and can be found under accession no. AF512997.
3167
TABLE 1. 16S rRNA wild type and mutants, MICs of tetracycline (determined by Etest), and melting temperatures of the 16S rRNA gene hybridization probes Strain or mutanta c
AGA GGAd ATA TGC ATC TTA GTA GGC TTC Strain 181 (TTC) KC617 (TTC) Brazilian isolatese (TTC)
Tmg (°C) with the following probe:
MICb (mg/liter)
16S-AGA-Sensor
16S-TTC-Sensor
0.19 ⬍0.75 1.5 1.5 2.0 1.5 1.5 1.0 6.0 6.0 8.0 4.0–6.0f
61.1 57.4 52.3 50.5 49.9 51.2 52.7 51.0 49.3 49.3 49.3 49.3
54.8 56.5 53.9 59.4 58.1 58.1 55.8 59.4 63.0 63.0 63.0 63.0
a Genotype at positions 926 to 928 (substituted residues are underlined); mutants exhibiting the ATA, TGC, ATC, TTA, and TTC genotypes were described previously (2). b Strains showing MICs ⱖ1 mg/liter were classified as resistant; strains showing MICs ⬍1 mg/liter were classified as sensitive to tetracycline. c Representative of 142 independent isolates analyzed. d Representative of eight independent isolates analyzed plus one artificial mutant. e Representative of the five independent isolates tested. f M. Ribeiro (Sao Francisco, Brazil), personal communication. g Tm, melting temperature.
RESULTS Mutations affecting nucleotides 926 to 928 of the 16S rRNA genes of H. pylori confer resistance to tetracycline. The seven Tetr clinical isolates (Dutch isolate 181, the five Brazilian isolates, and the Canadian isolate) and one of the artificial mutants, all of which harbored the TTC926-928 triple mutation, showed MICs of 4 to 8 mg/liter and were classified as resistant to tetracycline (Table 1). Five artificial mutants exhibiting double-base-pair mutations (TGC926-928, ATC926-928, TTA926-928, GTA926-928, and GGC926-928) were also resistant to tetracycline and showed MICs between 1 and 2 mg/liter (Table 1). The influence of a single-base-pair mutation appeared to be different. Whereas the ATA926-928 mutation was associated with an MIC of 1.5 mg/liter, the mutant with the GGA926-928 mutation showed a tetracycline-sensitive phenotype, resulting in an MIC of 0.75 mg/liter (Table 1). Sensitivity and specificity of the 16S real-time PCR. If it is assumed that one H. pylori genome corresponds to about 1.8 fg or 1.7 Mbp (21), the lowest bacterial DNA concentration that would render a positive signal defined by a melting curve was 20 fg, which roughly equals 10 H. pylori genomes per l (data not shown). No amplification and no melting curve were observed when H. pylori-negative gastric tissue samples or the DNA of Helicobacter mustelae, Campylobacter spp., Lactobacillus spp., and Escherichia coli were used as the template DNA. Although Helicobacter felis and Helicobacter pullorum were positive by the 16S rRNA PCR, the resultant melting curves were clearly distinguishable from those obtained with the different H. pylori isolates (data not shown). Screening for mutations associated with tetracycline resistance. Upon analysis of the melting curves of all Tets clinical isolates tested, two melting temperatures corresponding to two
3168
GLOCKER ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 1. Detection of the TTC mutant among AGA wild-type H. pylori isolates using a mixture of DNAs (wild-type and TTC mutant DNAs). The DNAs were mixed at ratios (wild type to mutant) of 2:1, 5:1, and 10:1. Wild-type and TTC mutant DNAs alone were included as controls. The melting peaks of the AGA wild type and the TTC mutant were readily detectable up to a ratio of 5:1. From a ratio of 10:1 onward, only the wild-type melting peak could be recognized.
different genotypes were found: 142 strains showed melting temperatures of 61.1°C and exhibited an AGA926-928 wild type, as confirmed by subsequent sequencing analysis (Table 1). For eight isolates we found a melting temperature of 57.4°C (Table 1). Sequencing of these isolates revealed a GGA926-928 genotype that fit the decreased temperature well due to the singlenucleotide exchange (Table 1). In parallel, we also investigated the DNA preparations of the corresponding gastric tissue samples and obtained identical results (Table 1). As expected, the seven Tetr clinical isolates and the artificial TTC mutant showed a markedly decreased melting temperature of 49.3°C (Table 1). The DNA preparation of the original biopsy specimen containing H. pylori isolate 181 yielded the same melting temperature as that of its pure culture (data not
shown), thus indicating that the direct detection of the tetracycline resistance of this isolate in a routine clinical sample is feasible, even in samples stored for prolonged periods of time. Mutants with a single mutation were identified by lower melting temperatures of 57.4°C (GGA mutant; Table 1) and 52.3°C (ATA mutant; Table 1). The five artificial mutants with double-base-pair mutations exhibited two mismatches to the 16S-AGA-sensor probe, which was also reflected by a decrease of the melting temperature compared to that for the Tets wild-type strains. The GTA mutant showed a melting temperature of 52.7°C, followed by the TTA mutant with a melting temperature of 51.2°C, the GGC mutant with a melting temperature of 51.0°C, the TGC mutant with a melting temperature of 50.5°C, and the ATC mutant with a melting tempera-
SCREENING FOR TETRACYCLINE RESISTANCE IN H. PYLORI
VOL. 49, 2005
ture of 49.9°C (Table 1). Unexpectedly, the GTA mutant with a double mutation revealed a higher melting temperature than the ATA mutant with a single mutation. A second probe (the 16S-TTC-Sensor probe) was used for the more accurate discrimination of the mutants with doublebase-pair mutations and for the better differentiation of the ATC mutant from the TTC mutant. The sequences of the TTC mutants perfectly matched that of this second probe and showed melting points of 63.0°C, whereas both the TGC and the GGC mutants (melting temperatures, 59.4°C) as well as the ATC and the TTA mutants (melting temperatures, 58.1°C) were associated with lower melting temperatures (Table 1). The use of this second probe also allowed a clear distinction between the ATA mutant and the GTA mutant (melting temperatures, 53.9°C versus 55.8°C). Furthermore, the 16S PCR assay was also capable of detecting mixed infections when the 16S-AGA-Sensor probe was used. Up to a ratio of 1:5 (TTC to AGA), both the TTC mutant and the AGA wild-type were readily detected. When the amount of the wild-type genotype was further increased, the melting peak of the mutant genotype was no longer detectable (Fig. 1). All strains tested, including the artificial mutants, showed a homozygous genotype (i.e., the same mutations in the 16S rrnA and rrnB genes), which was visible by only one peak in the melting curve analysis. DISCUSSION Due to the increased double resistance of H. pylori to clarithromycin and metronidazole in Germany (12), alternative second-line strategies that include tetracyclines might be used more often. Recent publications provide data on the successful eradication of H. pylori by the use of tetracycline-based treatments (1, 2, 6). The more frequent use of tetracyclines for the eradication of H. pylori in Germany is likely to be associated with increasing numbers of tetracycline-resistant H. pylori strains in this country in the future. Thus, before such a new antibiotic regimen can be applied to overcome initial treatment failure, it is critical to test for the potential loss of its efficacy due to resistance to these compounds. In our hands conventional antimicrobial susceptibility testing fails in approximately 10% of the attempts due to overgrowth by contaminating bacteria or the lack of live bacteria. As demonstrated previously for clarithromycin and ciprofloxacin, molecular techniques such as real-time PCRs can be used as diagnostic rescue methods (7, 15, 16). This study describes the development of a real-time PCR method that correctly recognized Tets strains in 150 clinical specimens as well as a set of seven natural and eight artificially constructed TetR strains exhibiting a multitude of mutations in the 16S rRNA gene. Of the 150 clinical samples, 142 contained phenotypically Tets H. pylori strains that harbored the AGA926-928 triplet; 8 of these 150 strains, however, showed a GGA926-928 triplet. As expected, the melting temperatures of the artificial TTC mutant and the naturally occurring Tetr strains were significantly decreased due to the triple-base-pair exchange. The other mutants yielded intermediate melting peaks that, for the most part, could be distinguished from each other. Because of the
3169
marginal differences between the TTC and the ATC mutants and the TTA and the GGC mutants, we applied a second mutation probe (the 16S-TTC-Sensor probe) that facilitated the discrimination of these four genotypes. The differentiation between the TGC mutant and the GGC mutant remained problematic and would require an additional probe; but as both mutants with double-base-pair mutations are classified as tetracycline resistant and showed the same MIC, there is no immediate clinical relevance for their distinction. The 16S rRNA gene-based real-time PCR assay presented here was able to detect about 10 bacteria/l. We believe that this is a very satisfactory sensitivity, because in our experience gastric tissue samples from H. pylori-positive patients usually harbor considerably higher numbers of bacteria. One shortcoming of the assay is its lack of specificity for H. pylori. The PCR primers applied are not specific for H. pylori per se; they also amplify the 16S rRNA genes of several other Helicobacter spp. However, melting curve analysis clearly allowed discrimination between H. pylori, Helicobacter felis, and Helicobacter pullorum. No PCR signal was obtained when the DNA of Helicobacter mustelae or other bacteria, such as Campylobacter spp., Escherichia coli, and Lactobacillus spp., was applied. Nevertheless, in order to exclude false-positive results, we strongly recommend that all samples be tested by an H. pylori-specific PCR assay (e.g., one that amplifies the vacA or ureC gene) and to subject only positive samples to the16S rRNA gene realtime PCR (9, 19). In contrast to other methods based on PCR-restriction fragment length polymorphism analysis (17), this novel real-time PCR was able to distinguish not only between the AGA wild type and resistant strains harboring the TTC triple-base-pair mutation but also between mutants exhibiting single- or double-base-pair mutations, thereby allowing a more accurate classification of the strains as Tets or Tetr. Furthermore, the realtime PCR method described here allowed detection of tetracycline resistance in cases of growth failure or contamination, thus obviating the need for live bacteria. Future studies need to address the question of whether additional mutations or mechanisms play a role in the resistance of H. pylori to tetracycline (3). ACKNOWLEDGMENTS This work was supported by the Robert-Koch-Institut by a grant to M. Kist (1369-239) of the German Ministry of Health. We are grateful to S. van Zanten, G. Cooper-Lesins, M. Ribeiro, and J. Pedrazzoli, Jr., for providing strains or DNA samples and to Christian Bogdan for critical reading of the manuscript. REFERENCES 1. Cammarota, G., A. Martino, G. Pirozzi, R. Cianci, G. Branca, E. C. Nista, A. Cazzato, O. Cannizzaro, L. Miele, A. Grieco, A. Gasbarrini, and G. Gasbarrini. 2004. High efficacy of 1-week doxycycline- and amoxicillin-based quadruple regimen in a culture-guided, third-line treatment approach for Helicobacter pylori infection. Aliment. Pharmacol. Ther. 19:789–795. 2. Chi, C. H., C. Y. Lin, B. S. Sheu, H. B. Yang, A. H. Huang, and J. J. Wu. 2003. Quadruple therapy containing amoxicillin and tetracycline is an effective regimen to rescue failed triple therapy by overcoming the antimicrobial resistance of Helicobacter pylori. Aliment. Pharmacol. Ther. 18:347–353. 3. Dailidiene, D., M. T. Bertoli, J. Miciuleviciene, A. K. Mukhopadhyay, G. Dailide, M. A. Pascasio, L. Kupcinskas, and D. E. Berg. 2002. Emergence of tetracycline resistance in Helicobacter pylori: multiple mutational changes in 16S ribosomal DNA and other genetic loci. Antimicrob. Agents Chemother. 46:3940–3946. 4. Gerrits, M. M., M. R. De Zoete, N. L. Arents, E. J. Kuipers, and J. G. Kusters. 2002. 16S rRNA mutation-mediated tetracycline resistance in Helicobacter pylori. Antimicrob. Agents Chemother. 46:2996–3000.
3170
GLOCKER ET AL.
5. Gerrits, M. M., M. Berning, A. H. M. Van Vliet, E. J. Kuipers, and J. G. Kusters. 2003. Effects of 16S rRNA gene mutations on tetracycline resistance in Helicobacter pylori. Antimicrob. Agents Chemother. 47:2984–2986. 6. Gisbert, J. P., and J. M. Pajares. 2001. Helicobacter pylori therapy: first-line options and rescue regimen. Dig. Dis. 19:134–143. 7. Glocker, E., and M. Kist. 2004. Rapid detection of point mutations in the gyrA gene of Helicobacter pylori conferring resistance to ciprofloxacin by a fluorescence resonance energy transfer-based real-time PCR approach. J. Clin. Microbiol. 42:2241–2246. 8. Glupczynski, Y., F. Me´graud, M. Lopez-Brea, and L. P. Andersen. 2001. European multicentre survey of in vitro antimicrobial resistance in Helicobacter pylori. Eur. J. Clin. Microbiol. Infect. Dis. 20:820–823. 9. He, Q., J. P. Wang, M. Osato, and L. B. Lachman. 2002. Real-time quantitative PCR for detection of Helicobacter pylori. J. Clin. Microbiol. 40:3720– 3728. 10. Heep, M., M. Kist, S. Strobel, D. Beck, and N. Lehn. 2000. Secondary resistance among 554 isolates of Helicobacter pylori after failure of therapy. Eur. J. Clin. Microbiol. Infect. Dis. 19:538–541. 11. Kist, M. 1991. Diagnostische Verfahrensrichtlinien der DGHM: Isolierung und Identifizierung von Bakterien der Gattungen Campylobacter und Helicobacter. Zentbl. Bakteriol. Parasitenkd. Infektkrankh. Hyg. Abt. 1 Orig. 276:124–139. 12. Kist, M., and E. Glocker. 2004. ResiNet—a nationwide German sentinel study for surveillance and analysis of antimicrobial resistance in Helicobacter pylori. Euro. Surveill. Q. 1:44–46 13. Leung, W. K., and J. J. Sung. 2002. Review article: intestinal metaplasia and gastric carcinogenesis. Aliment. Pharmacol. Ther. 16:209–216. 14. Malfertheiner, P., F. Me´graud, C. O’Morain, A. P. S. Hungin, R. Jones, A. Axon, D. Y. Graham, G. Tytgat, and The European Helicobacter pylori Study Group (EHPSG). 2002. Current concepts in the management of Helicobacter pylori infection. The Maastricht Consensus Report. Aliment. Pharmacol. Ther. 16:167–189.
ANTIMICROB. AGENTS CHEMOTHER. 15. Matsumura, M., Y. Hikiba, K. Ogura, G. Togo, I. Tsukuda, K. Ushikawa, Y. Shiratori, and M. Omata. 2001. Rapid detection of mutations in the 23S rRNA gene of Helicobacter pylori that confers resistance to clarithromycin treatment to the bacterium. J. Clin. Microbiol. 39:691–695. 16. Oleastro, M., A. Me´nard, A. Santos, H. Lamouliatte, L. Monteiro, P. Barthe´le´my, and F. Me´graud. 2003. Real-time PCR assay for rapid and accurate detection of point-mutations conferring resistance to clarithromycin in Helicobacter pylori. J. Clin. Microbiol. 41:397–402. 17. Ribeiro, M. L., M. M. Gerrits, Y. H. Benvengo, M. Berning, A. P. Godoy, E. J. Kuipers, S. Mendonca, S., A. H. van Vliet, J. Pedrazzoli, Jr., and J. G. Kusters. 2003. Detection of high-level tetracycline resistance in clinical isolates of Helicobacter pylori using PCR-RFLP. FEMS Immunol. Med. Microbiol. 40:57–61. 18. Stolte, M., E. Bayerdorffer, A. Morgner, B. Alpen, T. Wundisch, C. Thiede, and A. Neubauer. 2002. Helicobacter and gastric MALT lymphoma. Gut 50(Suppl. 3):19–24. 19. Strobel, S., S. Bereswill, P. Balig, P. Allgaier, H.-G. Sonntag, and M. Kist. 1998. Identification and analysis of a new vacA genotype variant of Helicobacter pylori in different patient groups in Germany. J. Clin. Microbiol. 36:1285–1290. 20. Suerbaum, S., and P. Michetti. 2002. Helicobacter pylori infection. N. Engl. J. Med. 347:1175–1186. 21. Tomb, J. F., O. White, A. R. Kerlavage, 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. Fitzegerald, N. Lee, M. D. Adams, J. C. Venter, et al. 1997. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388: 539–547. 22. Trieber, C. A., and D. E. Taylor. 2002. Mutations in the 16S rRNA genes of Helicobacter pylori mediate resistance to tetracycline. J. Bacteriol. 184:2131– 2140.
