1 Department of Microbiology and Institute of Biomedical Science, Hanyang University College of Medicine, Seoul, 2 Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, Korea
Received 9 July 2005; returned 7 August 2005; revised 22 August 2005; accepted 22 August 2005
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Abstract |
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Methods: One hundred and thirty-five strains of H. pylori (34 strains in 1987, 36 in 1994 and 65 in 2003) were isolated from antral gastric mucosal biopsy specimens. The determination of MICs for the H. pylori isolates of ciprofloxacin, levofloxacin and moxifloxacin was examined by using the serial 2-fold agar dilution method. DNA sequences of the gyrA gene in fluoroquinolone-resistant strains were determined.
Results: The distribution of fluoroquinolone MICs (ciprofloxacin, levofloxacin and moxifloxacin) shifted to higher concentrations during 19872003. All of the levofloxacin- or moxifloxacin-resistant strains were resistant to ciprofloxacin. Sequence analysis in fluoroquinolone-resistant strains showed point mutation of the gyrA gene at A272G and G271A, indicating mutations of the codon Asp-91 in the fluoroquinolone-resistance-determining region of the DNA gyrase.
Conclusions: These results suggest that resistance to fluoroquinolones has been increasing in the Korean population and the resistance is most likely mediated through point mutation in gyrA.
Keywords: gyrA , mutation , QRDR , resistance
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Introduction |
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Materials and methods |
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We isolated 135 strains of H. pylori from antral gastric mucosal biopsy specimens obtained in Seoul, Korea, in 1987 (Hanyang University Hospital, 34 strains), in 1994 (Seoul National University Hospital, 36 strains) and in 2003 (Seoul National University Hospital, 65 strains).3 None of the patients had taken antibiotics, PPI or non-steroidal anti-inflammatory drugs during the preceding 3 months. The distance between the two hospitals (Hanyang University Hospital and Seoul National University Hospital) located in Seoul is less than 5 km and these two are general and tertiary hospitals, indicating that the distribution of patient diseases is similar. The H. pylori strains were cultured under microaerophilic conditions (5% O2, 10% CO2, 85% N2) as previously described.3 All stock cultures were maintained at 70°C in Brucella broth supplemented with 15% glycerol. These preparations were thawed and subcultured for experiments.
Determination of MIC
The susceptibilities of the H. pylori isolates to ciprofloxacin (Sigma Chemical Co., St Louis, MO, USA), levofloxacin (Sigma) and moxifloxacin (Bayer AG Pharmaceuticals, Germany) were examined by using the serial 2-fold agar dilution method as described previously.3,4 Briefly, the bacteria were subcultured on MuellerHinton agar supplemented with 5% defibrinated sheep blood for 48 h. Bacterial suspension adjusted to 1 x 107 cfu was inoculated directly on each antibiotic-containing agar dilution plate. After incubation for 72 h, the MIC of each antibiotic was determined. The breakpoints for fluoroquinolone resistance were provisionally defined as >1.0 mg/L.
PCR amplification and nucleotide sequence
The extraction of H. pylori genomic DNA was performed as reported previously.3 To detect gene mutation of the quinolone resistance-determining regions (QRDRs) of the A subunit of the DNA gyrase (gyrA), we used the oligonucleotide primers: 5'-TTT AGC TTA TTC AAT GAG CGT-3' and 5'-GCA GAC GGC TTG GTA GAA TA-3'.5 The size of the amplified fragments of gyrA was 429 bp. The PCR profile consisted of 35 cycles of 1 min denaturation at 94°C, 1 min annealing at 57°C and 1 min extension at 72°C. Sequencing was performed on both strands of the non-restricted amplicons, using an ABI PRISM 377 DNA sequencer (Applied Biosystems, Foster City, CA, USA).
Transformation of H. pylori
Transformation of H. pylori was accomplished by using a modified version of the method previously described.6 H. pylori HP99, a clinical isolate, was chosen as a recipient for transformation experiments on the basis of prior experiments that revealed that it was readily transformed with DNA. The recipient strains (HP99, ciprofloxacin MIC 0.125 mg/L) were grown for 3 days on charcoal agar and then subcultured to a new plate in a 1 cm2 area. The plate was incubated overnight; donor DNAs [10 ng of PCR-amplified gyrA fragments of Korean resistant strain HP13 (ciprofloxacin MIC 32 mg/L, Asp-91Gly) and HP11 (ciprofloxacin MIC 32 mg/L, Asp-91
Asn) in Table 1] in a volume of 10 µL were applied to the cells, and the plate was incubated for a further 24 h. The cells were then scraped off the plate into PBS, pH 7.2, diluted, and plated onto chocolate agar containing ciprofloxacin 4 mg/L. After 3 days the colonies were counted, and five colonies from each transformation were purified and maintained on medium containing ciprofloxacin.
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Results and discussion |
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In conclusion, these results demonstrated that the prevalence of primary resistance to fluoroquinolones has been increasing in the Korean population and the resistance is most likely mediated through point mutation in gyrA.
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Acknowledgements |
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References |
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