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Real-Time PCR Detection of 16S rRNA Novel Mutations Associated with Helicobacter pylori Tetracycline Resistance in Iran

  • Dadashzadeh, Kianoosh (Department of Laboratory Sciences, Marand Branch, Islamic Azad University) ;
  • Milani, Morteza (Liver and Gastroenterology Diseases Research Cente, Tabriz University of Medical Sciences) ;
  • Rahmati, Mohammad (Drug Applied Research Center, Tabriz University of Medical Sciences) ;
  • Akbarzadeh, Abolfazl (Biotechnology Research Center, Tabriz University of Medical Sciences)
  • Published : 2014.11.06

Abstract

Background: Tetracycline is an antibiotic widely used for the treatment of Helicobacter pylori infection, but its effectiveness is decreasing due to increasing bacterial resistance. The aim of this study was to investigate the occurrence of 16S rRNA mutations associated with resistance or reduced susceptibility to tetracycline ofHelicobacter pylori by real-time PCR (RT-PCR) assays from culture. Materials and Methods: Tetracycline susceptibility and minimal inhibition concentration (MIC) was determined by the Epsilometer test (Etest) method. A LightCycler assay developed to detect these mutations was applied to DNA extracted from culture. The 16S rRNA of these isolates was sequenced and resistance-associated mutations were identified. From 104 isolates of H. pylori examined, 11 showed resistance to tetracycline. Results: LightCycler assay was applied to DNA extracted from 11 tetracycline-susceptible and 11 tetracycline resistance H. pylori isolates. In our study the sequencing of the H. pylori wild types in 16 s rRNA gene were AGA 926-928 with MIC (0.016 to $0.5{\mu}g/ml$), while the sequencing and MIC for resistant were GGA and AGC, (0.75 to $1.5{\mu}g/ml$), respectively. Also we found a novel mutation in 2 strains with $84^{\circ}C$ as their melting temperatures and exhibition of an A939C mutation. Conclusions: We conclude that real-time PCR is an excellent method for determination of H. pylori tetracycline resistance related mutations that could be used directly on biopsy specimens.

Keywords

Antimicrobial resistance mechanisms;light cycler;DNA polymorphisms

References

  1. Gisbert JP, Pajares JM (2001). Helicobacter pylori therapy: first-line options and rescue regimen. Digestive Diseases, 19, 134-43. https://doi.org/10.1159/000050668
  2. Abrams DN, Koslowsky I, Matte G (2000). Pharmaceutical interference with the [1v4C] carbon urea breath test for the detection of Helicobacter pylori infection. J Pharm Pharm Sci, 3, 228-33.
  3. Anoushiravani M, Falsafi, T, Niknam V (2009. Proton motive force-dependent efflux of tetracycline in clinical isolates of Helicobacter pylori. J Med Microbiol, 58, 1309-3. https://doi.org/10.1099/jmm.0.010876-0
  4. Dailidiene D, Bertoli MT, Miciuleviciene J (2002). Emergence of tetracycline resistance in Helicobacter pylori: multiple mutational changes in 16S ribosomal DNA and other genetic loci. Antimicrob Agents Chemother, 46, 3940-6. https://doi.org/10.1128/AAC.46.12.3940-3946.2002
  5. De Francesco V, Giorgio F, Hassan C, et al (2010). Worldwide H. pylori antibiotic resistance: a systematic review. J Gastrointestin Liver Dis, 19, 409-14.
  6. Gerrits MM, De Zoete MR, Arents NL, Kuipers EJ, Kusters, JG (2002). 16S rRNA mutation-mediated tetracycline resistance in Helicobacter pylori. Antimicrobial Agents Chemotherapy, 46, 2996-3000. https://doi.org/10.1128/AAC.46.9.2996-3000.2002
  7. Glocker E, Berning M, Gerrits MM, Kusters JG, Kist M (2005). Real-time PCR screening for 16S rRNA mutations associated with resistance to tetracycline in Helicobacter pylori. Antimicrobial Agents Chemotherapy, 49, 3166-70. https://doi.org/10.1128/AAC.49.8.3166-3170.2005
  8. Heep M, Kist M, Strobel S, Beck D, Lehn N (2000). Secondary resistance among 554 isolates of Helicobacter pylori after failure of therapy. Eur J Clin Microbiol Infect Dis, 19, 538-41. https://doi.org/10.1007/s100960000288
  9. Lawson A J, Elviss NC, Owen RJ (2005). Real-time PCR detection and frequency of 16S rDNA mutations associated with resistance and reduced susceptibility to tetracycline in Helicobacter pylori from England and Wales. Antimicrobial Agents Chemotherapy, 56, 282-6. https://doi.org/10.1093/jac/dki199
  10. Malekimsc P, Siavoshiphd F (2013). Helicobacter pylori vacA d1/-i1 Genotypes and Geographic Differ-entiation between High and Low Incidence Areas of Gastric Can-cer in Iran. Arch Iran med 16, 330.
  11. Malfertheiner P, Megraud F, O'morain C, et al (2007). Current concepts in the management of Helicobacter pylori infection: the Maastricht III Consensus Report. Gut, 56, 772. https://doi.org/10.1136/gut.2006.101634
  12. Talebi Bezmin Abadi A, Mobarez AM, Taghvaei T, Wolfram L (2010). Antibiotic resistance of Helicobacter pylori in Mazandaran, North of Iran. Helicobacter, 15, 505-9. https://doi.org/10.1111/j.1523-5378.2010.00795.x
  13. Mansour-Ghanaei F, Joukar F, Soati F, Mansour-Ghanaei A, Atrkar-Roushan Z (2013). Outcome of intestinal metaplasia in gastric biopsy of patients with dyspepsia in Guilan Province, North Iran. Asian Pac J Cancer Prev, 14, 3549-54. https://doi.org/10.7314/APJCP.2013.14.6.3549
  14. Mcnulty C, Owen R, Tompkins D, et al (2002). Helicobacter pylori susceptibility testing by disc diffusion. J Antimicrobial Chemotherapy, 49, 601. https://doi.org/10.1093/jac/49.4.601
  15. Milani M, Ghotaslou R, Akhi MT, et al (2012). The status of antimicrobial resistance of Helicobacter pylori in Eastern Azerbaijan, Iran: comparative study according to demographics. J Infect Chemother, 18, 848-52. https://doi.org/10.1007/s10156-012-0425-4
  16. Wang Y, Zhang FC, Wang YJ (2014). Helicobacter pylori and pancreatic cancer risk: a meta-analysis based on 2,049 cases and 2,861 controls. Asian Pac J Cancer Prev, 15, 4449-54. https://doi.org/10.7314/APJCP.2014.15.11.4449
  17. Wu H, Shi X, Wang H, Liu J (2000). Resistance of Helicobacter pylori to metronidazole, tetracycline and amoxycillin. J Antimicrobial Chemotherapy, 46, 121-3. https://doi.org/10.1093/jac/46.1.121
  18. Zullo A, Hassan C, Ridola L, De Francesco V, Vaira D (2012). Standard triple and sequential therapies for Helicobacter pylori eradication: An update. Eur J Int Med, 24, 16-9.

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