Article Text

Download PDFPDF

Rapid and specific detection of Helicobacter pylori macrolide resistance in gastric tissue by fluorescent in situ hybridisation
  1. K Trebesiusa,
  2. K Panthela,
  3. S Strobelb,
  4. K Vogtc,
  5. G Fallerd,
  6. T Kirchnerd,
  7. M Kistb,
  8. J Heesemanna,
  9. R Haasa
  1. aMax von Pettenkofer Institute for Hygiene and Medical Microbiology, Ludwig Maximilians University Munich, Germany, bInstitute for Medical Microbiology and Hygiene, University Freiburg, Germany, cDepartment of Microbiology, Hospital Virchow and Charite, Humboldt University of Berlin, Germany, dInstitute of Pathology, University of Erlangen-Nürnberg, Germany
  1. Rainer Haas, Max von Pettenkofer Institute for Hygiene and Medical Microbiology, Pettenkoferstr. 9a, D-80336 Munich, Germany. Email:haas{at}m3401.mpk.med.uni-muenchen.de

Abstract

BACKGROUND The development of macrolide resistance in Helicobacter pylori is considered an essential reason for failure of antibiotic eradication therapies. The predominant mechanism of resistance to macrolides, particularly clarithromycin, is based on three defined mutations within 23S rRNA, resulting in decreased binding of the antibiotic to the bacterial ribosome.

AIM To develop an rRNA based whole cell hybridisation method to detectHelicobacter species in situ within gastric tissue, simultaneously with its clarithromycin resistance genotype.

METHODS A set of fluorescent labelled oligonucleotide probes was developed, binding either to H pylori 16S rRNA or 23S rRNA sequences containing specific point mutations responsible for clarithromycin resistance. After hybridisation and stringent washing procedures, labelling of intact single bacteria was monitored by fluorescence microscopy. The new approach was compared with PCR based assays, histology, and microbiological culture.

RESULTS In comparison with the phenotypic resistance measurement by E test, the genotypic clarithromycin resistance correlated perfectly (100%) for 35H pylori isolates analysed. In a set of gastric biopsy specimens (27) H pyloriinfection was confirmed by histology (17/27) and correctly detected by whole cell hybridisation. Five clarithromycin resistant strains were identified in gastric tissue specimens directly. Furthermore, non-cultivable coccoid forms of H pyloriwere easily detectable by whole cell hybridisation.

CONCLUSIONS Whole cell hybridisation of rRNA holds great promise for cultivation independent, reliable, and rapid (three hours) genotypic determination of clarithromycin resistance in H pylori. Compared with PCR techniques it is independent of nucleic acid preparations, not prone to inhibition, and allows semiquantitative visualisation of the bacteria within intact tissue samples.

  • Helicobacter pylori
  • macrolide resistance
  • clarithromycin
  • in situ hybridisation
  • genotypic resistance
View Full Text

Statistics from Altmetric.com

Footnotes

  • Abbreviations used in this paper:
    MIC
    minimal inhibitory concentration
    rRNA
    ribosomal RNA
    H-E stain
    haematoxylin-eosin stain
    MALT
    mucosa associated lymphoid tissue
    FLUOS
    5(6)-carboxy-fluorescein-N-hydroxysuccinimide ester

Request Permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.