Use of peptide nucleic acid probes as a new method for the detection of clarithromycin resistance in Helicobacter pylori strains
Abstract number: P831
Cerqueira L., Azevedo N.F., Guimarães N., Figueiredo C., Keevil C.W., Vieira M.J.
Objectives: The treatment of patients infected with H. pylori is being seriously compromised due to increased antibiotic resistance patterns (e.g. amoxicillin, clarithromycin and metrodinazole). For that reason, the purpose of this study was to develop a fast and more efficient method to test clarithromycin resistance in clinical samples using fluorescence in situ hybridisation (FISH).
Methods: There are several phenotypic, cultivation-dependent methods able to identify the clarithromycin resistance in H. pylori strains, but they are all fastidious and growth is time-consuming. Three mutations in the 23S rRNA of H. pylori are strongly associated with clarithromycin resistance. In these mutations, an adenine is replaced by a guanine at positions 2142 and 2143, or by a cytosine at position 2142. Hence, we developed a set of peptide nucleic acid (PNA) probes for the identification of target sequences for the different clarithromycin resistance polymorphisms. PNA molecules are DNA synthetic mimics with a non-charged backbone, due to their chemical configuration. As such, they present a lack of electrostatic repulsion, resulting in improved thermal stability compared with DNA/DNA duplexes.
Results: After probe design, an optimisation of the hybridisation conditions, like temperature, pH, ionic strength and formamide concentrations, was performed. To ensure specificity and sensitivity, probes have been tested against resistant and susceptible strains of H. pylori.
Conclusions: This novel PNA FISH method will facilitate a more prompt (<3 h) diagnosis of H. pylori clarithromycin resistance in clinical samples such as gastric biopsies, thus allowing a more rational patient treatment.
|Session name:||19th European Congress of Clinical Microbiology and Infectious Diseases|
|Location:||Helsinki, Finland, 16 - 19 May 2009|
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