Genotypic characterization of clarithromycin-resistant Helicobacter pylori strains.

Helicobacterpylori (Hp) resistance to clarithromycin, one of the antibiotics most used to eradicate infection, is connected with the presence of a point mutation on the level of adenine at position 2143 or 2144 of 23S rRNA.The aim of the study is to evaluate of the presence of these mutation vs control clarithromycin resistant Hp strains present in North Sardinia; to verify the real association between the type of mutation and the resistance-level; to use easier molecular biology methods to quickly locate the resistance-associated mutations beginning with the bioptic material. The clarithromycin susceptibility of Hp isolates was tested by the E-test method (antibiotic assay). Genomic DNA of Hp strains was amplified using specific primers for the domain V. of ribosomic 23S rRNA and sequenced after the reaction with a primer within the fragment 23S. At the same time PCR-RFLP reliability was examined underlining the presence of these mutations with BsaI, BbsI, MboII restriction enzymes. Two mutations in 2143 (A- - G) and 2144 (A- - G) were found by domain V sequencing. The strains with mutation 2143 are characterized by a greater resistance level (MIC>64 g/ml) than those with mutation 2144 (MIC <64 g/ml). Restriction endonucleases BbsI and MboII recognise the site containing the mutation 2143 (A- - G), while BsaI recognise the mutation 2144 (A- - G). These methods might enable us to identify the presence of Hp directly from bioptic material and possible clarithromycin resistance and plan a suitable therapeutic strategy and consequently a better control of the infection.