Detection of clarithromycin-resistant Helicobacter pylori in stool samples

Abstract

The recognition of the role of Helicobacter pylori in gastric diseases has led to the widespread use of antibiotics in the eradication of this pathogen. The most advocated therapy, triple therapy, often includes clarithromycin. It is well known that clarithromycin resistance is one of the major causes of eradication failure. The development of a rapid noninvasive technique that could easily be performed on fecal samples and that could also provide information about the antibiotic resistance of this microorganism is therefore advisable. Previous findings have demonstrated that clarithromycin resistance is due to a single point mutation in the 23S rRNA. All the mutations described have been associated with specific restriction sites, namely BsaI (A2143G), MboII (A2142C/G), and HhaI (T2717C). On this basis we have developed a new method, a seminested PCR, allowing screening for clarithromycin resistance of H. pylori directly on stool samples. This method furnished a 783-bp fragment of the 23S rRNA, which was subsequently digested by MboII, BsaI, and HhaI, in order to identify single point mutations associated with clarithromycin resistance. Of a total of 283 stool samples examined, 125 were H. pylori positive and two of them were shown to contain clarithromycin-resistant strains due to the presence of a mutation at position 2717, whereas no PCR products contained mutations at position 2142 or 2143. In order to evaluate the reliability of the new system, we compared the results of restriction analysis of the PCR products with the MICs shown by the H. pylori isolates by culturing gastric biopsies from the same patients.

FIG. 1.

Lane MW, size markers (in base pairs). Lanes A to E, amplicons of β-actin for some fecal samples.

FIG. 2.

Specificity assay. Lane MW, size markers (in base pairs); lane A, negative control; lane B, Staphylococcus aureus ATCC 25923; lane C, Helicobacter heilmannii ATCC 49286; lane D, Campylobacter jejuni ATCC 33291; lane E, Escherichia coli ATCC 25922.

FIG. 3.

Lanes b to i, amplicons from stool samples from infected patients (after restriction endonuclease digestion); lane MW, size markers; lanes a, amplicon from H. pylori ATCC 43504. In the part of the figure illustrating HhaI digestion, lanes c and g correspond to the restriction patterns of two samples containing H. pylori with the T2717C transition in the 23S RNA gene (low-level Cla^r phenotype). In the part of the figure illustrating BsaI digestion, lane h corresponds to the restriction pattern of a positive control for H. pylori with a mutation at position 2143. In the part of the figure showing MboII digestion, lane h correspond to the restriction pattern of a positive control for H. pylori with a mutation at position 2142.