Rapid and specific detection of Helicobacter pylori macrolide resistance in gastric tissue by fluorescent in situ hybridisation

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 detect Helicobacter 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 35 H pylori isolates analysed. In a set of gastric biopsy specimens (27) H pylori infection 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 pylori were 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. (Gut 2000;46:608-614)

Prevalence of A2143G and A2144G Point Mutations Responsible for Clarithromycin Resistance among Helicobacter pylori Strains in Bushehr, Iran

“…These probes have been formerly described and evaluated by Trebesius et al (8). Probe Hpy-1 (5'-CAC ACC TGA CTG ACT ATC CCG-3') specifically hybridizes a 16S rRNA region of the species H. pylori and detects this organism.…”

“…For the first step, hybridization of all of the samples was performed using a mixture of five probes: Hpy-1, ClaR1, ClaR2, ClaR3, and ClaWT. Hybridization was done at 46°C for 90 minutes with a hybridization buffer (8), and then stringent washing was performed using a washing buffer (8). Thereafter, DNA was stained with 4 , 6-diamidine-2 -phenylindole dihydrochloride (DAPI; Roche, Mannheim, Germany).…”

“…FISH was carried out according to a protocol described elsewhere (8,11,15). For the first step, hybridization of all of the samples was performed using a mixture of five probes: Hpy-1, ClaR1, ClaR2, ClaR3, and ClaWT.…”

“…Resistance to clarithromycin in H. pylori is caused predominantly by point mutations in the peptidyl transferase center of 23S rRNA (5,8,9). These mutations prevent the binding of clarithromycin to the 50S subunit of the ribosome (6).…”

“…These mutations prevent the binding of clarithromycin to the 50S subunit of the ribosome (6). It has been proven that fluorescent in situ hybridization (FISH) by the use of rRNA-targeted oligonucleotide probes is an accurate and convenient molecular technique for the detection of three 23S rRNA point mutations responsible for clarithromycin resistance, in which the adenine residues at positions 2143 and 2144 are replaced by guanine (A2143G and A2144G) or cytosine (A2143C) (8,10,11). The FISH technique was used in this study.…”