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. 2000 May;46(5):608–614. doi: 10.1136/gut.46.5.608

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

K Trebesius 1, K Panthel 1, S Strobel 1, K Vogt 1, G Faller 1, T Kirchner 1, M Kist 1, J Heesemann 1, R Haas 1
PMCID: PMC1727914  PMID: 10764702

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.


Keywords: Helicobacter pylori; macrolide resistance; clarithromycin; in situ hybridisation; genotypic resistance

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Figure 1  .

Figure 1  

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Specificity testing of oligonucleotide probe Hpy-1 for discrimination of H pylori from related helicobacters. (A) A mixture of H pylori and H mustelae, as seen by phase contrast microscopy. (B) Detection of the bacterial mixture with two probes, a H pylori specific probe Hpy-1-Cy3 (red) and a bacterial specific probe Eub-FLUOS (green). Both helicobacters bound the bacterial probe (green) but H pylori bound Hpy-1-Cy3 (red) in addition, resulting in the yellow appearance of H pylori (mixed colour). Bar represents 10 µm.

Figure 2  .

Figure 2  

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Specific detection of clarithromycin sensitive and resistant H pylori isolates. (A) Phase contrast microscopy of a mixture of clarithromycin sensitive and resistant H pylori isolates. (B) The same microscopic field after hybridisation with probes ClaWT-FLUOS (green) and ClaR1-Cy3 (red). (C) Phase contrast micrograph showing coccoid forms of clarithromycin resistant H pylori. (D) Whole cell hybridisation of coccoid forms with probe Hpy-1-FLUOS. Bar represents 10 µm.

Figure 3  .

Figure 3  

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Specific detection of clarithromycin sensitive and resistant H pylori isolates within gastric antrum sections of H pylori infected patients. (Left) Detection of H pylori and clarithromycin resistance by whole cell hybridisation with fluorescent probes Hpy-1-FLUOS and ClaR1-Cy3 simultaneously. (Right) Antrum sections from the same biopsy material (Warthin-Starry stain). (A) Clarithromycin resistant H pylori are visible in yellow (mixed colour between green and red). (C) Mixed population of clarithromycin resistant (yellow) and sensitive (green) H pylori in the same biopsy specimen of a patient. (B, D) Antrum sections showing H pylori by the Warthin-Starry stain, which does not allow resistance determination.

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