A NOVEL FLOW CYTOMETRIC ASSAY FOR QUANTITATING ADHERENCE OF HELICOBACTER-PYLORI TO GASTRIC EPITHELIAL-CELLS

Adherence may be an important virulence factor for Helicobacter pylori . Current methods available for quantitation of adherence are time con suming and liable to observer error. A new direct technique for fluore scent labelling of bacteria has been developed to quantitate adherence of H. pylori to epithelial cells by fluorescence activated cell sorti ng (FACS). Type strains of H. pylori, H. mustelae, H. cinaedi and H. f ennelliae were grown microaerobically in broth culture for 24 h and fl uorescently labelled by incubation with carboxyfluorescein diacetate s uccinimidyl ester (CFDA-SE) at 37 degrees C. After washing to remove e xcess CFDA-SE, bacteria were co-incubated (ratio 10:1) with gastric ep ithelial cells at 37 degrees C for up to 24 h. After washing to remove non-adherent bacteria, epithelial cells were detached with EDTA (2 mM ) and fixed with formaldehyde for flow cytometry, Adherence was quanti tated both in terms of the proportion of cells with adherent H. pylori and as the mean number of adherent bacteria per cell. All H. pylori s trains adhered to gastric-type epithelial cells. The proportion of cel ls with bound bacteria varied from 40-99% and the number of bacteria p er cell from 1-50, both of which correlated with microscopy (r = 0.6, and r = 0.8 respectively, n = 35). Time course studies demonstrated sa turation of binding by H. pylori within 90 min. For H. mustelae, H. ci naedi and H. fennelliae the proportion of cells with bound bacteria va ried from 5-15% and the mean number of bacteria per cell was < 4. Bind ing of H. pylori to epithelial cells could be partly blocked by pre-in cubation with polyclonal anti-sera or using oligosaccharides against p otential binding epitopes of gastric mucus. Fluorescent labelling of H . pylori with CFDA-SE in combination with flow cytometry provides a qu ick, specific, and sensitive method to quantitate in vitro the adheren ce of H. pylori. (C) 1998 Elsevier Science B.V. All rights reserved.