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 consuming and liable to observer error. A new direct technique for fluorescent labelling of bacteria has been developed to quantitate adherence of H. pylori to epithelial cells by fluorescence activated cell sorting (FACS). Type strains of H. pylori, H. mustelae, H. cinaedi and H. fennelliae were grown microaerobically in broth culture for 24 h and fluorescently labelled by incubation with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) at 37 degrees C. After washing to remove excess CFDA-SE, bacteria were co-incubated (ratio 10:1) with gastric epithelial 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 quantitated 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 strains adhered to gastric-type epithelial cells. The proportion of cells with bound bacteria varied from 40-99% and the number of bacteria per cell from 1-50, both of which correlated with microscopy (r = 0.6, and r = 0.8 respectively, n = 35). Time course studies demonstrated saturation of binding by H. pylori within 90 min. For H. mustelae, H. cinaedi and H. fennelliae the proportion of cells with bound bacteria varied from 5-15% and the mean number of bacteria per cell was < 4. Binding of H. pylori to epithelial cells could be partly blocked by pre-incubation with polyclonal anti-sera or using oligosaccharides against potential binding epitopes of gastric mucus. Fluorescent labelling of H. pylori with CFDA-SE in combination with flow cytometry provides a quick, specific, and sensitive method to quantitate in vitro the adherence of H. pylori.
Logan
R P
RP
Division of Gastroenterology, University Hospital, Nottingham, UK. robert.logan@nottingham.ac.uk
Robins
A
A
Turner
G A
GA
Cockayne
A
A
Borriello
S P
SP
Hawkey
C J
CJ
D000907:Antibodies, Bacterial / Q000276:immunology; D001422:Bacterial Adhesion; D002460:Cell Line; D004847:Epithelial Cells / Q000382:microbiology; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D016480:Helicobacter pylori / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D015203:Reproducibility of Results; D013270:Stomach / Q000166:cytology / Q000382:microbiology*; D013388:Succinimides
C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D000907:Antibodies, Bacterial; D005452:Fluoresceins; D005456:Fluorescent Dyes; D013388:Succinimides