Quantitation of bacterial adherence by image analysis.

In studies of the adherence of pathogenic bacteria to host eukaryotic cells in vitro, the counting of the bacteria is often challenging, especially if many experiments are involved. We developed a method to use digital imaging and computer-aided recognition for the quantitation of bacteria attached to cultured cells. We employed an immunocytochemical method to stain the bacteria and leave the hosts cells relatively unstained. We describe this method for use with five species of bacteria, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus, and Chlamydia pneumoniae. To demonstrate an application of this method, we studied the attachment of H. influenzae and S. pneumoniae to target epithelial cell lines derived from the respiratory tract.

Adherence of Streptococcus pneumoniae to respiratory epithelial cells is inhibited by sialylated oligosaccharides.

To study carbohydrate-mediated adherence of Streptococcus pneumoniae to the human airway, we measured binding of live S. pneumoniae organisms to a cultured cell line derived from the lining of the conjunctiva and to primary monolayers of human bronchial epithelial cells in the presence and absence of oligosaccharide inhibitors. Both encapsulated and nonencapsulated strains of S. pneumoniae grown to mid-logarithmic phase in suspension culture adhered to cultured primary respiratory epithelial cells and the conjunctival cell line. Adherence of nine clinically prevalent S. pneumoniae capsular types studied was inhibited preferentially by sialylated oligosaccharides that terminate with the disaccharide NeuAc alpha2-3(or 6)Galbeta1. Adherence of some strains also was weakly inhibited by oligosaccharides that terminate with lactosamine (Galbeta1-4GlcNAcbeta1). When sialylated oligosaccharides were covalently coupled to human serum albumin at a density of approximately 20 oligosaccharides per molecule of protein, the molar inhibitory potency of the oligosaccharide inhibitor was enhanced 500-fold. The above-mentioned experiments reveal a previously unreported dependence upon sialylated carbohydrate ligands for adherence of S. pneumoniae to human upper airway epithelial cells. Enhanced inhibitory potencies of polyvalent over monovalent forms of oligosaccharide inhibitors of adherence suggest that the putative adhesin(s) that recognizes the structure NeuAc alpha2-3(or 6)Galbeta1 is arranged on the bacterial surface in such a manner that it may be cross-linked by oligosaccharides covalently linked to human serum albumin.

Inhibition of Helicobacter pylori binding to gastrointestinal epithelial cells by sialic acid-containing oligosaccharides.

Helicobacterpylori, the ulcer pathogen residing in the human stomach, binds to epithelial cells of the gastric antrum. We have examined binding of 13 bacterial isolates to epithelial cell lines by use of a sensitive microtiter plate method in which measurement of bacterial urease activity provides the means for quantitation of bound organisms. Several established human gastrointestinal carcinoma cell lines grown as monolayers were compared for suitability in these assays, and the duodenum-derived cell line HuTu-80 was selected for testing bacterial binding inhibitors. When bacteria are pretreated with oligosaccharides, glycoproteins, and glycolipids, a complex picture of bacterial-epithelial adherence specificities emerges. Among the monovalent inhibitors tested, 3'-sialyllactose (NeuAc alpha2-3Gal beta1-4Glc; 3'SL) was the most active oligosaccharide, inhibiting adherence for recent clinical isolates of H. pylori with a millimolar 50% inhibitory concentration (IC50). Its alpha2-6 isomer (6'SL) was less active. Most of the recent clinical isolates examined were inhibited by sialyllactose, whereas long-passaged isolates were insensitive. Among the long-passaged bacterial strains whose binding was not inhibited by 3'SL was the strain ATCC 43504, also known as NCTC 11637 and CCUG 17874, in which the proposed sialyllactose adhesin was recently reported to lack surface expression (P. G. O'Toole, L. Janzon, P. Doig, J. Huang, M. Kostrzynska, and T. H. Trust, J. Bacteriol. 177:6049-6057, 1995). Pretreatment of the epithelial monolayer with neuraminidase reduced the extent of binding by those bacteria that are sensitive to inhibition by 3'SL. Other potent inhibitors of bacterial binding are the glycoproteins alpha1-acid glycoprotein, fetuin, porcine gastric and bovine submaxillary mucins, and the glycolipid sulfatide, all of which present multivalent sialylated and/or sulfated galactosyl residues under the conditions of the binding assay. Consistent with this pattern, a multivalent neoglycoconjugate containing 20 mol of 3'SL per mol of human serum albumin inhibited bacterial binding with micromolar IC50. The H. pylori isolate most sensitive to inhibition by 3'SL was least sensitive to inhibition by sulfatide, gastric mucin, and other sulfated oligosaccharides. Bacteria that have been allowed to bind epithelial cells are also effectively detached by 3'SL. These results describe a heterogeneous adherence repertoire for these bacteria, but they also confirm the critical role of the 3'SL structure on human gastric epithelial cells as an adherence ligand for recent isolates of H. pylori.