Method for testing adherence of Haemophilus influenzae to human buccal epithelial cells

A method for testing adherence of Haemophilus influenzae strains to buccal mucosal cells is described. Bacteria grown in broth for 4 h were mixed with buccal mucosal cells. After elimination of unattached bacteria by repeated cycles of centrifugation and resuspension in PBS, the number of attached bacteria was counted microscopically. Optimal results were obtained with an early log-phase bacterial culture at a concentration of 10(9) bacteria/ml mixed with 2 X 10(4) cells/ml and incubated at 37 degrees C for 60 min. This assay showed an at least ten times higher rate of adherence for Haemophilus influenzae than previous studies. Nontypeable strains attached in higher numbers than strains with the type b capsule. Adherence was related to the frequency of nontypeable strains rather than to the site of isolation or type of infection. Thus all the isolates from middle ear fluid were nontypeable, and all but one adhered. The results suggest a difference in virulence mechanisms between type b and nontypeable Haemophilus influenzae strains.