In Vitro Multi-Bacterial Species Adhesion Assay to Saliva-Coated Hydroxyapatite Discs

Objectives: To develop an in vitro assay to measure adhesion of multiple bacterial species to saliva-coated hydroxyapatite (HA) discs. Methods: Unstimulated saliva was clarified by centrifugation and filtration and stored at -80°C. Mixtures of bacteria from saliva or plaque were washed by centrifugation and the OD adjusted to 1.0 at 600 nm. A sterile, 5 mm diameter HA disc was placed into each well of a 96-well ultra low binding microtiter plate. 150 µl of saliva was added to each well together with 10⁷ cells of bacteria derived from saliva or plaque. The suspension was gently mixed for 5 min and unattached cells removed by gentle washing with 3 changes of PBS buffer. Numbers of each of 40 test species adhering to each disc was determined using checkerboard DNA-DNA hybridization. Samples of each source suspension were analyzed in adjacent lanes to determine species that were present and proportions that were removed by the HA disk. Results: There was minimal non-specific binding of bacteria to the microtiter wells. Binding assays were performed at 5, 15, 30 and 60 min, with maximal binding detected by 5 min. Optimal discrimination of binding occurred when 10⁷ total bacterial cells were inoculated per well. Selective adhesion of bacterial taxa such as Streptococcus mitis, Streptococcus oralis, Eikenella corrodens, Capnocytophaga gingivalis from saliva samples mimicked that observed in early biofilm formation in vivo. Extent of bacterial binding differed markedly among saliva samples from different subjects. Bacteria from one subject's saliva bound differently to HA discs coated with saliva from other subjects. Conclusions: This new assay permits measurement of adhesion in vitro of multiple species in purposeful mixtures or directly from plaque or saliva. It should facilitate ecological studies of mechanisms of initial bacterial attachment to tooth surfaces. Supported by NIDCR grant DE17400.