Whole unstimulated saliva and supragingival plaque samples were collected concurrently from five healthy adult volunteers on at least two occasions. Volunteers were asked to refrain from brushing their teeth for 24 hours before each sample collection. Saliva was collected by expectoration into tubes on ice, and 20 ml from each donor was centrifuged for 10 min at 4°C at 1000g (Beckman SX4750 rotor) to remove large debris and bacterial aggregates. The supernatants (~15 ml) were collected, and the planktonic bacteria were pelleted by centrifugation at 13,000g for 1 min at 4°C and washed once with prechilled sterile phosphate-buffered saline (PBS). Supragingival plaque was obtained by scraping from the buccal surface of first and second maxillary molars using sterile micropipette tips. The plaque samples were immediately transferred to 1.7-ml microcentrifuge tubes containing 1 ml of prechilled sterile PBS, pelleted by centrifugation at 13,000g (Eppendorf 5415D) for 1 min at 4°C, and washed once with prechilled sterile PBS. Sessile plaque and planktonic salivary bacterial community samples were stored at −80°C in 500 μl of TRIzol Reagent (Ambion) until RNA isolation. Total RNA was purified using the Direct-zol RNA MiniPrep Kit (Zymo Research) according to the manufacturer’s protocol. RT-qPCR was performed as described below, and relative expression of adhesin genes was calculated using the ΔΔCt method.

For experiments testing biofilm formation in the presence of human saliva, stimulated whole saliva was collected from two to five donors on ice and pooled. Saliva and plaque collections were performed using a protocol that was reviewed and approved by the University of Minnesota Institutional Review Board.

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