Development of a flow method for susceptibility testing of oral biofilms in vitro.

Bacteria in biofilms are known to be more resistant than bacteria in batch cultures to antimicrobial agents. The purpose of the present study was to develop a flow method for formation of oral biofilms permitting susceptibility testing of plaque bacteria. A brain heart infusion (BHI) Streptococcus sanguis 804 culture was pumped through a modified Robbins Device (MRD) with 25 exchangeable silicone disks at 40 ml/h. After 24-48 h disks were removed and biofilm cells dispersed by vortex mixing and low-output ultrasonication. Colony forming units (cfu)/cm2 were determined after aerobic incubation on blood agar plates. Optimal biofilm formation was found after growth for 48 h at 37 degrees C in BHI + 1% sucrose, using saliva-coated silicone disks in inverted MRDs, yielding on average 4.4 x 10(5) cfu/cm2. Similar results were obtained for S. sanguis ATCC 10556 and five clinical isolates. Testing the susceptibility of S. sanguis to chlorhexidine gluconate showed increased resistance of biofilms compared to batch culture. Thus an appropriate biofilm model for susceptibility testing of oral microorganisms has been established.