Effect of Antibiotics on Subgingival Plaque in a Biofilm Model

It has been hypothesized that antibiotic concentrations necessary to inhibit bacteria in a biofilm may be several orders of magnitude greater than in planktonic cultures. However, the degree of magnitude for the bacteria in subgingival plaque is unknown. Objectives: To determine the inhibitory effects of antibiotics in a subgingival biofilm model and compare these to the inhibitory effect on pure planktonic cultures. Methods: Biofilms of subgingival plaque were anaerobically grown on saliva-coated hydroxyapatite disks in supplemented T-soy broth for 10-12 days. The effect of four antibiotics, tetracycline, amoxicillin, clindamycin, and erythromycin, were independently determined on mature biofilms cultivated from 5 individuals. The antibiotics were tested at increasing 2-fold concentrations from 4 -2048 µg/ml. Individual disks were placed in the broth containing the antibiotic concentration and left for 24 and 48 h. The major colony morphotypes surviving each concentration were subcultured, purified, identified, and susceptibilities of the planktonic cultures were determined. Results: Both tetracycline and amoxicillin at 256 µg/ml reduced the total cell counts from 10¹³ to around 10⁶ CFUs. However, some bacterial species demonstrated a rebound effect and were not inhibited by concentrations as high as 2048 µg/ml. Clindamycin gave a reduction of 10³ CFUs at 32 µg/ml. Higher concentrations had no apparent effect on the survivors. Erythromycin demonstrated the least effect and gave approximately a single log reduction in bacterial mass at 8-16 µg/ml. As expected, the antibiotic concentration required to inhibit the bacteria in the biofilm was greater than in planktonic culture. Depending on the particular species, 2 to >500-fold were required for inhibition in the biofilm relative to planktonic cultures. Conclusions: Substantially higher antibiotic concentrations are required to inhibit subgingival bacteria in a biofilm. This effect is likely dependent on the cell mass, the bacterial species, and the bacteria’s location within the biofilm.