Antibiotic Resistance in a Subgingival Biofilm Model

Objectives: Several theories have been proposed to account for the increase in antibiotic resistance associated with biofilm-grown bacteria relative to the same strains grown planktonically. One of these is the age of the biofilm. In this study, we investigated the effect that biofilm age had on survival rate following antibiotic exposure.

Methods: Biofilms, initiated with subgingival plaque samples from subjects with and without chronic periodontitis, were grown anaerobically on hydroxyapatite supports in T-soy broth for up to 10 days with the medium changed at 48-h intervals. Biofilms of 2, 5, 7, 8, 9 and 10 days were exposed to therapeutic levels for each of 7 antibiotics or antibiotic combinations for a period of 48-h. The bacteria were harvested and the percent survivors were determined relative to untreated controls.

Results: The tetracyclines inhibited roughly 90% of the growth in biofilms for 2 days. From day 2 through 10, the inhibitory effect progressively decreased. At day 10, the tetracycline-treated biofilms yielded 75-85% of the CFUs recovered from untreated controls. Amoxicillin, amoxicillin/clavulanic acid, and amoxicillin/metronidazole inhibited at least 90-95% of the bacteria for up to 7 days. Between 7 and 10 days, there was a significant increase in the number of survivors recovered following exposure to the each of these 3 antibiotics. This increase occurred between 8 and 10 days of biofilm growth and resulted in the survival of 50-70% the bacteria relative to untreated controls. The untreated controls did not show a significant increase in total CFUs from 8 to 10 days.

Conclusions: As expected, biofilms became more resistant as bacterial mass increased. Bacterial mass alone, however, does not explain the significant increase noted in the number of bacteria that survived treatment with the latter 3 antibiotics between days 8 and 10.

Supported by NIDCR grant R01 DE014714