Methods: F. nucleatum biofilms were grown on Calgary Biofilm Device for 6 days and exposed to different concentrations of amoxicillin to determine the biofilm inhibitory concentration (BIC). An inoculum of planktonically grown F. nucleatum with the same number of cells as the biofilm was used to test the minimum inhibitory concentration (MIC). Conversion of chromogenic nitrocefin was used to detect β-lactamase in the supernatant of the biofilms and planktonic cells. The number of cells in the biofilm and cells grown planktonically was determined using checkerboard DNA-DNA hybridization. The amoxicillin MIC of S. pyogenes was measured in presence or absence of a 7-day old F. nucleatum biofilm.
Results: 1) F. nucleatum amoxicillin BIC was 64μg/ml, while the MIC for the same number of planktonic cells was 0.125μg/ml. 2) F. nucleatum biofilms produced β-lactamase after 5 days of growth while F. nucleatum planktonic cells did not produce any β-lactamase even after 7 days of growth and at cell counts similar to the biofilms. 3) When exposed to 7-day old β-lactamase producing F. nucleatum biofilms, the amoxicillin MIC of planktonic S. pyogenes increased from 0.125μg/ml to 16μg/ml.
Conclusions: These results suggest that autoaggregation by F. nucleatum can trigger β-lactamase production. Further, β-lactamase producing F. nucleatum biofilms can confer β-lactam resistance to β-lactam sensitive neighboring species.
Keywords: Antimicrobial agents/inhibitors, Biofilm and Microbiology