Prevotella nigrescens and antimicrobial resistance: tetQ, biofilm metabolism and diffusion

Objectives:  Dental plaque biofilms are the prime etiological agent of both dental caries and periodontal disease. Biofilms are complex three-dimensional structures containing microorganisms, a protective barrier of exopolysaccharide, and are highly resistant to antimicrobial therapy. This resistance is hypothesized to be in part due to: slow metabolism, the presence of antibiotic resistance genes, and limited diffusion of antibiotics. This study was conducted using a Prevotella nigrescens biofilm model to determine the relative contributions of these factors to biofilm tenacity.  Methods:  Strains of P. nigrescens with or without the tetQ gene were grown as biofilms for five days then treated with varying concentrations of tetracycline for 24 hours.  Bacterial viability was determined by live dead staining with BacLight Live/Dead Stain (Molecular Probes), and quantitative recovery of viable bacteria on culture media. These results were compared to Minimal Inhibitory Concentrations (MIC) determined from bacterial planktonic logarithmic and stationary cultures. Molecular diffusion into biofilms was imaged with confocal microscopy. Statistical comparisons were done using the unpaired T-test. Results:  The biofilms observed under a confocal microscope showed a complex network of bacterial chains with both live and dead bacteria suspended in a gel of exopolysaccharide. Topical application of small molecules resulted in diffusion through the entire biofilm within 5 minutes. The presence of the tetQ gene significantly increased the P. nigrescens MIC for tetracycline when bacteria were grown planktonically, but not when in a biofilm. Conclusions:  This study implies that the primary mechanism of P. nigrescens biofilm resistance to tetracycline is metabolic. The presence of tetQ had no significant impact on viability in biofilms. The biofilm was encased in exopolysaccharide, which did not significantly impede small molecule diffusion.