ANTIBIOTICS SUSCEPTIBILITY OF AGGREGATIBACTER ACTINOMYCETEMCOMITANS WITHIN BIOFILMS

Aim: Characterize the morphology of the biofilm formed by clinical isolates of Aggregatibacter actinomycetemcomitans (Aa) and evaluate the susceptibility to Ciprofloxacin, Amoxicillin and Tetracycline in planktonic cultures and biofilm in vitro. Materials and methods:  The Aa clinical isolates were obtained from periodontally affected individuals (PAA003 and PAA005). To characterize the biofilm, in both early and late formation stages, 60 mm plates holding two coverslips were inoculated with cultures of the strains analyzed after 24 and 48 h incubation at 37°C on capnofilic environment. Coverslips were stained with Syto9 and propidium iodide. Samples were studied in a confocal microscope. Then, to determine the minimum bactericidal concentration (MBC) for the different antibiotics in planktonic culture, the antibiotic dilutions were inoculated (1:1) with a culture of Aa previously grown. Also, we evaluated the MBC during early biofilm formation (24 h) and late (48 h) on polystyrene plates. For both, planktonic and biofilm, the cultures were incubated for 24 h with the antibiotic and visualized by spots on BHI agar plates. Results:  The characterization of the biofilm developed by clinical isolates of Aa revealed a very different biofilm phenotype, serotype c forms a cluster-like biofilm, whereas serotype b forms a lawn-like biofilm. Also we observed a particular distribution of the biofilm structure, in which bacteria found in external areas would be dead (red or orange) and toward the center a higher percentage of them would be alive (green). Moreover, the evaluation of the Ciprofloxacin, Amoxicillin and Tetracycline action during the progress of the biofilm revealed that both clinical strains showed a significant MBC increase respect to that determined on planktonic cultures. Conclusion:  The biofilm of Aggregatibacter actinomycetemcomitans strains gives greater protection from antimicrobial agents Ciprofloxacin, Amoxicillin and Tetracycline in an in vitro model.