Avaliação da susceptibilidade de aggregatibacter actinomycemcomitans a compostos de inclusão com amoxicilina e doxiciclina em um modelo de biofilme dentário in vitro

Abstract

The Aggregatibacter actinomycemcomitans (Aa) is one of the main bacteria associated with periodontitis and their ability to form biofilms is a virulence factor that contributed to the aggravation of the disease. This study aimed to prepare inclusion compounds with amoxicillin (AMOX) and doxycycline (DOX) with hydroxypropyl--cyclodextrin (HpCd) in 1:1 molar ratio and characterize them physico-chemically in liquid and solid state by Fourier transformed infrared spectroscopy (FTIR), and nuclear magnetic resonance (NMR), respectively. The antimicrobial activity of the inclusion compounds was evaluated in planktonic form and in a biofilm model of Aa (ATCC 29522) in vitro. The minimum inhibitory biofilm concentration (MBIC) was determined by crystal violet assay after exposure to 4, 16 and 24h to the antimicrobial agents. Antimicrobial agents were considered effective when values of biofilm biomass were found to be equal or lower than 50% of the positive control (MBIC50). Also, Scanning Electron Microscopy (SEM) was used to verify biofilm bacterial growth and to assess cellular membrane integrity and morphologic changes caused by the drug treatments. Our data have shown that amoxicillin/hydroxypropyl-- cyclodextrin (AMOX/Hp!Cd, 1:1 molar ratio) and doxycycline/hydroxypropyl-- cyclodextrin (DOX/HpC, 1:1 molar ratio) were more effective in reducing biofilm biomass after 24 h of biofilm exposure when compared with the antibiotics alone in all concentrations tested. AMOX/HpCd achieved 42.2% of biofilm reduction at 2g/mL (p<0.05) while DOX/Hpd was extremely effective achieving 62.1% of biofilm reduction at 1g/mL (p<0.05) after 24 h. SEM images demonstrated that cell membrane damage is one of the important mechanism that contributes to the antimicrobial effects of AMOX/Hp!Cd and DOX/Hp!Cd compounds. It can be concluded that there is a greater susceptibility of Aa biofilms to the inclusion compounds of amoxycillin and doxycycline when complexed in Hp!Cd, inhibiting quantitatively the biofilm biomass in vitro.