Effect of extracellular DNA on the formation of Streptococcus mutans biofilm under sucrose environment

Abstract

To study the effect of extracellular DNA(eDNA) on the formation of Streptococcus mutans(Sm) biofilms during different growth periods in sucrose environment. Sm biofilms were established on smooth glass surfaces under the environment of 1% sucrose and cultured in the condition of 37 ℃, 5% O2, 85% N2 and 10% CO2. Samples were randomly divided into four groups based on fourculture time(6,12, 24 and 48 h), respectively. Each group was further divided into two subgroups: control group(without deoxyribonuclease Ⅰ[DNaseⅠ] treatment) and test group(with DNaseⅠtreatment). DNaseⅠ was added 1 h advance in the treatment group to a final concentration of 100 U/ml. Each sample was stained with mixed SYTO-9/PI fluorescent dye. Confocal laser scanning microscopy was used for biofilm observation and scanning. The total biomass, the thickness and the volume of red fluorescence of each biofilm sample were measured following three-dimensional reconstruction using the softwear of Imaris 7.2.3. Under the environment of 1% sucrose, the Sm bacterial adhesion and distribution density increased over time, the quantity of eDNA and membrane-damaged bacteria which were indicated by red fluorescence also increased within 24 h but dropped later. The biofilm biomasses of Sm biofilm in 6, 12, 24 and 48 h DNaseⅠ treatment group reduced significantly(P<0.05) compared to those in the corresponding control groups by 81.3%, 85.0%, 90.1% and 12.4%, respectively. The biofilm thicknesses in each DNase Ⅰ treatment group (except 6 h group) also reduced significantly(P<0.05) compared to those in the corresponding control group by 34.4%, 45.6% and 23.6%, respectively. The quantities of eDNA and membrane-damaged bacteria reduced in each treatment group except 48 h group compared to that in the corresponding control group. Under the environment of 1% sucrose, eDNA plays an important role in promoting the formation of Sm biofilm.