Secondary adhesion of Streptococcus mutans to disinfected biofilm structure

Objectives: Biofilm structure may remain intact after bacterial eradication using therapeutically available antimicrobials. This study aimed to examine whether remaining biofilm structure promotes bacterial secondary adhesion using an in vitro oral biofilm model. Methods: The rotating-disc biofilm reactor was used to develop standardized 3-day Streptococcus mutans ATCC 25175 biofilm on resin composite discs (Premise flowable, Kerr). The biofilm was disinfected with 70% isopropyl alcohol for 120 min, washed and returned to the reactor. The same bacterial strain in the logarithmic phase were then allowed to develop for 4 h. The discs without remaining structure served as the control. The samples were fluorescence-stained with either SYTO9/propidium iodide or calcein-AM/rhodamine B, and subjected to confocal laser scanning microscopy, either directly or after longitudinal cryosectioning, and three-dimensional reconstruction using MetaMorph software. Plate counts and PCR-Invader assay were performed to quantify viable and total bacteria, respectively. Results: Isopropyl alcohol treatment resulted in complete absence of viable bacteria. Three-dimensional reconstruction revealed that viable bacteria appear to get caught to upstream edges of disinfected biofilm clusters. The cryosectioning provided higher resolution of the deeper layer, and demonstrated stratified patterns of viable cells beside disinfected biofilm structure. SYTO9/propidium iodide and calcein-AM/rhodamine B staining revealed that 19.1 ± 11.3% and 20.1 ± 14.4%, respectively, of bacteria were viable following the secondary adhesion experiment. Mean viable count in the experimental group was significantly higher than that of the control group (7.3 vs, 6.4 log CFU/ml; P<0.05, Mann-Whitney U-test). Linear regression analysis of the PCR-Invader-derived bacterial counts and plate counts, exhibited a high correlation (r²= 0.999 for the experimental group, 0.998 for the control group). Conclusion: The remaining biofilm structure following complete disinfection favored the secondary bacterial adhesion, suggesting that degradation and/or detachment of biofilm matrix enhance the chemical control of oral biofilms.