Aims The aim of this study was to investigate the effects of tea catechin epigallocatechin gallate (EGCg) on established biofilms and biofilm formation by Porphyromonas gingivalis, a major pathogen of periodontal disease. Methods and Results Biofilm cell survival was measured using adenosine triphosphate (ATP) bioluminescence. In the presence of EGCg, the ATP level in cells of established biofilms was significantly decreased compared to the controls (P < 0·0001). Transmission electron microscopy revealed that EGCg damaged the cell membrane and cell wall of P. gingivalis. Confocal laser‐scanning microscopy revealed that the proportion of dead cells was higher in biofilms treated with EGCg. Moreover, the effects of subminimal inhibitory concentrations (MICs) of EGCg on P. gingivalis biofilm formation were dose‐dependent (P < 0·0001). Conclusion Our results suggest that EGCg destroys established P. gingivalis biofilms and inhibits biofilm formation. Significance and Impact of the Study Development of chemical control agents against oral biofilms is necessary, because oral biofilms can be only removed using mechanical debridement. This article indicates that EGCg may represent a novel antibiofilm agent that prevents infections involving bacterial biofilms such as periodontitis.

Scanning electron microscopy (SEM) has been successfully used to image biofilms because of its high resolution and magnification. However, conventional SEM requires dehydration and metal coating of biological samples before observation, and because biofilms consist mainly of water, sample dehydration may influence the biofilm structure. When coated with an ionic liquid, which is a kind of salt that exists in the liquid state at room temperature, biological samples for SEM observation do not require dehydration or metal coating because ionic liquids do not evaporate under vacuum conditions and are electrically conductive. This study investigates the ability of ionic liquids to allow SEM observation of Streptococcus mutans biofilms compared with conventional coating methods. Two hydrophilic and two hydrophobic ionic liquids, all of which are electronic conductors, are used. Compared with samples prepared by the conventional method, the ionic-liquid-treated samples do not exhibit a fibrous extracellular matrix structure and cracking on the biofilm surface. The hydrophilic ionic liquids give clearer images of the biofilm structure than those of the hydrophobic ionic liquids. This study finds that ionic liquids are useful for allowing the observation of biofilms by SEM without preparation by dehydration and metal coating.

Numerous studies on oral biofilms have been performed in vitro, although it is difficult to mimic the oral environment. Here we used an in situ model to conduct a quantitative analysis and comprehensive identification of bacterial communities over time by performing deep sequencing of 16S rRNA genes. We show here that the number of viable bacteria in supragingival biofilms increased in two steps. Using scanning and transmission electron microscopy, as well as confocal laser scanning microscopy, we detected gram-positive cocci during the first 8 h. The biofilm was subsequently covered with a thick matrix-like structure composed of different bacterial morphotypes that diversified as the number of bacteria increased. Streptococcus accounted for >20% of the population until 16 h, and obligate anaerobes such as Fusobacterium, Prevotella and Porphyromonas predominated after 48 h, and this increase was statistically significant after 96 h (P<0.05). Together, our data demonstrate that an initial population of facultative anaerobic bacteria was replaced with a population of gram-negative anaerobic bacteria during oral biofilm formation. This study, therefore, contributes to a comprehensive understanding of the composition of the bacterial microbiota involved in the health of the human oral cavity.

Antibiotic resistance of biofilm-grown bacteria contributes to chronic infections, such as marginal and periapical periodontitis, which are strongly associated with Porphyromonas gingivalis . Concurrent azithromycin (AZM) administration and mechanical debridement improve the clinical parameters of periodontal tissue in situ . We examined the in vitro efficacy of AZM against P. gingivalis biofilms. The susceptibilities of adherent P. gingivalis strains 381, HW24D1, 6/26, and W83 to AZM, erythromycin (ERY), ampicillin (AMP), ofloxacin (OFX), and gentamicin (GEN) were investigated using a static model. The optical densities of adherent P. gingivalis cells were significantly decreased by using AZM and ERY at sub-MIC levels compared with those of the controls in all the strains tested, except for the effect of ERY on strain W83. AMP and OFX inhibited P. gingivalis adherent cells at levels over their MICs, and GEN showed no inhibition in the static model. The effects of AZM and ERY against biofilm cells were investigated using a flow cell model. The ATP levels of P. gingivalis biofilms were significantly decreased by AZM at concentrations below the sub-MICs; however, ERY was not effective for inhibition of P. gingivalis biofilm cells at their sub-MICs. Furthermore, decreased density of P. gingivalis biofilms was observed three-dimensionally with sub-MIC AZM, using confocal laser scanning microscopy. These findings suggest that AZM is effective against P. gingivalis biofilms at sub-MIC levels and could have future clinical application for oral biofilm infections, such as chronic marginal and periapical periodontitis.