Yoko Asahi

Effects of the tea catechin epigallocatechin gallate on Porphyromonas gingivalis biofilms.

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.

Antibiofilm Effects of Azithromycin and Erythromycin on Porphyromonas gingivalis

ABSTRACT 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.

Effects of N-acyl homoserine lactone analogues on Porphyromonas gingivalis biofilm formation

BACKGROUND AND OBJECTIVE The gram-negative anaerobic rod Porphyromonas gingivalis in oral biofilms is a primary etiological agent of periodontal disease. Biofilm formation of various gram-negative bacteria is regulated by a quorum-sensing circuit that relies on N-acyl homoserine lactones (HSLs). Some synthetic N-acyl HSL analogues act as quorum-sensing inhibitors and suppress biofilm formation in Pseudomonas aeruginosa. Development of chemical control agents against oral biofilms is necessary, because until now, biofilms have been removed only by mechanical debridement. The present study investigated the effect of N-acyl HSL analogues on P. gingivalis biofilm formation, with the aim of developing new drugs that inhibit oral biofilm formation. MATERIAL AND METHODS A flow-cell model was used for P. gingivalis biofilm formation. Seventeen synthetic N-acyl HSL analogues were quantitatively assessed by spectrophotometry. The effects of three antagonistic compounds against P. gingivalis biofilm formation were further examined by confocal laser scanning microscopy, and investigated for primary attachment using spectrophotometry and phase contrast microscopy. RESULTS Ten out of 17 analogues affected P. gingivalis biofilm formation. Three out of 10 analogues significantly decreased biofilm-forming cells (p < 0.05), and these biofilm structures were less well formed three-dimensionally. There were no quantitative or qualitative differences in cell attachment between the control and the three analogue-treated groups. CONCLUSION Three synthetic N-acyl HSL analogues inhibited biofilm formation in P. gingivalis. We suggest that these analogues influence the development stage of P. gingivalis biofilm formation.

Simple observation of Streptococcus mutans biofilm by scanning electron microscopy using ionic liquids.

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.

Synergistic effects of antibiotics and an N-acyl homoserine lactone analog on Porphyromonas gingivalis biofilms.

Aims:  To investigate the effects of the combined application of an N‐acyl homoserine lactone (HSL) analog and antibiotics on biofilms of Porphyromonas gingivalis, a major pathogen of periodontal disease.

Time course of gene expression during Porphyromonas gingivalis strain ATCC 33277 biofilm formation.

ABSTRACT Chronological gene expression patterns of biofilm-forming cells are important to understand bioactivity and pathogenicity of biofilms. For Porphyromonas gingivalis ATCC 33277 biofilm formation, the number of genes differentially regulated by more than 1.5-fold was highest during the growth stage (312/2,090 genes), and some pathogen-associated genes were time-dependently controlled.

Temporal dynamics of bacterial microbiota in the human oral cavity determined using an in situ model of dental biofilms

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.

The sinR Ortholog PGN_0088 Encodes a Transcriptional Regulator That Inhibits Polysaccharide Synthesis in Porphyromonas gingivalis ATCC 33277 Biofilms

Biofilm-forming cells are distinct from well characterized planktonic cells and aggregate in the extracellular matrix, the so-called extracellular polymeric substances (EPS). The sinR gene of Bacillus subtilis encodes a transcriptional regulator that is known to be involved in the biosynthesis of EPS in biofilms. Porphyromonas gingivalis inhabits the subgingival and extraradicular biofilm of humans and is one of the primary pathogens that cause progressive marginal and refractory apical periodontitis. Furthermore, P. gingivalis possesses PGN_0088, which encodes a putative ortholog of B. subtilis sinR. Here, we investigated the role of PGN_0088 (sinR) on biofilm formation. P. gingivalis strains formed biofilms on saliva-coated glass surfaces in phosphate buffered saline. Quantitative analysis indicated that the biofilm of the sinR null mutant consisted of dense exopolysaccharide. Microscopic observations showed that the increased levels of exopolysaccharide produced by the sinR mutant changed the morphology of the EPS to a mesh-liked structure. Furthermore, physical analyses suggested that the enrichment of exopolysaccharide in the EPS enhanced the resistance of the biofilm to hydrodynamic shear force. The results presented here demonstrate sinR plays important roles in the ability of P. gingivalis strain ATCC 33277 to act as a negative mediator of exopolysaccharide accumulation and is indirectly associated with the structure of the EPS and the force of its adhesion to surfaces.

Inhibition of polysaccharide synthesis by the sinR orthologue PGN_0088 is indirectly associated with the penetration of Porphyromonas gingivalis biofilms by macrolide antibiotics.

Microbes commonly adhere to surfaces, aggregate in self-produced extracellular polymeric substances (EPS) and live in biofilms. Periodontitis is a serious oral infection that is initiated by the formation of biofilms by Porphyromonas gingivalis. EPS act as a barrier that protects biofilm-forming cells against sources of stress, including those induced by host immune cells and antimicrobial agents. Therefore, drugs intended to kill such micro-organisms cannot be used for the treatment of biofilm infections. Our previous studies revealed that subminimal inhibitory concentrations (subMIC) of two macrolide antibiotics (azithromycin, AZM and erythromycin, ERY) reduced P. gingivalis biofilms. Furthermore, we demonstrated that the Bacillus subtilis sinR orthologue (PGN_0088) inhibits the synthesis of carbohydrates that are components of EPS in P. gingivalis biofilms. Here, we constructed a novel sinR mutant from P. gingivalis ATCC 33277 and reveal that the increased abundance of carbohydrate in EPS of the mutant led to a reduced infiltration rate of AZM and ERY through EPS, and consequently elevated biofilm resistance to these macrolides. Detailed elucidation of the interaction between the product of the sinR gene and EPS will assist in the development of novel approaches that target EPS to prevent and inhibit the formation of biofilms.