Soybean peptide inhibits the biofilm of periodontopathic bacteria via bactericidal activity.

OBJECTIVE: This study aimed to clarify the antibacterial mechanism and antibiofilm effect of soybean-derived peptide BCBS-11 against periodontopathic bacteria. DESIGN: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of BCBS-11 against Porphyromonas gingivalis (P. gingivalis), Fusobacterium nucleatum (F. nucleatum), and Streptococcus mitis (S. mitis) were determined for the antibacterial mechanism. The effect of BCBS-11 on membrane permeability and depolarization activity were investigated using propidium iodide (PI) staining and 3, 3'-dipropylthiadicarbocyanine iodide (DiSC3-(5)) analysis. Monospecies and multispecies biofilms were cultured on 96-well plates. The amount of biofilm was determined using crystal violet staining to determine the inhibition of biofilm formation and the eradication of established biofilm using BCBS-11. The cytotoxicity of BCBS-11 was evaluated using 3-(4, 5-Dimethylthiazol-2-yl)- 2, 5-diphenyltetrazolium bromide (MTT) assay. RESULTS: The MIC and MBC indicated the bactericidal activity of BCBS-11 against P. gingivalis and F. nucleatum. The PI staining revealed that BCBS-11 disrupted the bacterial membrane integrity. The DiSC3-(5) analysis indicated that BCBS-11 depolarized the bacterial cytoplasmic membrane. These results indicate the antimicrobial action of BCBS-11 through membrane disruption and the collapse of membrane electrochemical gradient. BCBS-11 significantly inhibited the monospecies biofilm formation of P. gingivalis and F. nucleatum and also inhibited dual-species biofilm. BCBS-11 was not cytotoxic toward human oral epithelial cells. CONCLUSIONS: BCBS-11 inhibits the monospecies and multispecies biofilm formation of P. gingivalis and F. nucleatum, and their bactericidal activity results from membrane disruption.

Oral Pathobiont-Induced Changes in Gut Microbiota Aggravate the Pathology of Nonalcoholic Fatty Liver Disease in Mice.

Background & Aims: Periodontitis increases the risk of nonalcoholic fatty liver disease (NAFLD); however, the underlying mechanisms are unclear. Here, we show that gut dysbiosis induced by oral administration of Porphyromonas gingivalis, a representative periodontopathic bacterium, is involved in the aggravation of NAFLD pathology. Methods: C57BL/6N mice were administered either vehicle, P. gingivalis, or Prevotella intermedia, another periodontopathic bacterium with weaker periodontal pathogenicity, followed by feeding on a choline-deficient, l-amino acid-defined, high-fat diet with 60 kcal% fat and 0.1% methionine (CDAHFD60). The gut microbial communities were analyzed by pyrosequencing the 16S ribosomal RNA genes. Metagenomic analysis was used to determine the relative abundance of the Kyoto Encyclopedia of Genes and Genomes pathways encoded in the gut microbiota. Serum metabolites were analyzed using nuclear magnetic resonance-based metabolomics coupled with multivariate statistical analyses. Hepatic gene expression profiles were analyzed via DNA microarray and quantitative polymerase chain reaction. Results: CDAHFD60 feeding induced hepatic steatosis, and in combination with bacterial administration, it further aggravated NAFLD pathology, thereby increasing fibrosis. Gene expression analysis of liver samples revealed that genes involved in NAFLD pathology were perturbed, and the two bacteria induced distinct expression profiles. This might be due to quantitative and qualitative differences in the influx of bacterial products in the gut because the serum endotoxin levels, compositions of the gut microbiota, and serum metabolite profiles induced by the ingested P. intermedia and P. gingivalis were different. Conclusions: Swallowed periodontopathic bacteria aggravate NAFLD pathology, likely due to dysregulation of gene expression by inducing gut dysbiosis and subsequent influx of gut bacteria and/or bacterial products.

Rice peptide with amino acid substitution inhibits biofilm formation by Porphyromonas gingivalis and Fusobacterium nucleatum.

OBJECTIVE: Rice peptide has antibacterial properties that have been tested in planktonic bacterial culture. However, bacteria form biofilm at disease sites and are resistant to antibacterial agents. The aim of this study was to clarify the mechanisms of action of rice peptide and its amino acid substitution against periodontopathic bacteria and their antibiofilm effects. DESIGN: Porphyromonas gingivalis and Fusobacterium nucleatum were treated with AmyI-1-18 rice peptide or its arginine-substituted analog, G12R, under anaerobic conditions. The amount of biofilm was evaluated by crystal violet staining. The integrity of the bacteria cytoplasmic membrane was studied in a propidium iodide (PI) stain assay and transmission electron microscopy (TEM). RESULTS: Both AmyI-1-18 and G12R inhibited biofilm formation of P. gingivalis and F. nucleatum; in particular, G12R inhibited F. nucleatum at lower concentrations. However, neither peptide eradicated established biofilms significantly. According to the minimum inhibitory concentration and minimum bactericidal concentration against P. gingivalis, AmyI-1-18 has bacteriostatic properties and G12R has bactericidal activity, and both peptides showed bactericidal activity against F. nucleatum. PI staining and TEM analysis indicated that membrane disruption by G12R was enhanced, which suggests that the replacement amino acid reinforced the electostatic interaction between the peptide and bacteria by increase of cationic charge and α-helix content. CONCLUSIONS: Rice peptide inhibited biofilm formation of P. gingivalis and F. nucleatum, and bactericidal activity via membrane destruction was enhanced by amino acid substitution.