Inhibitory effects of epigallocatechin-3-gallate on the pathogenic properties of P. gingivalis in vitro / 口腔疾病防治

Objective @#To explore the antibacterial activity of epigallocatechin-3-gallate (EGCG) on P. gingivalis and the inhibitory effects on matrix metalloproteinases (MMPs) production induced by P. gingivalis.@*Methods@# The antimicrobial effect of EGCG against planktonic cultures and biofilms of P. gingivalis was evaluated using microplate dilution assays. The microstructural changes in biofilms were studied using scanning electron microscopy (SEM). The inhibitory effect of EGCG on arginine gingipain (Rgp) and lysine gingipain (Kgp) activity of P. gingivalis was evaluated using synthetic chromogenic peptides and fluorogenic substrates. Enzyme-linked immunosorbent assay (ELISA) and qRT-PCR analysis were used to assess MMP-1 and MMP-2 mRNA expression and secretion by human gingival fibroblasts (HGFs) stimulated with P. gingivalis in the presence or absence of EGCG, respectively. @*Results @# The MIC and MBC of EGCG against P. gingivalis were 62.5 μg/mL and 500 μg/mL, respectively. EGCG can not only inhibit the biofilm formation of P. gingivalis but also has a scavenging effect on mature biofilms and can affect their viability. Additionally, 10 μg/mL and 50 μg/mL of EGCG inhibited the proteinase activities of Rgp and Kgp, respectively (P < 0.05). Finally, the mRNA expression and secretion of MMP-1 and MMP-2 by HGFs stimulated by P. gingivalis were significantly inhibited by 50 μg/mL of EGCG (P < 0.05). @*Conclusion@#EGCG exhibits antimicrobial effects against P. gingivalis and reduces the expression of MMPs by HGFs.

Association of fimA genotype to kgp genotype in Porphyromonas gingivalis from infected root canals of primary apical periodontitis / 中华口腔医学杂志

Objective@#To investigate the distribution of fimA and kgp genotypes as well as the common genotype combination of Porphyromonas gingivalis (Pg) in infected root canals of primary apical periodontitis for virulent isolates screening in future.@*Methods@#Thirty-four samples harboring Pg were selected from infected root canals of primary apical periodontitis from patients of the Department of Endodontics, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine from June 2013 to September 2015. FimA type-specific primers were used to amplify the samples, revealing the distribution of various fimA genotypes. The genotypes of kgp were obtained by using Mse Ⅰ restriction endonuclease. The prevalence of each genotype and common genotype combinations were then calculated. Pearson's chi-squared test was performed to analyze the correlation between genotype combinations and clinical symptoms and major signs of apical periodontitis. In addition, the bioflim architectures between Pg isolates with different fimA and kgp genotype combinations were observed compared using confocal laser scanning microscope.@*Results@#Among the 34 Pg-positive samples, fimA Ⅱ was the most prevalent genotype [47% (16/34)] followed by fimA Ⅰ [26% (9/34)], while fimA Ⅴ was detected in only one sample. The prevalence of kgp Ⅰ [56% (19/34)] was slightly higher than that of kgp Ⅱ [44% (15/34)]. Both fimA Ⅱ+kgp Ⅰ and fimAⅡ+kgp Ⅱ were the most prevalent genotype combinations [24% (8/34) each]. No significant correlation was found between specific genotype combination and such major clinical manifestations as gingival swelling and sinus tract of dental origin (P>0.05). Three Pg isolates with different genotype combinations were acquired. Isolate A (fimAⅠ+kgpⅠ) formed densest biofilm, while the biofilm of isolate C (fimAⅤ+kgp Ⅰ) was much looser. The biofilm feature of isolate B (fimAⅢ+kgp Ⅱ) fell in between A and C.@*Conclusions@#Pg with fimA Ⅱ was most frequently detected in infected root canals of primary apical periodontitis. The prevalence of Pg with kgp Ⅰ was slightly higher than that with kgp Ⅱ, and fimAⅡ+kgp Ⅰ as well as fimA Ⅱ+kgp Ⅱ were the commonest genotype combinations. According to the comparison of Pg biofilms formed by clinical isolates, it might be possible that different genotype combinations may lead to distinct biofilm architectures.

Aspergillus oryzae S-03 Produces Gingipain Inhibitors as a Virulence Factor for Porphyromonas gingivalis

Oral infection with Porphyromonas (P.) gingivalis causes periodontitis that is manifested by the destruction of gingival connective tissues. Although a few types of antibiotics are effective against the infection, its use induces the appearance of drug-resistant bacteria. The present study shows that the fermented product of Aspergillus (A.) oryzae S-03, cultivated on the fat-removed soybean, inhibits the cell growth of the P. gingivalis. Likewise, the fermented product of the S-03 strain cultured for 26~42 h displays an inhibitory activity to gingipain as a virulence factor of P. gingivalis. The activity is not lost even with heat treatment at 100degrees C for 15 min. We also demonstrate that the S-03 strain exhibits high protease activity. In addition, the strain does not produce aflatoxin because of the loss of a regulatory gene, aflR, necessary for the toxin biosynthesis.

CLONING AND SEQUENCING ANALYSIS OF GINGIPAIN K OF PORPHYROMONAS GINGIVALIS / 微生物学通报

The desired DNA product of KGPcd and KGP-hag was obtained from the total DNA of Porphyromonas gingivalis by PCR with two pairs of gene specific primers. The segment of KGPcd and KGP-hag (about 1.5kb and 1.6kb) was inserted into pGEM-T easy Vector. The double-stranded DNA of the postitive clone was analyzed by restriction endonuclease mapping and DNA sequenceing. The sequences of KGPcd and KGP-hag were consistent with those of the references appeared. The proteins of KGPcd and KGP-hag will be obtained for further study.