Effect of Lonicera caerulea var. emphyllocalyx Fruit on Biofilm Formed by Porphyromonas gingivalis.

Porphyromonas gingivalis is an important pathogenic anaerobic bacterium that causes aspiration pneumonia. This bacterium frequently forms biofilms in the oral cavity and in respiratory tract-associated medical devices. Bacterial colonization that occurs in association with this biofilm formation is the main reason for incurable aspiration pneumonia. The Lonicera caerulea var. emphyllocalyx (LCE) fruit has been used in folk medicine in Hokkaido, the northern part of Japan. The aim of this study was to elucidate one of the antimicrobial mechanisms of LCE methanol extract (LCEE)-the inhibitory effect of LCEE on biofilm formation by P. gingivalis. Our results show that LCEE significantly reduced biofilm formation by three different P. gingivalis isolates in a concentration- and time-dependent manner that were quantified by the adsorption of safranin red. When LCEE was added to biofilms already formed by P. gingivalis, LCEE did not degrade the biofilm. However, treatment with LCEE significantly promoted the removal of existing biofilm by vibration compared to that of control. We also confirmed biofilm formation in LCEE-treated P. gingivalis in tracheal tubes using scanning electron microscopic (SEM) analysis. Cyanidin 3-O-glucoside (C3G), one of the components of LCE, also inhibited the formation of biofilm by P. gingivalis in a concentration-dependent manner. Our results reveal that LCEE may be an effective antibacterial substance for P. gingivalis-induced aspiration pneumonia because of its role in the suppression of bacterial biofilm formation in the oral cavity.

Repurposing Toremifene for Treatment of Oral Bacterial Infections.

The spread of antibiotic resistance and the challenges associated with antiseptics such as chlorhexidine have necessitated a search for new antibacterial agents against oral bacterial pathogens. As a result of failing traditional approaches, drug repurposing has emerged as a novel paradigm to find new antibacterial agents. In this study, we examined the effects of the FDA-approved anticancer agent toremifene against the oral bacteria Porphyromonas gingivalis and Streptococcus mutans We found that the drug was able to inhibit the growth of both pathogens, as well as prevent biofilm formation, at concentrations ranging from 12.5 to 25 µM. Moreover, toremifene was shown to eradicate preformed biofilms at concentrations ranging from 25 to 50 µM. In addition, we found that toremifene prevents P. gingivalis and S. mutans biofilm formation on titanium surfaces. A time-kill study indicated that toremifene is bactericidal against S. mutans Macromolecular synthesis assays revealed that treatment with toremifene does not cause preferential inhibition of DNA, RNA, or protein synthesis pathways, indicating membrane-damaging activity. Biophysical studies using fluorescent probes and fluorescence microscopy further confirmed the membrane-damaging mode of action. Taken together, our results suggest that the anticancer agent toremifene is a suitable candidate for further investigation for the development of new treatment strategies for oral bacterial infections.

α-Amylase is a potential growth inhibitor of Porphyromonas gingivalis, a periodontal pathogenic bacterium.

BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis is a major etiological agent in the development and progression of periodontal diseases. In this study, we isolated a cell growth inhibitor against P. gingivalis species from rice protein extract. MATERIAL AND METHODS: The cell growth inhibitor active against P. gingivalis was purified from polished rice extract using a six-step column chromatography process. Its antimicrobial properties were investigated through microscope analysis, spectrum of activity and general structure. RESULTS: The inhibitor was identified as AmyI-1, an α-amylase, and showed significant cell growth inhibitory activity against P. gingivalis species. Scanning electron microscopy micrograph analysis and bactericidal assay indicated an intriguing possibility that the inhibitor compromises the cell membrane structure of the bacterial cells and leads to cell death. Moreover, α-amylases from human saliva and porcine pancreas showed inhibitory activity similar to that of AmyI-1. CONCLUSIONS: This is the first study to report that α-amylases cause cell death of periodontal pathogenic bacteria. This finding highlights the potential importance and therapeutic potential of α-amylases in treating periodontal diseases.

Oral mucosal lipids are antibacterial against Porphyromonas gingivalis, induce ultrastructural damage, and alter bacterial lipid and protein compositions.

Oral mucosal and salivary lipids exhibit potent antimicrobial activity for a variety of Gram-positive and Gram-negative bacteria; however, little is known about their spectrum of antimicrobial activity or mechanisms of action against oral bacteria. In this study, we examine the activity of two fatty acids and three sphingoid bases against Porphyromonas gingivalis, an important colonizer of the oral cavity implicated in periodontitis. Minimal inhibitory concentrations, minimal bactericidal concentrations, and kill kinetics revealed variable, but potent, activity of oral mucosal and salivary lipids against P. gingivalis, indicating that lipid structure may be an important determinant in lipid mechanisms of activity against bacteria, although specific components of bacterial membranes are also likely important. Electron micrographs showed ultrastructural damage induced by sapienic acid and phytosphingosine and confirmed disruption of the bacterial plasma membrane. This information, coupled with the association of treatment lipids with P. gingivalis lipids revealed via thin layer chromatography, suggests that the plasma membrane is a likely target of lipid antibacterial activity. Utilizing a combination of two-dimensional in-gel electrophoresis and Western blot followed by mass spectroscopy and N-terminus degradation sequencing we also show that treatment with sapienic acid induces upregulation of a set of proteins comprising a unique P. gingivalis stress response, including proteins important in fatty acid biosynthesis, metabolism and energy production, protein processing, cell adhesion and virulence. Prophylactic or therapeutic lipid treatments may be beneficial for intervention of infection by supplementing the natural immune function of endogenous lipids on mucosal surfaces.

Further characterization of a bacteriocin produced by Lactobacillus paracasei HL32.

AIMS: Purification, identification and partial characterization of bacteriocin produced by Lactobacillus paracasei HL32. It has been shown to have activity against Porphyromonas sp. METHODS AND RESULTS: The purification of bacteriocin consisting of gel exclusion followed by anion exchange chromatography produced a single band upon an electrophoresis gel with a molecular weight corresponding to 56 kDa. The isolated protein contained 171 amino acids and the first 151 were sequenced. The bacteriocin contained a high percentage of cationic amino acids near the N-terminus, hydrophobic amino acids in the central region (Leu, Ile, Val, Phe, Trp and Gly) and hydrophilic residues (Ser, Asn and Gln) at the C-terminus. This structure did not match with that of previously reported bacteriocins. The antimicrobial activity of the bacteriocin was determined against some pathogens and normal microbiota (P. gingivalis, P. intermedia, T. forsythensis, S. salivarius and S. sanguinis) found in saliva and crevicular fluid. The bacteriocin was found to inhibit P. gingivalis at the minimum bactericidal concentration (MBC) of 0.14 mmol l(-1), but was found not to inhibit the other oral micro-organisms. The bacteriocin was found from transmission electron microscopy studies to cause pore formation in the cytoplasmic membranes of P. gingivalis at the pole and induce potassium efflux. Bacteriocin concentrations of two to four times of MBC were shown to induce haemolysis. The bacteriocin was heat-stable, surviving at 110 degrees C under pressure and possessed activity over a pH range of 6.8-8.5. Only a small reduction of activity was found to occur after incubation in biological fluids (saliva and crevicular fluid). CONCLUSIONS: A novel bacteriocin has been identified that has selective activity against Porphyromonas sp. associated with periodontal disease. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work gained the knowledge of specific antibacterial activity of bacteriocin against Porphyromonas gingivalis.

Antibacterial activity of a bacteriocin from Lactobacillus paracasei HL32 against Porphyromonas gingivalis.

Porphyromonas gingivalis infections cause problems in periodontal diseases and in certain systemic diseases. There is evidence that Lactobacillus spp. can control populations of P. gingivalis, but there are few data on the effects of purified bacteriocins from Lactobacillus paracasei HL32 on P. gingivalis. The objective of this study was to examine the antibacterial activity of a bacteriocin from L. paracasei HL32 and to relate this activity to its composition. A bacteriocin was purified from culture supernatants of Lactobacillus spp. using a dialysis technique followed by gel-permeation chromatography. Composition of the bacteriocin was characterised by ninhydrin tests, ultraviolet spectrophotometry, thin-layer chromatography, sodium-dodecyl sulphate-polyacrylamide gel electrophoresis, electrospray ionisation mass spectrometry and amino acid analysis. The amino acid sequence from the N-terminal of the bacteriocin was determined. Antibacterial activity was examined by the cylinder plate method, microtitre assay and scanning electron microscopy as compared with standard antibiotics. The bacteriocin had a molecular weight of approximately 56kDa, was comprised of 68% carbohydrate and 32% protein, and showed maximum peak absorbance at 214 and 254nm. The bacteriocin was found to be effective against P. gingivalis; it caused swelling and pore formation on the cell envelope at a minimum bactericidal concentration of 0.14mM, and caused death within 2h. Metronidazole killed P. gingivalis but did not affect the envelope, whereas tetracycline affected P. gingivalis with cell deformation. In conclusion, the bacteriocin from L. paracasei HL32 had the ability to kill P. gingivalis, suggesting that it could be a promising alternative chemotherapeutic agent for P. gingivalis infections.