• Journal of Periodontal and Implant Science
• /
• 제48권1호
• /
• pp.12-21
• /
• 2018

Purpose: The goal of this study was to develop and validate a standardized in vitro pathogenic biofilm attached onto saliva-coated surfaces. Methods: Fusobacterium nucleatum (F. nucleatum) and Porphyromonas gingivalis (P. gingivalis) strains were grown under anaerobic conditions as single species and in dual-species cultures. Initially, the bacterial biomass was evaluated at 24 and 48 hours to determine the optimal timing for the adhesion phase onto saliva-coated polystyrene surfaces. Thereafter, biofilm development was assessed over time by crystal violet staining and scanning electron microscopy. Results: The data showed no significant difference in the overall biomass after 48 hours for P. gingivalis in single- and dual-species conditions. After adhesion, P. gingivalis in single- and dual-species biofilms accumulated a substantially higher biomass after 7 days of incubation than after 3 days, but no significant difference was found between 5 and 7 days. Although the biomass of the F. nucleatum biofilm was higher at 3 days, no difference was found at 3, 5, or 7 days of incubation. Conclusions: Polystyrene substrates from well plates work as a standard surface and provide reproducible results for in vitro biofilm models. Our biofilm model could serve as a reference point for studies investigating biofilms on different surfaces.

• Journal of Korean Neurosurgical Society
• /
• 제30권sup1호
• /
• pp.13-19
• /
• 2001

Objective : We investigated the feasibility of a double suicide gene/prodrug therapy, involving direct introduction of the herpes simplex virus Type 1 thymidine kinase(TK) gene and the Escherichia coli cytosine deaminase(CD) gene, via a recombinant adenoviral vector and ganciclovir(GCV) and/or 5-fluorocytosine(5-FC) treatment, in C6 glioma cells. Methods : Efficient gene transfer and transduction of C6 glioma cells via a recombinant adenovirus were evaluated by infecting cells with adenovirus bearing the ${\beta}$-galactosidase gene and then staining cells with 5-bromo-4-chloro-3-indolyl-13-D-galactoside. CD/TK expression in cells infected with adenovirus bearing the CD/TK gene(ad-CD/TK) was examined by immunoblotting analysis. For in vitro cytotoxicity experiments, the cells were infected with ad-CD/TK or ad-${\Delta}E1$(as a control). After addition of a variety of concentrations of GCV and 5-FU, either separately or in combination, cell viability was determined by staining the cells with crystal violet solution 6 days after infection. Result : C6 glioma cells were efficiently transduced with recombinant adenoviral vector at multiplicities of infection of 200 or more. In vitro cytotoxicity of GCV and/or 5-FC, either alone or in combination, was exclusively observed in the cells transduced with ad-CD/TK. Obvious cytotoxicity(>50% inhibition) was observed in the presence of 5-FC at concentrations greater than 30ug/ml or GCV at concentrations greater than 0.3ug/ml at a multiplicity of infection of 100. Additionally, cytotoxicity in the presence of both GCV and 5-FC was greater than that after sinlge-prodrug treatments, indicating additive effects of the prodrug treatments. Conclusion : The administration of a double-suicide gene/prodrug therapy might have great potential in the treatment of brain tumors.

• Journal of Microbiology and Biotechnology
• /
• 제28권12호
• /
• pp.2057-2063
• /
• 2018

Laccases can oxidize a variety of phenolic and non-phenolic substrates including synthetic dyes. In this research, a laccase gene Lcc9 from Laccaria bicolor was chemically synthesized and optimized to heterogeneous expression in Pichia pastoris and Arabidopsis thaliana. The properties of recombinant laccase expressed by P. pastoris were investigated. The laccase activity was optimal at 3.6 pH and $40^{\circ}C$. It exhibited $K_m$ and $V_{max}$ values of $0.565mmol\;l^{-1}$ and $1.51{\mu}mol\;l^{-1}\;min^{-1}$ for ABTS respectively. As compared with untransformed control plants, the laccase activity in crude extracts of transgenic lines exhibited a 5.4 to 12.4-fold increase. Both laccases expressed in transgenic P. pastoris or A. thaliana could decolorize crystal violet. These results indicated that L. bicolor laccase gene may be transgenically exploited in fungi or plants for dye decolorization.

• Journal of Microbiology and Biotechnology
• /
• 제29권8호
• /
• pp.1177-1183
• /
• 2019

Grapefruit seed extract (GSE) is a safe and effective preservative that is used widely in the food industry. However, there are few studies addressing the anti-biofilm effect of GSE. In this study, the anti-biofilm effect of GSE was investigated against biofilm-forming strains of Staphylococcus aureus and Escherichia coli. The GSE minimum inhibitory concentration (MIC) for S. aureus and E. coli were $25{\mu}g/ml$ and $250{\mu}g/ml$, respectively. To investigate biofilm inhibition and degradation effect, crystal violet assay and stainless steel were used. Biofilm formation rates of four strains (S. aureus 7, S. aureus 8, E. coli ATCC 25922, and E. coli O157:H4 FRIK 125) were 55.8%, 70.2%, 55.4%, and 20.6% at $1/2{\times}MIC$ of GSE, respectively. The degradation effect of GSE on biofilms attached to stainless steel coupons was observed (${\geq}1$ log CFU/coupon) after exposure to concentrations above the MIC for all strains and $1/2{\times}MIC$ for S. aureus 7. In addition, the specific mechanisms of this anti-biofilm effect were investigated by evaluating hydrophobicity, auto-aggregation, exopolysaccharide (EPS) production rate, and motility. Significant changes in EPS production rate and motility were observed in both S. aureus and E. coli in the presence of GSE, while changes in hydrophobicity were observed only in E. coli. No relationship was seen between auto-aggregation and biofilm formation. Therefore, our results suggest that GSE might be used as an anti-biofilm agent that is effective against S. aureus and E. coli.

• Restorative Dentistry and Endodontics
• /
• 제44권1호
• /
• pp.4.1-4.10
• /
• 2019

Objectives: Biofilm formation is critical to dental caries initiation and development. The aim of this study was to investigate the effects of nicotine exposure on Streptococcus mutans (S. mutans) biofilm formation concomitantly with the inhibitory effects of sodium chloride (NaCl), potassium chloride (KCl) and potassium iodide (KI) salts. This study examined bacterial growth with varying concentrations of NaCl, KCl, and KI salts and nicotine levels consistent with primary levels of nicotine exposure. Materials and Methods: A preliminary screening experiment was performed to investigate the appropriate concentrations of NaCl, KCl, and KI to use with nicotine. With the data, a S. mutans biofilm growth assay was conducted using nicotine (0-32 mg/mL) in Tryptic Soy broth supplemented with 1% sucrose with and without 0.45 M of NaCl, 0.23 M of KCl, and 0.113 M of KI. The biofilm was stained with crystal violet dye and the absorbance measured to determine biofilm formation. Results: The presence of 0.45 M of NaCl, 0.23 M of KCl, and 0.113 M of KI significantly inhibited (p < 0.05) nicotine-induced S. mutans biofilm formation by 52%, 79.7%, and 64.1%, respectively. Conclusions: The results provide additional evidence regarding the biofilm-enhancing effects of nicotine and demonstrate the inhibitory influence of these salts in reducing the nicotine-induced biofilm formation. A short-term exposure to these salts may inhibit S. mutans biofilm formation.

• International Journal of Oral Biology
• /
• 제45권4호
• /
• pp.143-151
• /
• 2020

Streptococcus mutans and Streptococcus sobrinus play important roles in dental caries. Coptis chinensis is a natural product with antimicrobial activity against enterobacteria; however, its effects on oral streptococci are still unknown. Therefore, the effects of C. chinensis on the growth and biofilm formation of the representative cariogenic bacteria S. mutans and S. sobrinus were investigated for the possible use of C. chinensis as an anticaries agent. The C. chinensis extract was diluted with sterile distilled water, and 0.1-2.5% of the extract was used in the experiment. The effects of the C. chinensis extract on the growth and glucan formation of S. mutans and S. sobrinus were measured by viable cell counting and spectrophotometry at 650 nm absorbance, respectively. Crystal violet staining was also carried out to confirm the C. chinensis extract's inhibitory effect on biofilm formation. The C. chinensis extract significantly inhibited the growth of S. mutans and S. sobrinus at concentrations of ≥ 0.3% as compared with the control group. The viable cell count of colonies decreased by 1.7-fold and 1.2-fold at 2.5% and 1.25%, respectively, compared with the control group. The biofilm formation of S. mutans and S. sobrinus was inhibited by > 20-fold at C. chinensis extract concentrations of ≥ 1.25% as compared with the control group. In summary, the C. chinensis extract inhibited the growth and biofilm and glucan formation of S. mutans and S. sobrinus. Therefore, C. chinensis might be a potential candidate for controlling dental caries.

• Journal of Microbiology and Biotechnology
• /
• 제32권2호
• /
• pp.263-267
• /
• 2022

The aim of this study was to determine whether the antibacterial activity of chitosan-modified Fe₃O₄ (CS@Fe₃O₄) nanomaterials against Acinetobacter baumannii (A. baumannii) is mediated through changes in biofilm formation and reactive oxygen species (ROS) production. For this purpose, the broth dilution method was used to examine the effect of CS@Fe₃O₄ nanoparticles on bacterial growth. The effects of CS@Fe₃O₄ nanoparticles on biofilm formation were measured using a semi-quantitative crystal violet staining assay. In addition, a bacterial ROS detection kit was used to detect the production of ROS in bacteria. The results showed that CS@Fe₃O₄ nanoparticles had a significant inhibitory effect on the colony growth and biofilm formation of drug-resistant A. baumannii (p < 0.05). The ROS stress assay revealed significantly higher ROS levels in A. baumannii subjected to CS@Fe₃O₄ nanoparticle treatment than the control group (p < 0.05). Thus, we demonstrated for the first time that CS@Fe₃O₄ nanoparticles had an inhibitory effect on A. baumannii in vitro, and that the antibacterial effect of CS@Fe₃O₄ nanoparticles on drug-resistant A. baumannii was more significant than on drug-sensitive bacteria. Our findings suggest that the antibacterial mechanism of CS@Fe₃O₄ nanoparticles is mediated through inhibition of biofilm formation in drug-resistant bacteria, as well as stimulation of A. baumannii to produce ROS. In summary, our data indicate that CS@Fe₃O₄ nanoparticles could be used to treat infections caused by drug-resistant A. baumannii.

• Journal of Microbiology and Biotechnology
• /
• 제32권4호
• /
• pp.522-530
• /
• 2022

In this study we aimed to develop novel ZnO-NP/chitosan/β-glycerophosphate (ZnO-NP/CS/β-GP) antibacterial hydrogels for biomedical applications. According to the mass fraction ratio of ZnO-NPs to chitosan, mixtures of 1, 3, and 5% ZnO-NPs/CS/β-GP were prepared. Using the test-tube inversion method, scanning electron microscopy and Fourier-transform infrared spectroscopy, the influence of ZnO-NPs on gelation time, chemical composition, and cross-sectional microstructures were evaluated. Adding ZnO-NPs significantly improved the hydrogel's antibacterial activity as determined by bacteriostatic zone and colony counting. The hydrogel's bacteriostatic mechanism was investigated using live/dead fluorescent staining and scanning electron microscopy. In addition, crystal violet staining and MTT assay demonstrated that ZnO-NPs/CS/β-GP exhibited good antibacterial activity in inhibiting the formation of biofilms and eradicating existing biofilms. CCK-8 and live/dead cell staining methods revealed that the cell viability of gingival fibroblasts (L929) cocultured with hydrogel in each group was above 90% after 24, 48, and 72 h. These results suggest that ZnO-NPs improve the temperature sensitivity and bacteriostatic performance of chitosan/β-glycerophosphate (CS/β-GP), which could be injected into the periodontal pocket in solution form and quickly transformed into hydrogel adhesion on the gingiva, allowing for a straightforward and convenient procedure. In conclusion, ZnO-NP/CS/β-GP thermosensitive hydrogels could be expected to be utilized as adjuvant drugs for clinical prevention and treatment of peri-implant inflammation.

• Restorative Dentistry and Endodontics
• /
• 제48권1호
• /
• pp.1.1-1.11
• /
• 2023

Objectives: This study evaluated the dentinal penetration depth of 2.5% sodium hypochlorite (NaOCl) in root canals with and without preparation and different irrigant activation protocols. Materials and Methods: Sixty-three bovine mandibular incisors were randomly allocated to 6 groups (n = 10): G1, preparation + conventional needle irrigation (CNI); G2, preparation + passive ultrasonic irrigation (PUI); G3, preparation + Odous Clean (OC); G4, no preparation + CNI; G5, no preparation + PUI; G6, no preparation + OC; and CG (negative control; n = 3). Samples were filled with crystal violet for 72 hours. Irrigant activation was performed. Samples were sectioned perpendicularly along the long axis, 3 mm and 7 mm from the apex. Images of the root thirds of each block were captured with a stereomicroscope and analyzed with an image analysis software. One-way analysis of variance, followed by the Tukey post hoc test, and the Student's t-test were used for data analysis, with a significance level of 5%. Results: The NaOCl penetration depth was similar when preparation was performed, regardless of the method of irrigation activation (p > 0.05). In the groups without preparation, G6 showed greater NaOCl penetration depth (p < 0.05). The groups without preparation had a greater NaOCl penetration depth than those with preparation (p = 0.0019). Conclusions: The NaOCl penetration depth was similar in groups with root canal preparation. Without root canal preparation, OC allowed deeper NaOCl penetration. The groups without preparation had greater NaOCl penetration than those undergoing root canal preparation.

• Advances in nano research
• /
• 제14권4호
• /
• pp.391-397
• /
• 2023

This manuscript describes the one-step eco-friendly green fabrication of silver nanoparticles (AgNPs) through the in-situ bio-reduction of an aqueous solution of silver nitrate using Syzygium aromaticum leaf extract. UV-vis spectroscopy shows a characteristic SPR peak around 442 nm. FTIR spectroscopy showed that the AgNPs were capped with bioactive phyto-molecules. TEM images revealed oval and spherical particles with a mean diameter of ~12.6 nm. XRD analysis revealed crystalline and face-cantered cubic AgNPs. The phytosynthesized AgNPs showed broad-spectrum anti-microbial activity against two foodborne pathogenic bacteria, Listeria monocytogenes and Staphylococcus aureus. The AgNPs showed a prominent ability to inhibit biofilms formed by L. monocytogenes and S. aureus in laboratory conditions through a crystal violet assay. The results suggest that the AgNPs could be a novel nanotool to develop effective antimicrobial and anti-biofilm agents in food preservation.