Effective microbial molecular diagnosis of periodontitis-related pathogen Porphyromonas gingivalis from salivary samples using rgpA gene

Importance of accurate molecular diagnosis and quantification of particular disease-related pathogenic microorganisms is highlighted as an introductory step to prevent and care for diseases. In this study, we designed a primer/probe set for quantitative real-time polymerase chain reaction (qRT-PCR) targeting rgpA gene, known as the specific virulence factor of periodontitis-related pathogenic bacteria ‘Porphyromonas gingivalis’, and evaluated its diagnostic efficiency by detecting and quantifying relative bacterial load of P. gingivalis within saliva samples collected from clinical subjects. As a result of qRT-PCR, we confirmed that relative bacterial load of P. gingivalis was detected and quantified within all samples of positive control and periodontitis groups. On the contrary, negative results were confirmed in both negative control and healthy groups. Additionally, as a result of comparison with next-generation sequencing (NGS)–based 16S metagenome profiling data, we confirmed relative bacterial load of P. gingivalis, which was not identified on bacterial classification table created through 16S microbiome analysis, in qRT-PCR results. It showed that an approach to quantifying specific microorganisms by applying qRT-PCR method could solve microbial misclassification issues at species level of an NGS-based 16S microbiome study. In this respect, we suggest that P. gingivalis–specific primer/probe set introduced in present study has efficient applicability in various oral healthcare industries, including periodontitis-related microbial molecular diagnosis field.

Antimicrobial activity of candidate probiotic Streptococcus salivarius against Gram-positive bacteria in oral cavity / 대한구강보건학회지

Objectives@#The aim of this study is to investigate antimicrobial activity in isolated Streptococcus salivarius against Gram-positive bacteria related oral diseases. @*Methods@#S. salivarius was used in G2, G7, K12, and ATCC 7073 strains and tryptic soy broth supplemented with glucose was cultivated. Actinomyces israelii, Actinomyces viscosus, and Enterococcus faecalis were cultivated with brain heart infusion broth. Streptococcus mutans and Streptococcus sobrinus were maintained using tryptic soy broth. The antimicrobial activity of S. salivarius was performed by minimum inhibitory concentration using the spent culture medium. @*Results@#All S. salivarius have antimicrobial activity against Gram-positive bacteria in oral cavity. When comparing antimicrobial activity, S. salivarius G2 and G7 as isolated strain showed stronger antimicrobial activity against Gram-positive microbe than type K12 strain. @*Conclusions@#S. salivarius G2 and G7 have strong antimicrobial activity and may be prevent oral disease by Gram-positive bacteria in oral cavity.

Comparison of Cariogenic Characteristics between Fluoride-sensitive and Fluoride-resistant Streptococcus mutans / 대한소아치과학회지

The aim of this study is to compare cariogenic characteristics of fluoride-sensitive Streptococcus mutans [fluoride-sensitive (FS) S. mutans ] and fluoride-resistant Streptococcus mutans [fluoride-resistant (FR) S. mutans] in the presence of sucrose, and to evaluate its effect on cariogenic biofilm formation. S. mutans ATCC 25175 was continuously cultured in trypticase soy broth (TSB) containing NaF (70 ppm) for 40 days to generate FR S. mutans . FS and FR S. mutans were inoculated in TSB with or without 2% sucrose, and optical density and pH were measured every hour. An oral biofilm was formed using saliva bacteria and analyzed through confocal laser scanning microscopy and CFU count. Finally, the expression of glucosyltransferases genes of both S. mutans was investigated through RT-PCR. FR S. mutans exhibited slower growth and lower acidogenicity in the presence of sucrose compared to FS S. mutans . Both cariogenic and single species biofilm formation was lower in the presence of FR S. mutans , along with reduced number of bacteria. FR S. mutans showed significantly low levels of gtfB, gtfC, and gtfD expression compared to FS S. mutans . On the basis of results, FR S. mutans may be less virulent in the induction of dental caries.

Antifungal effect of electrolyzed hydrogen water on Candida albicans biofilm

PURPOSE: Candida albicans can cause mucosal disease in many vulnerable patients. Also they are associated with denture-related stomatitis. Electrolyzed water is generated by electric current passed via water using various metal electrodes and has antimicrobial activity. The aim of this study was to investigate antifungal activity of electrolyzed water on C. albicans biofilm. MATERIALS AND METHODS: C. albicans was cultured by sabouraud dextrose broth and F-12 nutrient medium in aerobic and 5% CO2 condition to form blastoconidia (yeast) and hyphae type, respectively. For formation of C. albicans biofilm, C. albicans was cultivated on rough surface 6-well plate by using F-12 nutrient medium in CO2 incubator for 48 hr. After electrolyzing tap water using various metal electrodes, the blastoconidia and hyphal type of C. albicans were treated with electrolyzed water. C. albicans formed blastoconidia and hyphae type when they were cultured by sabouraud dextrose broth and F-12 nutrient medium, respectively. RESULTS: The electrolyzed water using palladium electrode (EWP) exhibited antifungal effect on blastoconidia of C. albicans. Also, the EWP significantly has antifungal activity against C. albicans biofilm and hyphae. In the electrolyzed water using various metal electrodes, only the EWP have antifungal activity. CONCLUSION: The EWP may use a gargle solution and a soaking solution for prevention of oral candidiasis and denture-related stomatitis due to antifungal activity. (

Beneficial Effects of Lactobacillus casei ATCC 334 on Halitosis Induced by Periodontopathogens

Halitosis is caused by consumption of certain foods or drinks and production of volatile sulfur compounds (VSCs) by periodontopathogens. VSCs-related halitosis is not easily removed using mechanical or chemical therapies such as dental floss, plaque control and mouth rinse. Lactobacillus are known to be probiotics and stimulate immune systems of human. Furthermore, L. casei ATCC 334 and L. rhamnosus GG have an effect on protection of dental caries in vitro studies. The aim of this study was to investigate effect of Lactobacillus on halitosis by Fusobacterium nucleatum- and Porphyromonas gingivalis- producing VSCs and to analyze inhibitory mechanism. The periodontopathogens were cultivated in the presence or the absence Lactobacillus, and the level of VSCs was measured by gas chromatograph. For analysis of inhibitory mechanisms, the susceptibility assay of the spent culture medium of Lactobacillus against F. nucleatum and P. gingivalis was investigated. Also, the spent culture medium of Lactobacillus and periodontopathogens were mixed, and the emission of VSCs from the spent culture medium was measured by gas chromatograph. L. casei and L. rhamnosus significantly reduced production of VSCs. L. casei and L. rhamnosus exhibited strong antibacterial activity against F. nucleatum and P. gingivalis. The spent culture medium of L. casei inhibited to emit gaseous hydrogen sulfide, methyl mercaptan and dimethyl sulfide from the spent culture medium of periodontopathogens. However, the spent medium of L. rhamnosus repressed only dimethyl sulfide. L. casei ATCC 334 may improve halitosis by growth inhibition of periodontopathogens and reduction of VSCs emission.

Antibacterial Activity of Hydrogen-rich Water Against Oral Bacteria

There are estimated to be about 700 species of bacteria in the oral cavity. Based on epidemiological investigations, some of these strains have been proposed as the pathogens responsible for oral diseases such as dental caries, gingivitis and periodontitis. Since electrolyzed hydrogen-rich water has been shown to have beneficial effects on human immunity, its use has increased. In our study, the antibacterial activity of hydrogen-rich water for oralagainst bacteria associated with oral disease was evaluated. The bacterial strains Streptococcus mutans, Fusobacterium nucleatum, Porphyromonas gingivalis and Tannerella forsythia were cultured in specific growth medium. S. mutans, F. nucleatum and P. gingivalis were soaked to thein both hydrogen water and tap water for 30 sec and then inoculated onto mitis-salivarius agar and brain heart infusion agar including supplemented withvitamin K and hemin, respectively. The numbers of bacterial colonies were then measured after cultivation for 48 hours. In the case of T. forsythia, which does not grow well on agar plates, inoculations into modified new oral spirochete (NOS) broth were performed and growth curve analysis was undertaken every day with a spectrophotometer. Hydrogen water showed antibacterial activity against all four bacterial strains in comparison with tap-water. We conclude from this that hydrogen water may have a positive impact on oral hygiene by helping to remove cariogenic bacteria and periodontopathogens.

Effects of Microbial Communication on The Growth of Periodontopathogens

Most oral microorganisms exist as biofilms which initiate formation via the attachment of an early colonizer to host proteins on the tooth surface. Fusobacterium nucleatum act as a bridge between early and late colonizers. Dental biofilms eventually comprise dental pathogens such as Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia. To evaluate the effects of mutual interactions between oral bacteria on the growth of biofilms, periodontopathogens were co-cultured with a 0.4 microm barrier. Streptococcus gordonii inhibited the growth of F. nucleatum and periodontopathogens. However, F. nucleatum, P. gingivalis and T. denticola activated the growth of other bacteria. A co-culture system of early and late colonizers could be a useful tool to further understand bacterial interactions during the development of dental biofilm.

Inhibitory Effect of Pentose on Biofilm Formation by Oral Bacteria

A number of bacterial species coexist in oral cavities as a biofilm rather than a planktonic arrangement. By forming an oral biofilm with quorum sensing properties, microorganisms can develop a higher pathogenic potential and stronger resistance to the host immune system and antibiotics. Hence, the inhibition of biofilm formation has become a major research issue for the future prevention and treatment of oral diseases. In this study, we investigated the effects of pentose on biofilm formation and phenotypic changes using wild type oral bacteria obtained from healthy human saliva. D-ribose and D-arabinose were found to inhibit biofilm formation, but have no effects on the growth of each oral bacterium tested. Pentoses may thus be good candidate biofilm inhibitors without growth-inhibition activity and be employed for the future prevention or treatment of oral diseases.

Detection of Prevotella intermedia and Prevotella nigrescens using Pn17 and Pn34 DNA Probes

The DNA probes Pn17 and Pn34 were evaluated for their ability to specifically detect clinical strains of P. intermedia and P. nigrescens from a Korean population by dot blot hybridization. These probes were sequenced by extension termination and their specificity was determined by Southern blot analysis. The results revealed that the Pn17 sequence (2,517 bp) partially encodes an RNA polymerase beta subunit (rpoB) and that Pn34 (1,918 bp) partially encodes both rpoB (1-169 nts) and the RNA polymerase beta subunit (rpoB'; 695-1918 nts). These probes hybridized with both HindIII- and PstI-digested genomic DNAs from the strains of P. intermedia and P. nigrescens used in this study. Interestingly, each of the hybrid bands generated from the HindIII-digested genomic DNAs of the two bacterial species could be used to distinguish between them via restriction fragment length polymorphism. These results thus indicate that Pn17 and Pn34 can simultaneously detect P. intermedia and P. nigrescens.

The effect of nutraceutical containing PRF-K2 on periodontal condition during maintenance phase / 대한치주과학회지

The long term success of periodontal treatment is dependent upon the effectiveness of the maintenance care program after active treatment. The purpose of this study was to evaluate whether nutraceutical containing PRF-K2 as natural product from plant and seaweed has beneficial effects on clinical parameters, gingival crevicular fluid (GCF) volume and GCF cytokine levels during maintenance phase after periodontal treatment. Among the generally healthy and non-smoking, moderate to severe chronic periodontitis patients during maintenance phase in Department of Periodontics, Chonnam National University Hospital, twenty eight patients took nutraceutical containing PRF-K2 (Oscotec Inc. Cheonan, Korea) for 3 months as experimental group and sixteen patients received only maintenance care as control group. Clinical examination and GCF collection were performed at baseline, 1, 2 and 3 months of experiment. Total amounts and concentrations of GCF IL-1beta, IL-1ra and PGE2 were evaluated using ELISA kit. In probing pocket depth, experimental group showed the tendency of more reduction than control group after 3 months of experiment. Sulcus bleeding index (SBI) and GCF volume were significantly decreased in experimental group(p<0.05), whereas they were increased in control group. GCF IL-1betalevel tended to decrease in both experimental and control group and IL-1ra concentration tended to increase in experimental group and to decrease in control group. IL-1ra/IL-1beta ratio tended to increase in experimental group and to decrease in control group during experimental period. GCF PGE2 amount did not show any change in experimental group and tended to increase in control group. These results suggest that nutraceutical supplement which contain PRF-K2 could improve periodontal condition during maintenance phase after periodontal therapy.

Effects of Interleukin-1beta and Tumor Necrosis Factor-alpha on the Release of Collagenase and Gelatinase from Osteoblasts

A large number of factors such as osteotropic hormones, cytokines, or growth factors are related to the bone remodeling which is characterized by the coupling of osteoclast-mediated bone resorption and osteoblast-mediated bone formation. Recent investigations have indicated that cytokines such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) play a potential role in the bone resorption associated with a variety of pathological conditions such as inflammatory osteolytic disease. Collagen is the most abundant protein of the extracellular matrix of bone, and the participation of collagenase in bone resorption has been widely investigated. In this study, effects of IL-1beta and TNF-alpha on the release of collagenase from osteoblastic cells were measured. The gelatinase activity was also measured by gel substrate analysis (zymography) after electrophoresis of conditioned media of osteoblastic cell culture. IL-1beta increased the collagenase activity in ROS17/2.8 and HOS cell culture. TNF-alpha also increased the collagenase activity of osteoblastic cells. When two kinds of cytokines were treated simultaneously in the culture of osteoblastic cells, synergistic increase of collagenase activity was seen in ROS17/2.8 cells. IL-1beta and TNF-alpha significantly increased the collagenase activity after 6 hour treatment in the osteoblastic cell culture, and there was no additional increase according to the culture period. Osteoblastic cells released the gelatinase and molecular weight of this enzyme was measured about 70 KDa as assessed by zymogram. IL-1beta and TNF-alpha showed increase of the gelatinase activity produced by ROS17/2.8 and HOS cells. Taken together, this study suggested that IL-1beta and TNF-alpha can modulate bone metabolism, at least in part, by increased release of collagenase and gelatinase from osteoblasts.