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Extracellular vesicles (EVs) have been recognized as a universal method of cellular communications and are reportedly produced in bacteria, archaea, and eukaryotes. Bacterial EVs are often called “Outer Membrane Vesicles” (OMVs) as they were the result of a controlled blebbing of the outer membrane of gram-negative bacteria such as Porphyromonas gingivalis (P. gingivalis). Bacterial EVs are natural messengers, implicated in intra- and inter-species cell-to-cell communication among microorganism populations present in microbiota. Bacteria can incorporate their pathogens into OMVs; the content of OMVs differs, depending on the type of bacteria. The production of distinct types of OMVs can be mediated by different factors and routes. A recent study highlighted OMVs ability to carry crucial molecules implicated in immune modulation, and, nowadays, they are considered as a way to communicate and transfer messages from the bacteria to the host and vice versa. This review article focuses on the current understanding of OMVs produced from major oral bacteria, P. gingivalis: generation, characteristics, and contents as well as the involvement in signal transduction of host cells and systemic diseases. Our recent study regarding the action of P. gingivalis OMVs in the living body is also summarized.

In recent years, the exposure dose of the operator's eye lens during interventional radiology operations has become a problem. We therefore evaluated the feasibility of real-time lens dose measurement using scintillator with optical fiber (SOF) dosimeter. Given that the SOF dosimeter is calibrated for direct X-rays, we performed a calibration for scattered X-rays to investigate energy dependence and the accuracy of lens dose measurements. The detection limit was calculated using the Kaiser method. The SOF dosimeter and the radiophotoluminescence glass (RPLG) dosimeter were attached to the protective glasses worn by the operator, and the lens exposure dose of the operator during cardiac catheterization was measured. In the phantom experiment, the SOF dosimeter had an error rate of 5.45% based on the measured value of the ionization chamber dosimeter. The sensitivity characteristics of the SOF dosimeter were slightly reduced on the higher side of the effective energy. The difference in sensitivity was related to variations in the additional filter and energy dependency. The sensitivity difference was 18.5% at maximum. Furthermore, when the additional dose was displayed, the influence of noise on long-term measurement was considerable. Using the Kaiser method to obtain the detection limit, the accuracy of the integrated dose had SOF dosimeter error rates of 4.3% to 15.5% with respect to the integrated value of the RPLG dosimeter when calibrated by the ionization chamber dosimeter. The use of the SOF dosimeter allowed for the real-time visualization of the exposure status of the eye lens and measurements with a relatively high accuracy.

Objectives: The effects of 2-methacryloyloxyethyl phosphorylcholine (MPC)-polymer on the adherence of microorganisms such as non-Candida albicans Candida (NCAC) and methicillin-resistant Staphylococcus aureus (MRSA), frequently detected in oral infections in immunocompromised and/or elderly people, to denture resin material, are still unclear. Here, we report the effects of MPC-polymer on the adherence of C. albicans, NCAC, and MRSA to acrylic denture resin. Methods: Sixteen strains of C. albicans, seven strains of C. glabrata, two strains of C. tropicalis, one strain of C. parapsilosis, and six strains of MRSA were used. We cultured the fungal/bacterial strains and examined the cell growth and adherence of fungi/bacteria to mucin-coated acrylic denture resin plates (ADRP) with or without MPC-polymer coating, by scanning electron microscopy. The cell surface hydrophobicity of the fungal/bacterial strains was measured by the adsorption to hydrocarbons. Results: MPC-polymer did not affect the growth of all strains of Candida species and MRSA, but significantly suppressed adherence to ADRP in most strains of C. albicans and all strains of NCAC and MRSA. A significant positive correlation was found between cell hydrophobicity and the reduction rates of microbial adherence to ADRP treated with 5% of MPC-polymer. Conclusions: MPC-polymer treatment for acrylic resin material suppresses the adherence of C. albicans, NCAC and MRSA via their hydrophilicity interaction. Clinical significance: The application of MPC-polymer for denture hygiene is potent to prevent oral candidiasis, denture stomatitis and opportunistic infection, caused by Candida species and MRSA, via suppressing the adherence of those fungus/bacteria.

The oral cavity is suggested as the reservoir of bacterial infection, and the oral and pharyngeal biofilms formed by oral bacterial flora, which is comprised of over 700 microbial species, have been found to be associated with systemic conditions. Almost all oral microorganisms are non-pathogenic opportunistic commensals to maintain oral health condition and defend against pathogenic microorganisms. However, oral Streptococci, the first microorganisms to colonize oral surfaces and the dominant microorganisms in the human mouth, has recently gained attention as the pathogens of various systemic diseases, such as infective endocarditis, purulent infections, brain hemorrhage, intestinal inflammation, and autoimmune diseases, as well as bacteremia. As pathogenic factors from oral Streptococci, extracellular polymeric substances, toxins, proteins and nucleic acids as well as vesicles, which secrete these components outside of bacterial cells in biofilm, have been reported. Therefore, it is necessary to consider that the relevance of these pathogenic factors to systemic diseases and also vaccine candidates to protect infectious diseases caused by Streptococci. This review article focuses on the mechanistic links among pathogenic factors from oral Streptococci, inflammation, and systemic diseases to provide the current understanding of oral biofilm infections based on biofilm and widespread systemic diseases.

In this study, we have investigated the effects of the newly synthesized analog of Pseudomonas aeruginosa quorum-sensing autoinducer named AIA-1 (autoinducer analog) against antibiotic-resistant bacteria. In vitro susceptibility and killing assays for P. aeruginosa PAO1ΔoprD mutant and clinical isolates were performed by using antibiotics and AIA-1. In an in vivo assay, a luminescent carbapenem-resistant strain derived from PAO1ΔoprD was injected into neutropenic ICR mice and bioluminescence images were acquired after the treatment with antibiotics and AIA-1. Additionally, we investigated the effects of the combination use against carbapenem-resistant Enterobacteriaceae (CRE). Using killing assays in P. aeruginosa, the survival rates in the presence of antibiotics and AIA-1 significantly decreased in comparison with those with antibiotics alone. Furthermore, dual treatment of biapenem and AIA-1 was more effective than biapenem alone in a mouse infection model. AIA-1 did not change the MICs in P. aeruginosa, suggesting that AIA-1 acts on the mechanism of antibiotic tolerance. Conversely, the MICs of antibiotics decreased in the presence of AIA-1 in some CRE strains, indicating that AIA-1 may require additional mechanism to act on CRE. In conclusion, AIA-1 may be a potent drug for clinical treatment of infections caused by antibiotic-resistant bacteria. J. Med. Invest. 64: 101-109, February, 2017.

Dental biofilms that form in the oral cavity play a critical role in the pathogenesis of several infectious oral diseases, including dental caries, periodontal disease, and oral candidiasis. Houttuynia cordata (HC, Saururaceae) is a widely used traditional medicine, for both internal and external application. A decoction of dried HC leaves (dHC) has long been consumed as a health-promoting herbal tea in Japan. We have recently reported that a water solution of HC poultice ethanol extract (wHCP) exerts antimicrobial and antibiofilm effects against several important oral pathogens. It also exhibits anti-inflammatory effects on human keratinocytes. In our current study, we examined the effects of dHC on infectious oral pathogens and inflammation. Our results demonstrated that dHC exerts moderate antimicrobial effects against methicillin-resistant Staphylococcus aureus (MRSA) and other oral microorganisms. dHC also exhibited antibiofilm effects against MRSA, Fusobacterium nucleatum (involved in dental plaque formation), and Candida albicans and inhibitory effects on interleukin-8, CCL20, IP-10, and GROα productions by human oral keratinocytes stimulated by Porphyromonas gingivalis lipopolysaccharide (a cause of periodontal disease), without cytotoxic effects. This suggests that dHC exhibits multiple activities in microorganisms and host cells. dHC can be easily prepared and may be effective in preventing infectious oral diseases.

Objectives The biocompatible 2-methacryloyloxyethyl phosphorylcholine (MPC)-polymers, which mimic a biomembrane, reduce protein adsorption and bacterial adhesion and inhibit cell attachment. The aim of this study is to clarify whether MPC-polymer can suppress the bacterial adherence in oral cavity by a crossover design. We also investigated the number of Fusobacterium nucleatum, which is the key bacterium forming dental plaque, in clinical samples. Materials and methods This study was a randomized, placebo-controlled, single-blind, crossover study, with two treatment periods separated by a 2-week washout period. We conducted clinical trial with 20 healthy subjects to evaluate the effect of 5% MPC-polymer mouthwash after 5 h on oral microflora. PBS was used as a control. The bacterial number in the gargling sample before and after intervention was counted by an electronic bacterial counter and a culture method. DNA amounts of total bacteria and F. nucleatum were examined by q-PCR. Results The numbers of total bacteria and oral streptcocci after 5 h of 5% MPC-polymer treatment significantly decreased, compared to the control group. Moreover, the DNA amounts of total bacteria and F. nucleatum significantly decreased by 5% MPC-polymer mouthwash. Conclusions We suggest that MPC-polymer coating in the oral cavity may suppress the oral bacterial adherence. Clinical relevance MPC-polymer can be a potent compound for the control of oral microflora to prevent oral infection.

Bacteria attached to a surface are generally more tolerant to antibiotics than their planktonic counterparts, even without the formation of a biofilm. The mechanism of antibiotic tolerance in biofilm communities is multifactorial, and the genetic background underlying this antibiotic tolerance has not yet been fully elucidated. Using transposon mutagenesis, we isolated a mutant with reduced tolerance to biapenem (relative to that of the wild type) from adherent cells. Sequencing analysis revealed a mutation in the pslL gene, which is part of the polysaccharide biosynthesis operon. The Pseudomonas aeruginosa PAO1Δ pslBCD mutant demonstrated a 100-fold-lower survival rate during the exposure of planktonic and biofilm cells to biapenem; a similar phenotype was observed in a mouse infection model and in clinical strains. Transcriptional analysis of adherent cells revealed increased expression of both pslA and pelA , which are directly regulated by bis-(3′,5′)-cyclic dimeric GMP (c-di-GMP). Inactivation of wspF resulted in significantly increased tolerance to biapenem due to increased production of c-di-GMP. The loss of pslBCD in the Δ wspF mutant background abolished the biapenem-tolerant phenotype of the Δ wspF mutant, underscoring the importance of psl in biapenem tolerance. Overexpression of PA2133, which can catalyze the degradation of c-di-GMP, led to a significant reduction in biapenem tolerance in adherent cells, indicating that c-di-GMP is essential in mediating the tolerance effect. The effect of pslBCD on antibiotic tolerance was evident, with 50- and 200-fold-lower survival in the presence of ofloxacin and tobramycin, respectively. We speculate that the psl genes, which are activated by surface adherence through elevated intracellular c-di-GMP levels, confer tolerance to antimicrobials.

Objectives Antibiotic tolerance causes chronic, refractory and persistent infections. In order to advance the development of a new type of drug for the treatment of infectious diseases, we herein investigated the effects of a newly synthesized analogue of the Pseudomonas aeruginosa quorum-sensing autoinducer named AIA-1 ( a uto i nducer a nalogue) on antibiotic tolerance in P. aeruginosa . Methods A P. aeruginosa luminescent strain derived from PAO1 was injected into neutropenic ICR mice and bioluminescence images were acquired for a period of time after treatments with antibiotics and AIA-1. In vitro susceptibility testing and killing assays for the planktonic and biofilm cells of PAO1 were performed using antibiotics and AIA-1. The expression of quorum-sensing-related genes was examined using real-time PCR. Results In vivo and in vitro assays showed that AIA-1 alone did not exert any bactericidal effects and also did not affect the MICs of antibiotics. However, the combined use of AIA-1 and antibiotics exerted markedly stronger therapeutic effects against experimental infection than antibiotics alone. The presence of AIA-1 also enhanced the killing effects of antibiotics in planktonic and biofilm cells. Although AIA-1 did not inhibit the expression of lasB and rhlA genes, which are directly regulated by quorum sensing, it clearly suppressed expression of the rpoS gene. Conclusions The new compound, AIA-1, did not alter the antibiotic susceptibility of P. aeruginosa by itself; however, its addition enhanced the antibacterial activity of antibiotics. AIA-1 did not inhibit quorum sensing, but reduced the antibiotic tolerance of P. aeruginosa by suppressing rpoS gene expression.

Pseudomonas aeruginosa is a Gram-negative bacterium and causes respiratory infection especially in elderly patients. Royal jelly has been used worldwide as a traditional remedy and as a nutrient; however, the effect against P. aeruginosa is unclear. The aim of this study was to analyze antibacterial, antiadherent, and anti-inflammatory effects of royal jelly against P. aeruginosa. Wild-type strain PAO1 and clinical isolates of P. aeruginosa were used for antibacterial assay and antiadherent assay to abiotic surface and epithelial cells, which are pharynx (Detroit 562) and lung (NCI-H292) epithelial cells. In anti-inflammatory assay, epithelial cells were pretreated with royal jelly before bacterial exposure to investigate its inhibitory effect on interleukin (IL-8) and macrophage inflammatory protein-3α/CCL20 overproduction. Although royal jelly did not have antibacterial activity at concentration of 50% w/v, antiadherent activity was confirmed on the abiotic surface and epithelial cells under concentration of 25%. Pretreatment with royal jelly significantly inhibited overproduction of IL-8 and CCL20 from both cells. These results demonstrated that royal jelly inhibits P. aeruginosa adherence and protects epithelial cells from excessive inflammatory responses against P. aeruginosa infection. Our findings suggested that royal jelly may be a useful supplement as complementary and alternative medicine for preventing respiratory infection caused by P. aeruginosa.

Pseudomonas aeruginosais a Gram-negative bacterium and causes respiratory infection especially in elderly patients. Royal jelly has been used worldwide as a traditional remedy and as a nutrient; however, the effect againstP. aeruginosais unclear. The aim of this study was to analyze antibacterial, antiadherent, and anti-inflammatory effects of royal jelly againstP. aeruginosa. Wild-type strain PAO1 and clinical isolates ofP. aeruginosawere used for antibacterial assay and antiadherent assay to abiotic surface and epithelial cells, which are pharynx (Detroit 562) and lung (NCI-H292) epithelial cells. In anti-inflammatory assay, epithelial cells were pretreated with royal jelly before bacterial exposure to investigate its inhibitory effect on interleukin (IL-8) and macrophage inflammatory protein-3α/CCL20 overproduction. Although royal jelly did not have antibacterial activity at concentration of 50% w/v, antiadherent activity was confirmed on the abiotic surface and epithelial cells under concentration of 25%. Pretreatment with royal jelly significantly inhibited overproduction of IL-8 and CCL20 from both cells. These results demonstrated that royal jelly inhibitsP. aeruginosaadherence and protects epithelial cells from excessive inflammatory responses againstP. aeruginosainfection. Our findings suggested that royal jelly may be a useful supplement as complementary and alternative medicine for preventing respiratory infection caused byP. aeruginosa.

Aims: The major objective of the study was to evaluate the enhanced germicidal effects of low-frequency pulsed ultraviolet A (UVA)-light-emitting diode (LED) on biofilms. Methods and Results: The germicidal effects of UVA-LED irradiation (365 nm, 0·28 mW cm−2, in pulsed or continuous mode) on Candida albicans or Escherichia coli biofilms were evaluated by determining colony-forming units. The morphological change of microbial cells in biofilms was observed using scanning electron microscopy. After 5-min irradiation, over 90% of viable micro-organisms in biofilms had been killed, and pulsed irradiation (1–1000 Hz) had significantly greater germicidal ability than continuous irradiation. Pulsed irradiation (100 Hz, 60 min) almost completely killed micro-organisms in biofilm (>99·9%), and 20-min irradiation greatly damaged both microbial species. Interestingly, few hyphae were found in irradiated Candida biofilms. Moreover, mannitol treatment, a scavenger of hydroxyl radicals (OH•), significantly protected viable micro-organisms in biofilms from UVA-LED irradiation. Conclusions: The study demonstrated that pulsed UVA-LED irradiation has a strong germicidal effect (maximum at 100 Hz, over 5-min irradiation) and causes the disappearance of hyphal forms of Candida. Significance and Impact of the Study: This study can assist in developing a low-frequency pulsed UVA-LED system to be applied to pathogenic biofilms for disinfection.

Cyclosporine A (CsA) and tacrolimus (FK506), a member of calcineurin inhibitors, inhibit inflammation process as part of immune response. Nuclear activated T cells subfamily NFAT1 is a trascription factor responsible for the regulation of immune response genes. Streptococcus intermedius, an oral commensal bacterium, has been shown to strongly associate with liver abscess. The S. intermedius strains produce intermedilysin (ILY), which is responsible for the bacterial virulence. Cyclosporine A and FK506 have been widely used to control NFAT activation in most of cell types, however the ability of CsA and FK506 to inhibit ILY-induced NFAT1 activation remains to be investigated. The aim of this study was to investigate the effect of CsA and FK506 on NFAT1 activation caused by ILY. Human cholangiocellular cell line HuCCT1 was stimulated with various concentrations of ILY. The cell and nuclear morphological change was observed by microscopy analysis. The NFAT1 nuclear translocation that indicates its activation was detected by immunocytochemistry. The inhibitory effect of CsA and FK506 was tested after 30 min application before ILY treatment by using immunofluorescence microscope. The results showed cell and nuclear shrinkage in ILY-treated cells. The NFAT1 was translocated to the nuclei in HuCCT1 cells, and observed in dose dependent manner. Cyclosporine A and FK506 inhibited ILY-induced NFAT1 nuclear translocation. In conclusion, CsA and FK506 may act as potent inflammation control agents in S. intermedius ILY-infected cells.Keywords: Cyclosporine A, FK506, NFAT1, intermedilysin

Diagnosis by CTHA (CT during hepatic arteriography) and CTAP (CT during arterio-portography) is indispensable in the treatment of hepatocellular carcinoma (HCC). An IVR-CT system makes it possible to perform accurate diagnosis and treatment of HCC in a short period of time. In recent years, attention has been drawn to cone-beam CT (CBCT) using a flat panel detector (FPD) angio-system, and the application of CBCT for CTAP and CTHA has been reported. However, it is well known that CBCT easily generates artifacts on the images, so it is necessary to use CBCT according to the intended purpose. The purpose of this study was to evaluate 3-D images reconstructed by MDCT or CBCT, image noise, and low-contrast resolution in a phantom with a tumor mimic model. From our results, CBCT images showed distortion and blurring, and it was difficult to visualize a tumor model of 7 mm or less. In addition CBCT can create only a small area of 3-D vascular mapping. In conclusion, it is considered that CBCT cannot be used in place of conventional CTHA or CTA for the treatment of HCC.

Escherichia coli failed to survive after transformation with a Streptococcus intermedius histone-like protein gene (Si-hlp) and its promoter-harbored plasmid. The promoter function of Si-hlp in E. coli was determined using enhanced green fluorescence protein (egfp) gene as a reporter. The inhibitory effect of Si-HLP on E. coli viability was verified by a tetracycline-inducible gene expression system. Further study suggested that Si-HLP may alter the bacterial nucleoid structure, leading to the growth inhibition of E. coli.

International audience; Phase changes in Bacteroides fragilis, a member of the human colonic microbiota, mediate variations in a vast array of cell surface molecules, such as capsular polysaccharides and outer membrane proteins through DNA inversion. The results of the present study show that outer membrane vesicle (OMV) formation in this anaerobe is also controlled by DNA inversions at two distantly localized promoters, IVp-I and IVp-II that are associated with extracellular polysaccharide biosynthesis and the expression of outer membrane proteins. These promoter inversions are mediated by a single tyrosine recombinase encoded by BF2766 (orthologous to tsr19 in strain NCTC9343) in B. fragilis YCH46, which is located near IVp-I. A series of BF2766 mutants were constructed in which the two promoters were locked in different configurations (IVp-I/IVp-II = ON/ON, OFF/OFF, ON/OFF or OFF/ON). ON/ON B. fragilis mutants exhibited hypervesiculating, whereas the other mutants formed only a trace amount of OMVs. The hypervesiculating ON/ON mutants showed higher resistance to treatment with bile, LL-37, and human beta-defensin 2. Incubation of wild-type cells with 5% bile increased the population of cells with the ON/ON genotype. These results indicate that B. fragilis regulates the formation of OMVs through DNA inversions at two distantly related promoter regions in response to membrane stress, although the mechanism underlying the interplay between the two regions controlled by the invertible promoters remains unknown.

遠隔地間会議システム等の応用を想定して, 異なる可視化要件を持つ各種の表示オブジェクトを分散処理によりオブジェクト毎に独立に描画， これを重畳・立体視表示する手法を提案し， 実験システムにより機能・性能面で検証を行なった結果を報告する． 本手法では各オブジェクトが独立な可視化速度を持つ事が可能で， 遠隔地間会議システムにおいて高い可視化速度が要求される人物像と, 高速な可視化を要求しない他のオブジェクトを同時に表示する場合に有効である． 提案手法を実装した実験システムにより, 異なる可視化要件を持つ表示オブジェクトの仮想会議室空間への配置と立体視表示， 複数オブジェクトの同時表示機能を確認した. 性能面では， 複数オブジェクト同時表示時に相互の可視化速度へ与える影響が少ない事を確認し, 人物像の高速な可視化には, 描画処理と重畳処理間のデータ転送の高速化が必要である事を確認した.

Purpose: The purpose of this study was to investigate denture plaque microflora of geriatric inpatients and maxillary defect patients from the standpoint of infection control in the elderly.Methods: Denture plaque was collected from 80 elderly subjects who were categorized into 3 groups: group A, 21 healthy complete denture wearers; group B, 49 complete denture wearers residing at a geriatric hospital; and group C, 10 complete denture wearers with maxillary defects. The microflora was investigated by counting the total number of microorganisms, including streptococci, staphylococci, and Candida spp.Results: The colony-forming unit (CFUs) count and incidence, particularly those of Candida spp., were significantly higher in group B (P

The present study compares the retention of four species that are often isolated in association with biomedical device-related infections - Staphylococcus aureus, Streptococcus mutans, Pseudomonas aeruginosa, and Candida albicans - to three different surfaces. All four bacterial species were found to bind significantly less well to MPC-coated surfaces than to non-coated surfaces. We attribute this effect to the "superhydrophilicity" of MPC-coated surfaces, whereas hydrophobic surfaces are well known to reduce bacterial retention and thus to inhibit a crucial step in the formation of bacterial biofilms that lead to biomedical device-related infections and complications.

A biapenem-tolerant mutant of Pseudomonas aeruginosa was isolated by Tn 1737 KH insertion. The survival of the mutant 3 h after the addition of biapenem was about 1,000 times greater than that of the wild type. The mutant was also tolerant to other biapenems, such as imipenem, panipenem, and meropenem.

目的: 高齢者の全身感染症の予防, 特に誤嚥性肺炎の予防が重要な課題の1つとなっており, われわれは, 義歯が微生物のリザーバーとなって誤嚥性肺炎などの感染症を引き起こしている危険性を指摘している. また, これらの感染症は, 薬剤耐性菌の出現により治療が困難となっている. そこで, デンチャープラークと咽頭より分離した微生物叢の関連性とカンジダの薬剤感受性について検討した.方法: 老人病院入院患者と大学病院外来患者の義歯装着者を対象に, 上顎義歯床口蓋部粘膜面および咽頭粘膜面における口腔レンサ球菌, ブドウ球菌, カンジダ, 緑膿菌, 大腸菌・大腸菌群, MRSAの検出率を評価し, さらにカンジダに関しては, 薬剤感受性試験を行った.結果: デンチャープラークから微生物が検出されると, 咽頭からも同種の微生物が検出される傾向にあった. 多くの微生物は, 大学病院外来患者より老人病院入院患者のほうが有意に高い検出率を示した. カンジダが検出されると, 同部位からブドウ球菌も検出されるケースが多かった. アムホテリシンBに対しては, 菌種に関係なく, 高感受性の傾向を示した. フルコナゾールに対しては, Gandida albicansが高感受性の傾向を示したが, Cglabrata, C. tropicalis, C. kruseiが低感受性の傾向を示し, 耐性株もあった.結論: デンチャープラークが, 咽頭の微生物叢に与える影響は大きいと考えられる.

To clarify the availability of the dielectrophoretic impedance measurement (DEPIM) system as the evaluator for oral care, we evaluated the usefulness of DEPIM system by comparison with the standard plate counting (SPC) method. First, the relationship between the DEPIM results and bacterial concentration measured by SPC was clarified. Next, the measurement of the microorganism number in a mixed suspension was evaluated with DEPIM and SPC. The bacterial counts with DEPIM strongly correlated with those with SPC (r(2)=0.633-0.997) and this correlation was also shown in the measurement of a mixed bacterial suspension (ranging from 10(5) to 10(8) cfu/ml) of two bacterial species. Moreover, the experiments using dissociating enzymes to eliminate the influence of the size of the bacterial aggregates demonstrated that the microbial measurement results with DEPIM are unaffected by bacterial aggregates. This study demonstrated that bacterial counts with DEPIM strongly correlated with those with SPC and were unaffected by bacterial aggregates.

Dentures may act as a reservoir for bacteria which cause opportunistic infection such as aspiration pneumonia, and denture cleaning could prevent such an infection. Recently, the bacteriocidal effect of electrolyzed water (EW) on bacteria has been reported.In this study, the influence of EWs on the Candida albicans biofilm in vitro was investigated. C. albicans adhered avidly to type IV collagen. The biofilm was established on the cover slip coated with type IV collagen using Luria broth at 37°C for 5 days. The biofilms were treated with several kinds of EWs, and the efficacy of EWs was investigated.C. albicans well adhered to collagen-coated celldesk, produced glycocalyx-like substance and formed a biofilm after five-day incubation. Scanning electron microscopy revealed degradation of biofilm treated with EW. Vital CFU in the biofilm decreased time-dependently and logarithmically. Five treatments for one minute was more effective than one treatment for five minutes. The effect of EW synthesized by a home care device was equal to or higher than that synthesized by a medical device. These results suggested that EW, even if synthesized by the home care device, is a potent denture-cleanser.

Many bacteria cover denture surfaces as denture plaque in the form of a biofilm, especially dentures of geriatric hospital inpatients. Dentures may act as a reservoir of microorganisms which cause opportunistic infection such as aspiration pneumonia. Oral health care, including denture plaque control could prevent such infection. Overviews of Candida spp. and Helicobacter pylori in denture plaque and the approach to denture plaque control for the prevention of systemic infection are presented in this report.

We have previously shown the immunological mimicry of human sialyl-Lewis x (CD15s) by a surface antigen of Streptococcus pyogenes . This mimicking surface antigen may act as a ligand to the selectin family and may induce antibody production against CD15s on host cells, suggesting a possible role in the pathogenesis of S. pyogenes . In this study, the effects of antibiotics on the CD15s-related antigen expression of S. pyogenes were examined at a concentration below the MIC (sub-MIC). The amounts of CD15s on the surfaces of S. pyogenes cells and on the surfaces of S. pyogenes biofilms were determined by a whole-cell enzyme-linked immunosorbent assay and by laser scanning fluorescence microscopy, respectively, by using an anti-CD15s monoclonal antibody. At the sub-MICs, fosfomycin (1 R ,2 S -1,2-epoxypropyl phosphonic acid), its enantiomer (1 S ,2 R -1,2-epoxypropyl phosphonic acid), and benzylpenicillin significantly inhibited the CD15s expression of all strains studied. The effects of fosfomycin and its enantiomer on biofilms were also observed by scanning electron microscopy. Incubation of S. pyogenes with the sub-MIC of fosfomycin or its enantiomer, which has no antibacterial activity, reduced the amount of CD15s on the biofilm surface and made it smooth. These results suggest that fosfomycin or its enantiomer might be useful for preventing S. pyogenes adherence to human CD15s receptors and the resulting immunological pathogenicity.

Aims The aim of this study was to investigate the effects of genomic DNA purified from Candida albicans and pneumonia-related pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, on in vitro biofilm formation and morphological change of 3 Candida species (C. albicans, C. glabrata, and C. tropicalis). Methods and Results Biofilm formation was evaluated by the crystal violet assay and colony-forming unit counts. Morphological characteristics of biofilms were evaluated by scanning electron microscopy and fluorescence microscopy. Addition of DNA at a low concentration (

Dental treatment must meet the needs of the rapidly increasing elderly people in Japan. In addition, geriatric inpatients also increased with the increase of elderly people. Investigation was carried out to determine the oral condition and microflora in denture plaque of elderly patients in two geriatric hospitals and Tokushima Dental Hospital. The difference between healthy patients and geriatric inpatients was. discussed. The results of the preliminary survey were as follows:1. Inpatients in a geriatric hospital were more elderly, and the frequency of edentulous patients was fairly high. The prognosis of the denture was poor because of high frequencies of low residual ridge and oral diskinesia.2. Denture plaque of geriatric inpatients had the following characteristics compared to that of healthy elderly patients.1) Numbers of total bacteria, Streptococci, Candida, and Staphylococci in isolated denture plaque of the geriatric inpatients were larger than those of healthy patients.2) Linear relationships between numbers of total bacteria and Streptococci and between numbers of Candida and Staphylococci were found.3) The frequency of MRSA in the geriatric inpatients was higher than in the healthy patients.4) Numbers of total bacteria and Streptococci in denture plaque obtained in the inpatients with heart disease and cerebrovascular accident were much larger than those in the inpatients without these diseases.

The purpose of this study was to investigate efficacies of acidic and neutral electrolyzed water as denture cleansers from the bacteriological aspect.Twenty-five upper full dentures of patients of an elderly care center were used in this study. Nine dentures were immersed in acidic electrolyzed water, eight in neutral electrolyzed water and eight in denture cleanser (Pika®). Plaque samples were collected from fitting surface of the dentures by scrubbing before and after immersion, and the changes in the number of bacteria were observed.The results were as follows:1. Acidic and neutral electrolyzed water were effective as denture cleansers, and the rates of removal [log10 (bacteria number after immersion)/logic, (before immersion)] tended to be constant.2. MRSA decreased by immersion in acidic electrolyzed water and in denture cleanser (MRSA was isolated from four dentures. Two were immersed in acidic electrolyzed water and two in denture cleanser).

The expression of sialy-Lewis(x) (sLe(x); Neu5Ac alpha 2-3 Gal beta 1-4) (Fuc alpha 1-3) GlcNAc-R) on oral bacteria producing infective endocarditis was determined by a whole-cell enzyme linked immunosorbent assay and an immunoelectron microscopy using the well-characterized anti-sLe(x) monoclonal antibody SNH-3 (mAb SNH-3; IgM class). mAb SNH-3 reacted strongly with whole cells of oral bacteria: Streptococcus sanguis, Streptococcus mutans, Streptococcus mitis, Streptococcus salivarius, Streptococcus intermedius, Streptococcus constellatus, Streptococcus anginosus, Streptococcus pyogenes, Actinobacillus actinomycetemcomitans, Eikenella corrodens and Porphyromonas gingivalis. The negatively stained immuno-electron micrograph of Streptococcus pyogenes showed many reactive gold particles on the cell surface. Our findings demonstrated the existence of immunologic mimicry between the sLe(x) oligosaccharide and cell surface antigens of many species associated with infective endocarditis. We propose the hypothesis that if these bacteria escape their normal habitats, the surface components that mimic the sLe(x) oligosaccharide might bind to host antigens of the selectin family which could promote binding to endothelial cells and, consequently, initiation of the events leading to infective endocarditis.

ヒトの象牙質の頬舌と歯軸方向の6部位での自己蛍光強度の加齢的な変化を, 高速・時間分解・顕微蛍光測定装置を用いて検討した。静的な蛍光測定では, 蛍光強度はすべての部位で加齢に伴って増加した。動的な蛍光測定では, 蛍光スペクトルでピーク強度波長を示す440nmでの自己蛍光に少なくとも3つの蛍光成分の存在が示唆された。いずれの成分の蛍光強度も, すべての部位で加齢により増加したが, 増加の様子には差がみられた。蛍光の減衰速度が最も速い成分では, 蛍光強度が18歳以後加齢に比例して増加したのに対し, これより減衰速度の遅い他の2成分では, 蛍光強度の増加の度合が加齢とともにゆるやかになった。また, 静的な蛍光強度に対する3成分の蛍光強度の比率の加齢的な変化には, 歯軸方向で部位差がみられ, 加齢に伴い, 歯冠象牙質では減衰速度の最も速い成分の比率が増加したのに対し, 根尖象牙質では速度の最も遅い成分の比率の減少が特徴であった。

Bacterial infection has become a focus of attention in the pathogenesis of primary biliary cirrhosis (PBC). We earlier reported that the bacterial lipoteichoic acid was detected at the sites of inflammation around damaged bile ducts in the livers of PBC, and PBC patients' sera showed high titers against streptococcal histone-like protein. Here, we investigated whether chronic bacterial exposure could trigger PBC-like epithelial cell damage in normal mouse. BALB/c mice were repeatedly inoculated with various bacteria for 8 weeks. At 1 week (Group 1) and 3, 4, or 20 months (long term; Group 2) after the final inoculation, mice were killed to obtain samples. In the livers of the Streptococcus intermedius (S.i.)-inoculated mice in Group 1, cellular infiltration was predominantly observed around the bile ducts over the hepatic parenchyma. In the S.i.-inoculated mice in Group 2, portal but not parenchymal inflammation was observed in the livers, and periductal cellular infiltrates were detected in the salivary glands. Both S.i.-inoculated Groups 1 and 2 BALB/c mice sera had antibodies against HuCCT1 biliary epithelial cells, anti-nuclear antibodies, and anti-gp210 antibodies, but not anti-mitochondrial antibodies. Immunoreactivity to histone-like DNA-binding protein of S.i. (S.i.-HLP) was detectable around the sites of chronic nonsuppurative destructive cholangitis in the portal area in the livers of both S.i.-inoculated Groups 1 and 2 BALB/c mice. Furthermore, anti-S.i.-HLP antibody bound to synthetic gp210 peptide, as well. Bacteria triggered PBC-like cholangitis, multifocal epithelial inflammation, and autoantibody production. Bacteria are likely involved in the pathogenesis of PBC and of associated multifocal epithelial inflammation.

Groups of mice were fed for 2 weeks on isocaloric diets containing 5, 7, 20 and 40% (w/w) casein, respectively, then injected intraperitoneally with group B streptococci, and observed for their survival rates. The mice fed 7% or 20% casein had lower mortalities than those fed 5% or 40% casein. In order to explain the different survival rates, other groups of mice were fed on the experimental diets and examined for the number of leukocytes in the blood, of spleen cells and thymocytes, for IgG and IgM antibody titres to the streptococci, for haemolytic titres of sera, the amount of complement component 3 (C3), for chemiluminescence and opsonic activity of peritoneal exudate cells (PEC) and spleen cells (SP), production of superoxide anion from PEC and SP, and production of immunoglobulins from cultured SP. After 2 weeks on a 7 or 20% casein diet mice showed increased serum levels of IgM antibodies reactive with the whole bacterial cells on days 3-5 when they were immunised with a sublethal dose of group B streptococci. The mice fed on the 7% casein diet also showed a higher C3 titre than the other diet groups when assayed by enzyme immunoassay. Furthermore, opsonophagocytic activity was highest when PEC or SP taken from mice on the 20% case in diet were incubated with radiolabelled microorganisms in the presence of fresh serum taken from the 7% casein group. The production of superoxide anions from PEC and SP was lowest in the mice fed on 5% casein when activity was expressed as nano-mol per animal. It is suggested from these results that the greater activity of phagocytic cells in the presence of increased amounts of C3 and IgM explains the heightened resistance in the mice fed on a 7% casein diet, and that suppressed opsonophagocytic activity resulting from the decreased number of leukocytes in the blood and other phagocytic cells explains the lowest resistance in the 5% casein group. However, mice fed on a 40% casein diet showed all these immunological parameters untouched, and their lowered resistance could not be explained. Different factors seem to be operative in them.

The basis of the bactericidal action of antibiotics and the mechanisms of antibiotic tolerance are largely unknown. To elucidate one of the mechanisms of antibiotic tolerance, the present study investigated the role of Pseudomonas aeruginosa quorum sensing (QS) and the rpoS gene in antibiotic tolerance. The survival rates of the lasR and lasI mutants were observed to be lower than that of the parental strain in time-dependent killing studies with 8 microg mL(-1) ofloxacin, but the survival rates of the rhlR and rhlI mutants were not different from that of the parental strain. Moreover, a lasR-overexpressing strain was more tolerant to ofloxacin than the parental strain, but this was not the case for an rhlR-overexpressing strain. The mRNA expression levels of lasR, lasI, and rpoS in the wild-type strain in the presence of bactericidal concentration of ofloxacin were lower than that in the absence of ofloxacin. In addition, the significant loss of antibiotic tolerance in the lasR mutant was recovered by the overexpression of rpoS. These results suggest that the Las QS system in P. aeruginosa is involved in the development of ofloxacin tolerance, and the tolerance induced by the Las-system is regulated by rpoS gene.

Streptococcus intermedius histone-like DNA-binding protein (Si-HLP) is a homodimeric protein and, conserved with Escherichia coli HU, a well-documented nucleoid-associated protein (NAP). In E. coli, HU plays important roles as both structural and regulatory factors, but it is not essential for E. coli viability. Streptococcal HLP has been found to bind host cells and induce cytokine production, but its physiological role remains poorly defined. In the present study, using gene insertion knockout and tetracycline-regulated antisense RNA expression techniques, we determined whether Si-HLP is essential for bacterial viability and normal growth in S. intermedius. The Si-HLP-downregulated S. intermedius strain showed alterations in its morphology and surface properties. Downregulation of Si-HLP led to an expanded nucleoid to fill the intracellular space. Transcription levels of several genes, including virulence-associated factors, were found to be activated or repressed in the antisense Si-hlp RNA-expressing strain by real-time PCR and reverse-transcription PCR. Collectively, these data suggest that Si-HLP serves as an essential NAP governing the nucleoid architecture and controlling the gene transcription profile in S. intermedius.

紫外線照射されたヒト歯牙より発せられる螢光に関して, 従来より測定されている静的螢光に加えナノ秒域の動的螢光に着目し, 専用の時間分解顕微螢光測光装置を試作した。本装置を用いて, 加齢および齲蝕という老化に関する2大要素がヒト歯牙各組織の螢光特性に及ぼす影響について検討した。その結果, (1) ヒト歯牙の螢光物質としてヒドロオキシアパタイトと螢光性架橋コラーゲンの複合体が考えられた。 (2) 象牙質において螢光強度が加齢とともに増大し, 螢光減衰時間が短縮するという顕著な年齢依存性が認められた。 (3) 齲蝕部の螢光特性は健全部と異なり, ヒドロオキシアパタイトの螢光特性により近いものとなった。

Intermedilysin is a pore-forming cytolysin belonging to the streptolysin O gene family known as the 'Cholesterol-binding/dependent cytolysins' and is unique within the family in that it is highly humanspecific. This specificity suggests interaction with a component of human cells other than cholesterol, the proposed receptor for the other toxins of the gene family. Indeed, intermedilysin showed no significant degree of affinity to free or liposome-embedded cholesterol. Characterization of intermedilysin undecapeptide mutants revealed that this lack of affinity to cholesterol was a result of the substitutions of intermedilysin in this region. Absorption assays with erythrocyte membranes from various animals, competitive inhibition with domain 4 of intermedilysin and liposome-binding assays of streptolysin O and intermedilysin indicated that cell membrane binding is the human-specific step of intermedilysin action, that the host cell membrane-binding site is located within domain 4 in common with other members of the family and that the receptor for this toxin is not cholesterol. The species specificity of undecapeptide mutants of intermedilysin and streptolysin O and chimeric mutants between intermedilysin and streptolysin O, and intermedilysin and pneumolysin indicated that domain 4 of intermedilysin determines the human-specific action step and the cell-binding site of domain 4 lies within the 56 amino acids of the C-terminal, excluding the undecapeptide region.

Streptococcus intermedius belongs to the anginosus group of streptococci (AGS) and is associated with endogenous infections leading to abscesses in the oral cavity and at deep-seated sites, such as the brain and liver. Two other species, S. anginosus and S. constellatus, and some presently unnamed taxa, are also classified as AGS. Recently, S. constellatus subsp. pharyngis, a new subspecies with biochemical characteristics similar to S. intermedius, was described with the potential for causing confusion when trying to identify isolates of these two species routinely with commercial identification kits, such as Rapid ID32 Strep and Fluo-Card Milleri. To correctly identify S. intermedius, this study attempted to develop an accurate PCR identification system with the ily gene as a species marker. This approach relies on amplification of an 819-bp fragment of the ily gene and its 3′-flanking region and is shown here to be specific for S. intermedius strains among all other streptococcal species. Moreover, this PCR system was applicable in direct rapid PCR with whole bacterial cells and TaKaRa Z-Taq TM (TaKaRa), a highly efficient DNA polymerase, as the template and DNA amplification enzyme, respectively.

The susceptibility of viridans group streptococci to macrolides was determined. Thirteen isolates (17%) were resistant to erythromycin. Five strains carried an erm gene that was highly homologous to that in Tn 917 . Four strains had mefE genes that coded erythromycin efflux ability.

For 3 months, 4 patients in a nursing home for the elderly received supervised professional oral care in conjunction with oral hygiene instruction. Within the first month after the start of this program, plaque was reduced to 1/3 and gingivitis was reduced to 1/9 of their respective initial levels. In 5 patients who did not receive either oral hygiene instruction or professional oral care, plaque retention and gingivitis were unchanged. We conclude that professional oral care combined with oral hygiene instruction supervised by dental professionals can help to control gingivitis in the elderly.

Nowadays, a huge volume datasets has been generated from numerical simulations and measurement equipments. However the classification and reference has been usually performed manually, such as vision or experience of a measurement engineer. There still has not been revealed efficient techniques for searching similarity between volumetric datasets.In this report, we propose a technique of performing the classification and reference using the critical point graph which expresses the feature of volume datasets in the simple figure. For this purpose, critical point graphs need to be generated efficiently and it is necessary to calculate the critical point in volume datasets efficiently.

Gram-negative tannin-protein complex degrading bacteria (T-PCDB) were first isolated from animals except for the koala. The occurrence of T-PCDB in feces of 15 species of mammals with different feeding habits was investigated. T-PCDB occurred in 7 of 54 horses but they could not be isolated from other mammals tested. These T-PCDB comprised less than 0.1% of the facultative anaerobic microflora in horse feces and it was much less than that previously reported in koala feces ( > 60%). A total of 7 T-PCDB fecal isolates showed a range of phenotypic diversities. They were all Gram-negative rods of various sizes and shapes including coccoidal rod. Although all produced tannase, no strain showed to have gallate decarboxylase. A total of 23 representative strains belonging to the family Pasteurellaceae were also tested for tannase production. Two strains, Haemophilus actinomycetemcomitans NIAH-10202T and Haemophilus segnis NIAH-10183T which were isolated from human oral cavity were detected positive for tannase.

Streptococcus intermedius is a commensal associated with serious, deep-seated purulent infections in major organs, such as the brain and liver. Histone-like DNA binding protein (HLP) is an accessory architectural protein in a variety of bacterial cellular processes. In this study, we investigated the mechanisms of pro-inflammatory cytokine inductions in THP-1 cells by stimulation with recombinant HLP of S. intermedius (rSi-HLP). rSi-HLP stimulation-induced production of pro-inflammatory cytokines (IL-8, IL-1 beta and TNF-alpha) occurred in a time- and dose-dependent manner. In contrast with the heat-stable activity of DNA binding, the induction activity of rSi-HLP was heat-unstable. In subsequent studies, rSi-HLP acted cooperatively with lipoteichoic acid, the synthetic Toll-like receptor 2 agonist, Pam3CSK4, and the cytosolic nucleotide binding oligomerization domain 2 receptor agonist, muramyldipeptide. Furthermore, Western blot and blocking assays with specific inhibitors showed that rSi-HLP stimulation induced the activation of cell signal transduction pathways, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). In addition to its physiological role in bacterial growth through DNA binding, these results indicate that Si-HLP can trigger a cascade of events that induce pro-inflammatory responses via ERK1/2 and JNK signal pathways, and suggest that bacterial HLP may contribute to the activation of host innate immunity during bacterial infection.