Anti-Inflammatory and Protective Effects of Juncus effusus L. Water Extract on Oral Keratinocytes

Periodontitis is a chronic inflammatory disease caused by periodontopathogenic bacteria that form biofilms in periodontal pockets. The gingival epithelium acts as the first physical barrier in fighting attacks by periodontopathogenic pathogens, such as the primary etiological agent , and various exogenous chemicals, as well as regulates the local innate immune responses. Therefore, the development of novel oral care products to inhibit inflammatory reactions caused by bacterial infection and protect the gingival epithelium is necessary. L. has generally been used as an indigenous medicine, such as a diuretic, an antipyretic, and an analgesic, in ancient practice. In this study, we examined the effects of a water extract from L. on the inhibition of the inflammatory reaction elicited by bacterial infection and protection of the oral epithelium by chemical irritation. Pretreatment of oral epithelial cells with the water extract from L. significantly reduced or its lipopolysaccharide- (LPS-) mediated production of chemokines (interleukin-8 and C-C-chemokine ligand20) in a concentration-dependent manner with comparable to or greater effects than epigallocatechin gallate and protected oral epithelial cells from injury by chemical irritants, cetylpyridinium chloride, and benzethonium chloride. Moreover, the water extract from . L. in the presence of antimicrobial agents or antifibrinolytics already used as ingredients in mouthwash could significantly reduce the production of chemokines from LPS-stimulated oral epithelial cells in a concentration-dependent manner. These findings suggest that the water extract from . L. is potentially useful for oral care to prevent oral infections, such as periodontal infections, and maintain oral epithelial function.


Clinical Significance of Carbapenem-Tolerant Pseudomonas aeruginosa Isolated in the Respiratory Tract


Suppresive effects of 2-methacryloyloxyethyl phosphorylcholine (MPC)-polymer on the adherence of Candida species and MRSA to acrylic denture resin.


2-Methacryloyloxyethyl phosphorylcholine (MPC)-polymer suppresses an increase of oral bacteria: a single-blind, crossover clinical trial

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. 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. 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. We suggest that MPC-polymer coating in the oral cavity may suppress the oral bacterial adherence. MPC-polymer can be a potent compound for the control of oral microflora to prevent oral infection.


Anti-inflammatory effects of olanexidine gluconate on oral epithelial cells


The pathogenic factors from oral Streptococci for systemic diseases

International Journal of Molecular Sciences


Antifungal and Mechanical Properties of Tissue Conditioner Containing Plant-Derived Component --- An In Vitro Study

To evaluate the antifungal activity and mechanical properties of a novel antifungal tissue conditioner containing Juncus powder. Juncus powder was mixed with GC tissue conditioner at concentrations of 2.5%, 5.0%, and 10.0% by mass. The cylindrical specimens of Juncus-mixed tissue conditioner (dimensions: 10 mm in diameter and 2 and 6 mm in height for antimicrobial and mechanical tests, respectively) were prepared. The specimens placed on the bottom of the 24-well tissue culture plate were cultured with Candida albicans CAD1 for 2 and 4 days. The proliferation of the C. albicans in the wells was determined by measuring the optical density of fungal culture, and the surface of the specimens were also observed by scanning electron microscopy (SEM). To assess the mechanical properties of the specimens, the fluidity and hardness of Juncus-mixed tissue conditioner were measured using the methods certified according to ISO 10139-1. Juncus-mixed tissue conditioner significantly exhibited growth inhibitory effect in a Juncus concentration-dependent manner after both 2- and 4- day cultures. SEM observation showed that the amount of C. albicans on Juncus-mixed specimens drastically decreased, and biofilm formation was markedly inhibited. Moreover, both mechanical properties were found to be within the ranges regulated and specified by ISO. These findings demonstrated that the tissue conditioner including Juncus powder has a significant growth inhibitory effect against C. albicans, and it is suggested that the application of Juncus-mixed tissue conditioner may prevent denture stomatitis and oral candidiasis in denture wearers.


Royal jelly inhibits Pseudomonas aeruginosa adherence and reduces excessive inflammatory responses in human epithelial cells

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 is unclear. The aim of this study was to analyze antibacterial, antiadherent, and anti-inflammatory effects of royal jelly against . Wild-type strain PAO1 and clinical isolates of 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-3/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 adherence and protects epithelial cells from excessive inflammatory responses against infection. Our findings suggested that royal jelly may be a useful supplement as complementary and alternative medicine for preventing respiratory infection caused by .


Antibiofilm and Anti-Inflammatory Activities of+ Houttuynia cordata Decoction for Oral Care

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. (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 (MRSA) and other oral microorganisms. dHC also exhibited antibiofilm effects against MRSA, (involved in dental plaque formation), and and inhibitory effects on interleukin-8, CCL20, IP-10, and GRO productions by human oral keratinocytes stimulated by 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.

Evidence-Based Complementary and Alternative Medicine : eCAM


Effects of an autoinducer analogue on antibiotic tolerance in Pseudomonas aeruginosa

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 . 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. 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. 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.

The Journal of Antimicrobial Chemotherapy


Antibacterial and antibiofilm effects of Reuterin-Related Compounds to periodontopathic bacteria


Role of psl Genes in Antibiotic Tolerance of Adherent Pseudomonas aeruginosa.

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 PAO1 slBCD 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 spF mutant background abolished the biapenem-tolerant phenotype of the spF 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.


Synthesis and evaluation of c-di-4'-thioAMP as an artificial ligand for c-di-AMP riboswitch

Cyclic-di-adenosine monophosphate (c-di-AMP) is a bacterial second messenger that binds to an RNA receptor called riboswitch and regulates its downstream genes involving cell wall metabolism, ion transport, and spore germination. Therefore, the c-di-AMP riboswitch can be a novel target of antibiotics. In this study, we synthesized c-di-4'-thioAMP (1), which possesses a sulfur atom instead of an oxygen atom in the furanose ring, as a candidate of a bioisoster for natural c-di-AMP. The resulting 1 bound to the c-di-AMP riboswitch with a micromolar affinity (34.8M), and the phosphodiesterase resistance of 1 was >12-times higher than that of c-di-AMP. Thus, 1 can be considered to be a stable ligand against a c-di-AMP riboswitch.


RpoN Promotes Pseudomonas aeruginosa Survival in the Presence of Tobramycin.

Pseudomonas aeruginosa has developed diverse strategies to respond and adapt to antibiotic stress. Among the factors that modulate survival in the presence of antibiotics, alternative sigma factors play an important role. Here, we demonstrate that the alternative sigma factor RpoN ( (54)) promotes survival in the presence of tobramycin. The tobramycin-sensitive phenotype of logarithmic phase poN mutant cells is suppressed by the loss of the alternative sigma factor RpoS. Transcriptional analysis indicated that RpoN positively regulates the expression of RsmA, an RNA-binding protein, in the P. aeruginosa stationary growth phase in a nutrient-rich medium. The loss of RpoS led to the upregulation of gacA expression in the nutrient-limited medium-grown stationary phase cells. Conversely, in the logarithmic growth phase, the poS mutant demonstrated lower expression of gacA, underscoring a regulatory role of RpoS for GacA. Supplementation of tobramycin to stationary phase poN mutant cells grown in nutrient-rich medium resulted in decreased expression of gacA, relA, and rpoS without altering the expression of rsmA relative to wild-type PAO1. The observed downregulation of gacA and relA in the poN mutant in the presence of tobramycin could be reversed through the mutation of rpoS in the poN mutant background. The tobramycin-tolerant phenotype of the poN poS mutant logarithmic phase cells may be associated with the expression of relA, which remained unresponsive upon addition of tobramycin. The logarithmic phase poS and poN poS mutant cells demonstrated increased expression of gacA in response to tobramycin. Together, these results suggest that a complex regulatory interaction between RpoN, RpoS, the Gac/Rsm pathway, and RelA modulates the P. aeruginosa response to tobramycin.


Novel reuterin-related compounds suppress odour by periodontopathic bacteria

Halitosis is caused by volatile sulphur compounds including methyl mercaptan (CH3 SH) in the oral cavity and is a serious problem that limits interpersonal social communication. The aim of study was to evaluate the effects of reuterin-related compounds (RRCs) on halitosis-related periodontopathic bacteria in vitro. RRC-01, RRC-02 and RRC-03 (32 and 64 g ml(-1) ) in culture media containing Fusobacterium nucleatum JCM8523 and Porphyromonas gingivalis ATCC33277 were used. The effects of RRCs on CH3 SH production and detectable odour by F. nucleatum and P. gingivalis were examined by CH3 SH production assay and organoleptic test, respectively. The number of bacterial cells was also measured using an ATP assay. In P. gingivalis treated with RRCs, the expression of mgl gene, which is responsible for CH3 SH production, was examined by qRT-PCR. CH3 SH production and the score of detectable odour from F. nucleatum and P. gingivalis culture media containing RRCs were significantly lower than that without RRCs (P < 0.05). The expression of mgl gene in P. gingivalis was significantly downregulated by RRC-01 (P < 0.01), but not by RRC-02 or RRC-03. RRCs are potent oral care products for preventing halitosis via reducing CH3 SH production.


Explorative gene analysis of antibiotic tolerance-related genes in adherent and biofilm cells of Pseudomonas aeruginosa.

Antibiotic tolerance has attracted worldwide attention, as it leads to chronic, refractory, and persistent infections that are difficult to control. Bacterial biofilms are well known to be more tolerant to antibiotics compared to planktonic bacteria. We previously revealed that adherent bacteria on a solid surface also exhibited tolerance to antibiotics before forming a biofilm. However, little is known about the mechanisms of antibiotic tolerance for adherent or biofilm cells. We investigated the mechanisms of antibiotic tolerance in the biofilm life cycle using adherent and biofilm cells, and evaluated the possibility that common mechanisms operate at each stage. We constructed transposon mutants of Pseudomonas aeruginosa PAO1 and screened for low-tolerant mutants with two different methods, using adherent cells and biofilm cells. Fourteen and nine mutants exhibiting low antibiotic tolerance were detected in the adherent cells and biofilm cells, and 14 and 7 candidate genes linked to this tolerance were identified by sequencing, respectively. Eight of the 14 genes related to the antibiotic tolerance of the adherent cells were involved in biofilm formation. Two of the seven genes related to the antibiotic tolerance of biofilm cells participated in the antibiotic tolerance of adherent cells. The antibiotic tolerance of adherent cells and biofilm formation appear to be under the same regulation mechanism to promote survival in the presence of antibiotics. Antibiotic tolerance shows a complex regulation mechanism at each stage of biofilm formation.


Role of the interplay between quorum sensing regulator VqsR and the Pseudomonas quinolone signal in mediating carbapenem tolerance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa coordinates its response to environmental conditions through activation of a quorum sensing (QS) system. In this study, we investigated the regulatory interaction between the QS transcriptional regulator VqsR and the Pseudomonas quinolone signal (PQS) through integration of sigma factor RpoS, and we addressed whether one of the pathways controlling carbapenem tolerance can be attributed to VqsR. We demonstrate that vqsR expression at the transcriptional level is regulated by pqsA, pqsR, and pqsE. Assessment of the transcriptional expression of vqsR, lasI, rhlI, and qscR in qsA and qsA poS mutants provided insight into pqsA- and rpoS-dependent regulation of vqsR and vqsR-controlled genes. Exogenously supplemented PQS reversed expression of vqsR and vqsR-controlled genes in the qsA mutant to wild-type levels, but failed to increase expression levels of lasI and qscR in the qsA poS mutant to levels observed in wild-type PAO1. The qsR mutant showed reduced survival when challenged with carbapenems compared to wild-type PAO1. Introduction of a pqsA mutation into the qsR mutant completely abolished its carbapenem-sensitive phenotype. We conclude that a regulatory link between PQS and vqsR exists, and that RpoS is important in their interaction. We also demonstrate that VqsR affects carbapenem tolerance.


Evaluation of the effectiveness of the potent bis-quaternary ammonium compound, 4,4'-(α,ω-hexametylenedithio) bis (1-octylpyridinium bromide) (4DTBP-6,8) on Pseudomonas aeruginosa

Quaternary ammonium compounds (QACs), including benzalkonium chloride (BAC) and cetylpyridinium chloride (CPC) are cationic surfactants and have been used widely as general disinfectants in the medical field due to their strong antibacterial effects and low cytotoxicity to human cells. 4,4'-(α,ω-hexametylenedithio) bis (1-octylpyridinium bromide) (4DTBP-6,8) is one of the potent bis-QACs synthesized to improve the antimicrobial activities of mono-QACs such as BAC. This study aimed to assess the effectiveness of 4DTBP-6,8 against Pseudomonas aeruginosa, a prevalent hospital pathogen. The minimum inhibitory concentrations of 4DTBP-6,8, CPC and BAC against P. aeruginosa were measured. 4DTBP-6,8 exhibited strong antibacterial activity. We assessed the bactericidal effects of QACs against P. aeruginosa under certain conditions and their cytotoxicities in human epithelial cells using lactate dehydrogenase (LDH) release. 4DTBP-6,8 exerted excellent bactericidal effects against high concentrations of bacteria, biofilm cells and even in the presence of contaminated proteins. Cellular LDH was not released by the treatment with 4DTBP-6,8. 4DTBP-6,8 exhibited the strongest bactericidal activity against P. aeruginosa among the three QACs tested without any cytotoxicity. The potent bis-QAC, 4DTBP-6,8 has the potential to be an effective disinfectant in preventing hospital infections caused by P. aeruginosa.


A Pseudomonas aeruginosa Quorum-Sensing autoinducer analog enhances the activity of antibiotics against resistant strains.

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 PAO1 prD mutant and clinical isolates were performed by using antibiotics and AIA-1. In an in vivo assay, a luminescent carbapenem-resistant strain derived from PAO1 prD 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.

The Journal of Medical Investigation : JMI


Pathogenic factors in Candida biofilm-related infectious diseases.

Candida albicans is a commonly found member of the human microflora and is a major human opportunistic fungal pathogen. A perturbation of the microbiome can lead to infectious diseases caused by various micro-organisms, including C. albicans. Moreover, the interactions between C. albicans and bacteria are considered to play critical roles in human health. The major biological feature of C. albicans, which impacts human health, resides in its ability to form biofilms. In particular, the extracellular matrix (ECM) of Candida biofilm plays a multifaceted role and therefore may be considered as a highly attractive target to combat biofilm-related infectious diseases. In addition, extracellular DNA (eDNA) also plays a crucial role in Candida biofilm formation and its structural integrity and induces the morphological transition from yeast to the hyphal growth form during C. albicans biofilm development. This review focuses on pathogenic factors such as eDNA in Candida biofilm formation and its ECM production and provides meaningful information for future studies to develop a novel strategy to battle infectious diseases elicited by Candida-formed biofilm.


RpoN Modulates Carbapenem Tolerance in Pseudomonas aeruginosa through Pseudomonas Quinolone Signal and PqsE.

The ability of Pseudomonas aeruginosa to rapidly modulate its response to antibiotic stress and persist in the presence of antibiotics is closely associated with the process of cell-to-cell signaling. The alternative sigma factor RpoN ( (54)) is involved in the regulation of quorum sensing (QS) and plays an important role in the survival of stationary-phase cells in the presence of carbapenems. Here, we demonstrate that a poN mutant grown in nutrient-rich medium has increased expression of pqsA, pqsH, and pqsR throughout growth, resulting in the increased production of the Pseudomonas quinolone signal (PQS). The link between pqsA and its role in carbapenem tolerance was studied using a poN qsA mutant, in which the carbapenem-tolerant phenotype of the poN mutant was abolished. In addition, we demonstrate that another mechanism leading to carbapenem tolerance in the poN mutant is mediated through pqsE Exogenously supplied PQS abolished the biapenem-sensitive phenotype of the poN qsA mutant, and overexpression of pqsE failed to alter the susceptibility of the poN qsA mutant to biapenem. The mutations in the poN hlR mutant and the poN qsH mutant led to susceptibility to biapenem. Comparison of the changes in the expression of the genes involved in QS in wild-type PAO1 with their expression in the poN mutant and the poN mutant-derived strains demonstrated the regulatory effect of RpoN on the transcript levels of rhlR, vqsR, and rpoS The findings of this study demonstrate that RpoN negatively regulates the expression of PQS in nutrient-rich medium and provide evidence that RpoN interacts with pqsA, pqsE, pqsH, and rhlR in response to antibiotic stress.


Preventive Effects of Houttuynia cordata Extract for Oral Infectious Diseases.

Houttuynia cordata (HC) (Saururaceae) has been used internally and externally as a traditional medicine and as an herbal tea for healthcare in Japan. Our recent survey showed that HC poultice (HCP) prepared from smothering fresh leaves of HC had been frequently used for the treatment of purulent skin diseases with high effectiveness. Our experimental study also demonstrated that ethanol extract of HCP (eHCP) has antibacterial, antibiofilm, and anti-inflammatory effects against S. aureus which caused purulent skin diseases. In this study, we focused on novel effects of HCP against oral infectious diseases, such as periodontal disease and dental caries. We determined the antimicrobial and antibiofilm effects of water solution of HCP ethanol extract (wHCP) against important oral pathogens and investigated its cytotoxicity and anti-inflammatory effects on human oral epithelial cells. wHCP had moderate antimicrobial effects against some oral microorganisms and profound antibiofilm effects against Fusobacterium nucleatum, Streptococcus mutans, and Candida albicans. In addition, wHCP had no cytotoxic effects and could inhibit interleukin-8 and CCL20 productions by Porphyromonas gingivalis lipopolysaccharide-stimulated human oral keratinocytes. Our findings suggested that wHCP may be clinically useful for preventing oral infectious diseases as a mouthwash for oral care.


Anti-bacterial and anti-inflammatory effects of ethanol extract from Houttuynia cordata poultice.

Houttuynia cordata (HC) has been commonly used as many traditional remedies in local areas of Japan. Although many pharmacological activities of HC have been reported, the mechanism underlying the effect of HC remains unknown. We conducted the interview survey in Japan to verify how HC was actually used. The interview survey revealed that HC poultice (HCP) prepared from smothering fresh leaves of HC was most frequently used for the treatment of purulent skin diseases including furuncle and carbuncle with high effectiveness. Ethanol extract of HCP (eHCP) showed anti-bacterial effects against methicillin-resistant Staphylococcus aureus (MRSA), and showed an anti-biofilm activity against MRSA. eHCP showed dose-dependent inhibition of S. aureus lipoteichoic acid (LTA)-induced interleukin-8 and CCL20 production in human keratinocyte without any cytotoxicity. These results suggest that HCP is effective for skin abscess and its underlying mechanism might be the complicated multiple activities for both bacteria and host cells.

Bioscience, Biotechnology, and Biochemistry


DNA Inversion Regulates Outer Membrane Vesicle Production in Bacteroides fragilis.

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 β-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.


A proposed core curriculum for dental English education in Japan.

Globalization of the professions has become a necessity among schools and universities across the world. It has affected the medical and dental professions in terms of curriculum design and student and patient needs. In Japan, where medicine and dentistry are taught mainly in the Japanese language, profession-based courses in English, known as Medical English and Dental English, have been integrated into the existing curriculum among its 83 medical and 29 dental schools. Unfortunately, there is neither a core curriculum nor a model syllabus for these courses. This report is based on a survey, two discussion forums, a workshop, and finally, the drafting of a proposed core curriculum for dental English approved by consensus of the participants from each university. The core curriculum covers the theoretical aspects, including dental English terms and oral pathologies; and practical aspects, including blended learning and dentist-patient communication. It is divided into modules and is recommended to be offered for at least two semesters. The core curriculum is expected to guide curriculum developers in schools where dental English courses are yet to be offered or are still in their early development. It may also serve as a model curriculum to medical and dental schools in countries in Asia, Europe, Africa, and Central and South America, where English is not the medium of instruction.


Anti-inflammatory and protective effects of 2-methacryloyloxyethyl phosphorylcholine polymer on oral epithelial cells.

Periodontitis is a chronic inflammatory disease initiated by a microbial biofilm formed in the periodontal pocket. Gingival epithelium plays important roles as the first physical barrier to bacterial invasion and in orchestrating the innate immune reaction via toll-like receptors (TLRs), which recognize various bacterial products, and maintaining its function. Newly developed oral care products to inhibit bacterial adherence, subsequent inflammatory reaction and protect the gingival epithelium are expected. We previously reported that 2-methacryloyloxyethyl phosphorylcholine (MPC)-polymer coating decreased bacterial adhesion to human oral keratinocytes, RT-7, and mouth-rinsing with MPC-polymer inhibited the increase of oral bacteria. In this study, regarding the possibility of MPC-polymer application for preventing the adherence of periodontal pathogen, subsequent inflammatory reaction and protection of gingival epithelium, we examined the effects of MPC-polymer on the adherence of Porphyromonas gingivalis, major periodontitis-related pathogen, and TLR2 ligand to RT-7 and subsequent interleukin (IL)-8 production. MPC-polymer treatment significantly reduced P. gingivalis adherence by 44% and TLR2-mediated IL-8 production by blocking the binding of its specific-ligand in a concentration-dependent manner. Furthermore, MPC-polymer pretreatment protected RT-7 from injury by chemical irritants, cetylpyridinium chloride. These findings suggest that MPC-polymer is potentially useful for oral care to prevent oral infection and to maintain oral epithelial function. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2014.

Journal of Biomedical Materials Research. Part A


Effect of extracellular DNA on pyocyanin production from Pseudomonas aeruginosa


マウスを用いた緑膿菌性尿路バイオフィルム感染症モデルの作製

マウスを用いた緑膿菌性尿路バイオフィルム感染症モデルの作製


Effects of extracellular DNA from Candida albicans and pneumonia-related pathogens on Candida biofilm formation and hyphal transformation.

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). 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 (<1·0 μg ml(-1)) significantly increased biofilm mass of all three Candida species. In contrast, the addition of DNA at a high concentration (10 μg ml(-1)) decreased the biofilm mass. Interestingly, the formation of hyphae in a dense network of yeast cells was observed in C. albicans biofilms exposed to a low concentration of DNA (<1·0 μg ml(-1)). These findings demonstrated that extracellular DNA (eDNA) plays a crucial role in Candida biofilm formation and suggested that eDNA may induce the morphological transition from yeast to hyphal growth form during C. albicans biofilm development. A novel therapy targeting eDNA may be applicable for Candida infection to decrease biofilm formation and hyphal formation.


Draft Genome Sequence of Porphyromonas gingivalis Strain SJD2, Isolated from Periodontal Pocket of a Patient with Periodontitis in China

Porphyromonas gingivalis strain SJD2 was isolated from subgingival plaque of a patient in China with chronic periodontitis. Here, we report the draft genome of this strain, with a size of 2,328,850 bp, average G+C content of 48.3%, and 2,020 predicted protein-coding sequences.


Evaluation of a rapid oral bacteria quantification system using dielectrophoresis and the impedance measurement.

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.


付着及びバイオフィルム形成緑膿菌の抗菌薬抵抗性関連遺伝子の探索

付着及びバイオフィルム形成緑膿菌の抗菌薬抵抗性関連遺伝子の探索


Role of Extracellular DNA and DNA-binding protein in biofilm formation of Streptococcus intermeius


Effects of extracellular DNA and DNA-binding protein on the development of a Streptococcus intermedius biofilm.

The aim of this study was to clarify the effects of homologous and heterologous extracellular DNAs (eDNAs) and histone-like DNA-binding protein (HLP) on Streptococcus intermedius biofilm development and rigidity. Formed biofilm mass was measured with 0·1% crystal violet staining method and observed with a scanning electron microscope. The localizations of eDNA and extracellular HLP (eHLP) in formed biofilm were detected by staining with 7-hydoxyl-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) and anti-HLP antibody without fixation, respectively. DNase I treatment (200 U ml(-1) ) markedly decreased biofilm formation and cell density in biofilms. Colocalization of eHLP and eDNA in biofilm was confirmed. The addition of eDNA (up to 1 μg ml(-1) ) purified from Strep. intermedius, other Gram-positive bacteria, Gram-negative bacteria, or human KB cells into the Strep. intermedius culture increased the biofilm mass of all tested strains of Strep. intermedius, wild-type, HLP-downregulated strain and control strains. In contrast, the addition of eDNA (>1 μg ml(-1) ) decreased the biofilm mass of all Strep. intermedius strains. These findings demonstrated that eDNA and eHLP play crucial roles in biofilm development and its rigidity. eDNA- and HLP-targeting strategies may be applicable to novel treatments for bacterial biofilm-related infectious diseases.


Involvement of Commensal Bacteria may Lead to Dysregulated Inflammatory and Autoimmune Responses in a Mouse Model for Chronic Nonsuppurative Destructive Cholangitis.

BACKGROUND: We previously reported a mouse model of primary biliary cirrhosis (PBC)-like chronic nonsuppurative destructive cholangitis (CNSDC), in which frequent injections of Streptococcus intermedius induced CNSDC and autoantibody production. The present study was performed to verify the model by examining 1) the reappearance of the PBC-like CNSDC after lymphocyte transfer from model to naïve mice, 2) the involvement of autophagy, and 3) the influence of the strain difference. METHODS: Mice were inoculated with S. intermedius weekly for 8 weeks, then sacrificed to obtain samples. Spleen cells obtained from S. intermedius-inoculated mice were transferred to RAG2(-/-) mice. RESULTS: CNSDC and elevated serum level of anti-gp210 titers were observed in S. intermedius-inoculated C57BL/6 mice, similar to the results of our previous report using BALB/c mice. Portal inflammation was induced in the livers of RAG2(-/-) mice by the transfer of spleen cells from S. intermedius-inoculated C57BL/6 mice. Among the inflammatory cells in the RAG2(-/-) mice, CD3-positive cells were predominant. Autophagosome-like structures were detected histologically, in the cytoplasm of infiltrated cells around the bile ducts in the livers of S. intermedius-inoculated both C57BL/6 and BALB/c mice. In S. intermedius-inoculated C3H/HeJ mice, inflammation in the portal area was less extensive than that in the hepatic parenchyma. CONCLUSION: Bacterial component(s) and sequentially upregulated innate and acquired immune responses, accompanied by autophagy, might trigger CNSDC, via autoimmune mechanisms. Throughout the generation of bacteria-triggered PBC-like CNSDC, strain difference may influence the response to S. intermedius-inoculation in the liver.


Biofilm formation of Candida albicans on implant overdenture materials and its removal.

The purposes of this study were to clarify the surface characteristics of various implant overdenture materials and the capabilities of Candida albicans adherence and biofilm formation on these surfaces, and to investigate the role of salivary mucin in biofilm formation. Seven commonly used implant and restorative materials were assessed. The surface roughness averages of all materials were limited to 0.07-0.10μm. Contact angles and salivary mucin absorption were measured. After 90-min initial adhesion and 2-day biofilm formation, the amounts of C. albicans were determined by counting colony-forming units and the morphological characteristics were observed by scanning electron microscopy (SEM). The effects of saliva coating and the influences of material surface property on initial adhesion, biofilm formation and its removability were analysed by univariate two-way analysis of variance and multiple linear regression analysis. Surface contact angle of materials, the index of hydrophobicity, was found to be correlated positively with initial adhesion and biofilm formation of C. albicans. A negative correlation between mucin absorption and removability of Candida biofilm indicates that mucin plays an important role in biofilm formation and its rigidity. SEM observation also revealed fewer Candida cells on saliva-coated Ti than on saliva-coated hydroxyapatite or acrylic resin. The materials with different hydrophobic property and compositions display diverse manners of salivary mucin absorption, initial adhesion and biofilm formation. The hydrophobic materials encourage enhanced initial adhesion, subsequently resulting in the active biofilm formation. Mucin has decisive effects on Candida immobilization and biofilm development on the materials. Surface hydrophilic property and composition of materials and salivary proteins, especially mucin, affect the process of Candida biofilm formation and influence the amount and rigidity of formed biofilm. The present data may be applied as a reference for selecting materials in implant overdenture treatment from a microbiological point of view.


MPC-Polymer Reduces Adherence and Biofilm Formation by Oral Bacteria.

Oral biofilms such as dental plaque cause dental caries and periodontitis, as well as aspiration pneumonia and infectious endocarditis by translocation. Hence, the suppression of oral biofilm formation is an issue of considerable importance. Mechanical removal, disinfectants, inhibition of polysaccharide formation, and artificial sugar have been used for the reduction of oral biofilm. From the viewpoint of the inhibition of bacterial adherence, we investigated whether aqueous biocompatible 2-methacryloyloxyethyl phosphorylcholine (MPC)-polymer can reduce streptococcal colonization and biofilm formation. We examined the effects of MPC-polymer on streptococcal adherence to saliva-coated hydroxyapatite and oral epithelial cells, and the adherence of Fusobacterium nucleatum to streptococcal biofilm. MPC-polymer application markedly inhibited both the adherence and biofilm formation of Streptococcus mutans on saliva-coated hydroxyapatite and streptococcal adherence to oral epithelial cells, and reduced the adherence of F. nucleatum to streptococcal biofilms. A small-scale clinical trial revealed that mouthrinsing with MPC-polymer inhibited the increase of oral bacterial numbers, especially of S. mutans. These findings suggest that MPC-polymer is a potent inhibitor of bacterial adherence and biofilm development, and may be useful to prevent dental-plaque-related diseases. (UMIN Clinical Trial Registry UMIN000003471). Abbreviations: QOL, quality of life; MPC, 2-methacryloyloxyethyl phosphorylcholine; BHI, brain-heart infusion broth; PBS, phosphate-buffered saline; SEM, scanning electron microscopy; CFU, colony-forming unit.


Intermedilysin induces EGR-1 expression through calcineurin/NFAT pathway in human cholangiocellular carcinoma cells.

(Intermedilysin (ILY) is a cholesterol-dependent cytolysin produced by Streptococcus intermedius, which is associated with human brain and liver abscesses. Although intrahepatic bile duct cells play a valuable role in the pathogenesis of liver abscess, the molecular mechanism of ILY-treated intrahepatic bile duct cells remains unknown. In this study, we report that ILY induced a nuclear accumulation of intracellular calcium ([Ca(2+)]i) in human cholangiocellular cells HuCCT1. We also demonstrate that 10 ng/ml ILY induced NFAT1 dephosphorylation and its nuclear translocation in HuCCT1 cells. In contrast to the result that ILY induced NF-B translocation in human hepatic HepG2 cells, ILY did not affect NF-B localization in HuCCT1 cells. Dephosphorylation and nuclear translocation of NFAT1 caused by ILY were prevented by [Ca(2+)]i calcium chelator, BAPTA/AM, and calcineurin inhibitors, cyclosporine A and tacrolimus. ILY induced early growth response-1 (EGR-1) expression and it was inhibited by the pre-treatment with cyclosporine A, indicating that the calcineurin/NFAT pathway was involved in EGR-1 expression in response to ILY. ILY-induced calcineurin/NFAT1 activation and sequential EGR-1 expression might be related to the pathogenesis of S. intermedius in human bile duct cells.)

Biochemical and Biophysical Research Communications


Enhanced germicidal effects of pulsed UV-LED irradiation on biofilms.

This study can assist in developing a low-frequency pulsed UVA-LED system to be applied to pathogenic biofilms for disinfection.

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.


Long-term bacterial exposure can trigger nonsuppurative destructive cholangitis associated with multifocal epithelial inflammation.

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.

Laboratory Investigation; a Journal of Technical Methods and Pathology


Effect of substrate surface hydrophobicity on the adherence of yeast and hyphal Candida

A biofilm composed of various microorganisms including Candida is found on denture surfaces and is likely to be involved in the etiology of denture-induced stomatitis. The purpose of this study was to examine the role of hydrophobic interactions in candidal adherence to acrylic surfaces, particularly that of the hyphal form of Candida albicans. Candida clinical isolates were used. Acrylic plates coated with carrageenan and hydrocolloid (Hitachi chemical, Tokyo, Japan) were used as a hydrophilic substratum. A microbial suspension was placed on each acrylic plate and incubated. All plates were washed in phosphate-buffered saline containing CaCl(2) and MgCl(2) [PBS (+)] and cells still adhering to the acrylic surface were collected by 0.25% trypsin treatment. Cell-surface hydrophobicity was estimated using a modification of the technique used to measure adherence to hydrocarbons. When the acrylic plates were coated with hydrophilic materials, the adherence of hydrophobic clinical isolates of Candida and the hydrophobic hyphal C. albicans decreased, whereas the adherence of non-hydrophobic Candida was not affected or increased. We suggest that hydrophilic coating of denture surfaces could be a potent method for reduction of the adherence of relatively hydrophobic fungal cells, particularly hyphal C. albicans, which causes denture stomatitis and related infections.


The role of rpoS gene and quorum-sensing system in ofloxacin tolerance in Pseudomonas aeruginosa

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.


Heterologous expression of a histone-like protein from Streptococcus intermedius in Escherichia coli alters the nucleoid structure and inhibits the growth of E. coli

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.


Professional oral health care reduces the number of oropharyngeal bacteria

Silent aspiration of oropharyngeal pathogenic organisms is a significant risk factor causing pneumonia in the elderly. We hypothesized that regular oral hygiene care will affect the presence of oropharyngeal bacteria. Professional cleaning of the oral cavity and/or the gargling of a disinfectant liquid solution was performed over a five-month period in three facilities for the dependent elderly. Total oropharyngeal bacteria, streptococci, staphylococci, Candida, Pseudomonas, and black-pigmented Bacteroides species were monitored. The levels of these oropharyngeal bacteria decreased or disappeared after weekly professional oral health care, i.e., cleaning of teeth, dentures, tongue, and oral mucous membrane by dental hygienists. After lunch, gargling with povidone iodine was shown to be less effective than professional oral care. These findings indicate that weekly professional mechanical cleaning of the oral cavity, rather than a daily chemical disinfection of the mouth, can be an important strategy to prevent aspiration pneumonia in the dependent elderly.


The essentiality and involvement of Streptococcus intermedius histone-like DNA-binding protein in bacterial viability and normal growth

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.


A Possible role of histone-like DNA-binding protein of Streptococcus intermedius in the pathogenesis of bile duct damage in primary biliary cirrhosis

Bacterial infection has become a focus of attention in the pathogenesis of primary biliary cirrhosis (PBC). It was reported that anti-histone autoantibody was detected in PBC, suggesting that bacterial histone-like DNA-binding protein (HLP) may be involved in the pathogenesis of PBC. To identify bacterial species in PBC to confirm this possibility, serum reactivity to bacterial cells was studied by ELISA. The IgM class Streptococcus intermedius titers were significantly higher in PBC than chronic hepatitis due to hepatitis C virus (CH-C) and healthy subjects. Among the streptococci, S. intermedius was selected for further study. The antigenic peptide of S. intermedius of HLP was synthesized to examine the serum reactivity to Si-HLP. IgM class anti-Si-HLP peptide titers were significantly higher in PBC. Immunoreactivity to anti-Si-HLP was detected in the cytoplasm of biliary epithelial cells and inflammatory cells in the portal area in PBC patients' livers. Streptococci, especially S. intermedius, might play a key role in the pathogenesis of PBC, possibly involving HLP.


Histone-like DNA binding protein of Streptococcus intermedius induces the expression of pro-inflammatory cytokines in human monocytes via action of ERK1/2 and JNK pathways

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.


rpoN gene of Pseudomonas aeruginosa alters Its susceptibility to quinolones and cabapenems

The alternative sigma factor sigma54 has been implicated in diverse functions within the cells. In this study, we have constructed an rpoN mutant of Pseudomonas aeruginosa and investigated its importance as a target for antimicrobial agents, such as quinolones and carbapenems. The stationary-phase cells of the rpoN mutant displayed a survival rate approximately 15 times higher than that of the wild-type cells in the presence of quinolones and carbapenems. The stationary phase led to substantial production of pyoverdine by the P. aeruginosa rpoN mutant. Pyoverdine synthesis correlated with decreased susceptibility to antimicrobial agents. Quantitative real-time PCR revealed that stationary-phase cells of the rpoN mutant grown without an antimicrobial agent had approximately 4- to 140- and 2- to 14-fold-higher levels of transcripts of the pvdS and vqsR genes, respectively, than the wild-type strain. In the presence of an antimicrobial agent, levels of pvdS and vqsR transcripts were elevated 400- and 5-fold, respectively, in comparison to the wild-type levels. Flow cytometry assays using a green fluorescent protein reporter demonstrated increased expression of the vqsR gene in the rpoN mutant throughout growth. A pvdS mutant of P. aeruginosa, deficient in pyoverdine production, was shown to be susceptible to biapenem. These findings suggest that rpoN is involved in tolerance to antimicrobial agents in P. aeruginosa and that its tolerant effect is partly dependent on increased pyoverdine production and vqsR gene expression.


Functional Analysis of spoT, relA and dksA Genes on Quinolone Tolerance in Pseudomonas aeruginosa under Nongrowing Condition

To assess the contribution of ppGpp in antibiotic tolerance to quinolone in Pseudomonas aeruginosa, knockout mutants of the genes involved or linked with the stringent response, such as relA, spoT and dksA, were constructed and investigated for their antibiotic susceptibility to quinolones. The survival of the dksA and spoT mutants in the presence of 8 microg/ml of ofloxacin and 1 microg/ml of ciprofloxacin were shown to be approximately 20-180 and 10-40 times respectively, higher than the same for the wild type strain. The intracellular levels of ppGpp determined with high performance liquid chromatography (HPLC) demonstrated that spoT and dksA mutants possess higher basal levels of ppGpp. The data suggest that elevated basal levels of ppGpp may be responsible for rendering these mutants tolerant to quinolones and expand the importance of ppGpp as an antimicrobial target in P. aeruginosa.


CXCL12 and CXCR4 expression by human gingival fibroblasts in periodontal disease

CXCL12 is a CXC chemokine that is related to lymphocyte infiltration and angiogenesis in inflammatory sites such as arthritis. However, the expression and roles of CXCL12 in periodontal disease are uncertain. The aim of this study was to assess the expression of CXCL12 and its receptor, CXCR4, in periodontal tissue and to investigate the properties of CXCL12 and CXCR4 expression by human gingival fibroblasts (HGF). RT-PCR analysis revealed that CXCL12 and CXCR4 mRNA were expressed in both normal gingival tissues and periodontal diseased tissues. Immunohistochemistry disclosed that CXCL12 was expressed and CXCR4 positive cells were found in both normal and periodontal diseased gingival tissues. Our in vitro experiments elucidated that HGF constitutively produced CXCL12, and the levels were enhanced by stimulation with tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), transforming growth factor-beta (TGF-beta), regulated upon activation normal T cell expressed and secreted (RANTES) and macrophage inflammatory protein 3(alpha) (MIP-3(alpha)). On the other hand, heat killed Porphyromonas gingivalis (P. gingivalis) and P. gingivalis LPS reduced the CXCL12 production by HGF. Flow cytometry analysis clarified that CXCR4 was highly expressed on HGF, and CXCR4 expression was abrogated by TNF-alpha, IFN-gamma and P. gingivalis LPS. Moreover, CXCL12 induced vascular endothelial growth factor (VEGF) production by HGF. Our results demonstrated that CXCL12 might be related to CXCR4+ cells infiltration and angiogenesis both in normal periodontal tissues and periodontal diseased tissue. P. gingivalis, a known periodontal pathogen, inhibits the production of CXCL12 and the expression of CXCR4 by HGF. This fact means that P. gingivalis may inhibit CXCR4+ cells infiltration and neovascularization in periodontal tissue and escape from the immune response.


Coating of a surface with 2-methacryloyloxyethyl phosphorylcholine (MPC) co-polymer significantly reduces retention of human pathogenic microorganisms

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.


Intermedilysin Is Essential for the Invasion of Hepatoma HepG2 Cells by Streptococcus intermedius

Streptococcus intermedius causes endogenous infections leading to abscesses. This species produces intermedilysin (ILY), a human-specific cytolysin. Because of the significant correlation between higher ILY production levels by S. intermedius and deep-seated abscesses, we constructed ily knockout mutant UNS38 B3 and complementation strain UNS38 B3R1 in order to investigate the role of ILY in deep-seated infections. Strain UNS38 reduced the viability of human liver cell line HepG2 at infection but not of rat liver cell line BRL3A. Isogenic mutant strain UNS38 B3 was not cytotoxic in either cell line. Quantification of S. intermedius revealed that in infected HepG2 cells UNS38 but not UNS38 B3 increased intracellularly concomitantly with increasing cell damage. This difference between UNS38 and UNS38 B3 was not observed with UNS38 B3R1. Invasion and proliferation in BRL3A cells was not observed. Masking UNS38 or UNS38 B3R1 with ILY antibody drastically decreased adherence and invasion of HepG2. Moreover, coating strain UNS38 B3 with ILY partially restored adherence to HepG2 but without subsequent bacterial growth. At 1 day post-infection, many intact UNS38 were detected in the damaged phagosomes of HepG2 with bacterial proliferation observed in the cytoplasm of dead HepG2 after an additional 2 day incubation. These results indicate that surface-bound ILY on S. intermedius is an important factor for invasion of human cells by this bacterium and that secretion of ILY within host cells is essential for subsequent host cell death. These data strongly implicate ILY as an important factor in the pathogenesis of abscesses in vivo by this streptococcus.


Role for rpoS gene of Pseudomonas aeruginosa in antibiotic tolerance

The alternative sigma factor, RpoS has been described as a central regulator of many stationary phase-inducible genes and a master stress-response regulator under various stress conditions. We constructed an rpoS mutant in Pseudomonas aeruginosa and investigated the role of rpoS gene in antibiotic tolerance. The survival of the rpoS mutant cells in stationary phase was approximately 70 times lower when compared with that of the parental strain at 37 degrees C for 2 h after the addition of biapenem. For imipenem, the survival was approximately 40 times lower. Heat stress promoted an increase in the survival of the parental strain to biapenem, but the same was not found to be the case for the rpoS mutant. Our results indicate that rpoS gene is involved in tolerance to antibiotics in P. aeruginosa during the stationary phase and heat stress. However, under osmotic stress, tolerance to biapenem was not dependent on the rpoS gene.


The human-specific action of intermedilysin, a homolog of streptolysin O, is dictated by domain 4 of the protein.

ヒト特異的細胞溶解毒素インターメディリシン(ILY)はストレプトリシンO(SLO)と同様にコレステロール(CHL)結合性細胞溶解毒素遺伝子ファミリーに属するが，CHLとの直接結合能を持たず，そのドメイン4でヒト細胞と結合することを明らかとした．またILYとSLO等の同属毒素間で多数のキメラ毒素を作成してその活性を調べた結果，SLO等ではCHLを結合する11アミノ酸領域は直接ヒト細胞の認識には関わらず，ここを除くドメイン4のC末端側56アミノ酸残基中にヒト細胞の受容体を認識する部位があることを示した．


Novel Pseudomonas aeruginosa gene that suppresses tolerance to carbapenems

A biapenem-tolerant mutant of Pseudomonas aeruginosa was isolated by Tn1737KH insertion. The survival of the mutant 3 h after the addition of biapenem was about 1000 times greater than that of the wild type. The mutant was also tolerant to other biapenems, such as imipenem, panipenem, and meropenem.


Correlation between resistance of Pseudomonas aeruginosa to quaternary ammonium compounds and expression of outer membrane protein OprR

論文題目:緑膿菌の細胞膜タンパク質OprRの発現と第四アンモニウム塩に対する耐性化の相関 第四アンモニウム塩系消毒剤に耐性化した緑膿菌において顕著に発現量が増加する約25.6kDaの蛋白質を2次元電気泳動で分離し，そのN末端アミノ酸配列の解析によって，それが外膜蛋白質のOprRであることを明らかとした．またその耐性化株でOprRのノックアウト株を作成したところ，第四アンモニウム塩系消毒剤に対する感受性が野生株と同程度まで上昇することから，OprRがこの消毒薬耐性に重要な役割を果たしていることを示した．


Effect of varidase (streptodornase) on biofilm formed by Pseudomonas aeruginosa

The biofilm of Pseudomonas aeruginosa could be removed by Varidase (streptodornase) that was used as defibrinating drug. After cultivating the biofilm on a silicone chip in a modified alginate-producing medium in vitro, it was treated with Varidase or DNase I. In both treatments, the biofilm was removed depending on the concentration of the reagents. Varidase was apparently effective under the condition that the biofilm was exposed to more than 625 U/ml (a quarter of the concentration of the medical use) for 3 h, twice, at 37 degrees C. The result of this experiment indicates that (a) the DNases, DNase I and Varidase, were effective in destroying the biofilm of P. aeruginosa in vitro, and (b) in a clinical field, Varidase could be useful for P. aeruginosa focal infection, such as urinary tract infection, by removing the biofilm.


PBIIでフッ素イオン注入した口腔内装置用材料の細菌付着抑制効果に関する研究

PBIIでフッ素イオン注入した口腔内装置用材料の細菌付着抑制効果に関する研究


Rapid identification of Streptococcus intermedius by PCR with the ily gene as a species marker gene

ヒト特異的細胞溶解毒素インターメディリシン(ILY)遺伝子はアンギノーサス群連鎖球菌ではS. intermedius(SI)のみに特異的に存在したことから，ily遺伝子が本菌種にだけ分布する可能性が考えられた．そこでPCRやサザンドットハイブリダイゼーション法を用いて，連鎖球菌属や近縁菌種におけるily遺伝子の分布を調べ，ily遺伝子がSI特異的な菌種マーカーになり得ることを確認し，それを指標として本菌種による感染症診断に応用可能なPCR検出システムを構築した．


Identification of Helicobacter Pylori in Denture Plaque of Hospitalized Aged Patients


Suppression of gingival inflammation induced by Porphyromonas gingivalis in rats by leupeptin

In this study, we developed a procedure to produce gingivitis in rats by inoculation of Porphyromonas gingivalis and studied the contribution of the bacterial cysteine proteinases, Arg-gingipain (Rgp) and Lys-gingipain (Kgp), to the pathology in the gingiva. To adhere the bacterium to periodontal tissues, a cotton thread was inserted between the first and second molar of right maxillary sites of rats. Rats in group A were administered with vehicle alone after bacterial (strain W83) inoculation. In group B, the bacteria were inoculated in combination with leupeptin, a potent inhibitor of Rgp and Kgp, and then leupeptin alone was administered the week after. Rats in group C were administered leupeptin for 6 weeks after bacteria inoculation. All left maxillary gingiva in three groups showed no inflammatory changes. Right maxillary gingiva of group A showed most of the clinical landmarks of gingivitis. Leupeptin exhibited only a little inhibitory effect on this gingivitis in group B, whereas it had a strong inhibitory effect on the inflammation in group C. These results suggest that P. gingivalis-induced gingivitis is attributable to Rgp and Kgp and that leupeptin is more effective in the late phase than the early stage of gingivitis.


Susceptibilities of Oral and Nasal Isolates of Streptococcus mitis and Streptococcus oralis to Macrolides and PCR Detection of Resistance Genes

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 Tn917. Four strains had mefE genes that coded erythromycin efflux ability.


Effect of antibiotics on CD15s-related antigen expressed on the Streptococcus anginosus group


Effect of varidase (streptokinase) on biofilm formed by Staphylococcus aureus

Staphylococcus aureus forms a fibrin-rich biofilm in the presence of plasma which is highly resistant to attack by the human immune system and to chemotherapy. Varidase, composed mainly of streptokinase, is used for hydrolyzing clots. In this study, we attempted to destroy the biofilm of S. aureus with Varidase and to apply this drug in the treatment of staphylococcal infections. Four clinical isolates were used in the experiments. These organisms formed a several-millimeter-thick biofilm on type IV collagen coated coverslips in trypticase soy broth containing 50% human plasma. The biofilm was composed of bacterial cell which adhered to fibrillar fibers and of sediment derived from plasma. 10,000 U/ml of Varidase, the dose which is used clinically, removed the sediment and reduced the number of live bacteria in biofilms to less than 20% of control. 200 U/ml of Varidase was also effective against biofilms of the organisms. An equal combination of Varidase and ofloxacin had an additive effect on the bacteria. The results of this study demonstrate that Varidase is highly effective in destroying biofilms of S. aureus in vitro and suggest that this drug would be useful for treating staphylococcal infections.


Three-dimensional structural analysis of histone-like protein from streptococci

レンサ球菌のヒストン様蛋白質の構造解析


Study on oral health care and general health of elderly (in Japanese)

高齢者における口腔ケアと健康に関する基礎的研究


Relationships between activity of daily living, and oral cavity care and the number of oral cavity microorganisms in patients with cerebrovascular diseases

We examined the relationships among the activity of daily living (ADL), oral cavity care, and the number of oral cavity microorganisms in 40 patients with cerebrovascular diseases (CVD). The CVD patients were classified into 4 groups, I, II, III and IV based on their ADL and the method used for oral cavity care. The ADL was highest in group I and lowest in group III. Only the patients of only group III could not eat by themselves and were receiving naso-esophageal feeding. Oral cavity care was performed by the patients themselves in groups I and IV, but was performed by caregivers in groups II and III. The group IV patients had no teeth, but could eat by themselves using full dentures. The numbers of microorganisms in the pharyngeal swabs from the 4 groups were measured and expressed as colony-forming units (cfu). The numbers of both Staphylococci spp. and Candida spp. were significantly higher in group III than in the other groups. Moreover, Pseudomonas aeruginosa was isolated only from patients of group III (in about 66%). The oral cavity care by caregivers was almost the same in groups II and III, but the numbers of oral cavity microorganisms were significantly higher in group III than in group II. These results indicated that microorganisms grow more easily in the oral cavities of CVD patients with low ADL compared with CVD patients with higher ADL, and that eating is thought to be important for the prevention of an increase of microorganisms in the oral cavity.

The Journal of Medical Investigation : JMI


Fosfomycin reduces CD15s-related antigen expression of Streptococcus pyogenes

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 (1R,2S-1,2-epoxypropyl phosphonic acid), its enantiomer (1S,2R-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.


Effect of adherence on antimicrobial susceptivility of Pseudomonas aeruginosa, Serratia marcescens and {Proteus mirabilis}

A simple method was used for testing the antibiotic susceptibility of adherent bacteria to plastic surfaces. Pseudomonas aeruginosa, Serratia marcescens, and Proteus mirabilis cells adhering to the bottom of a plastic tissue culture plate were incubated in serially diluted antibiotic solutions. After 24-h incubation the solutions were removed and a fresh medium without antibiotics was added to each well. The viability of the cells was judged by their growth after a further 24-h incubation. In our assay system, we employed a short incubation time (1-h) involving adherence of bacteria to a surface for the purpose of minimizing the effect of the glycocalyx on antibiotic activity. Even if the bacteria did not form a biofilm, the minimal bactericidal concentrations for adherent bacteria (MBCADs) markedly elevated. The MBCADs of ofloxacin well correlated with the bacteriological eradication by ofloxacin treatment for urinary tract infections (UTIs) associated with indwelling urinary catheters, whereas the minimal inhibitory concentrations did not show a correlation. Kinetic studies showed that adherent Pseudomonas aeruginosa had a 2 h-lag time before logarithmically growing when these bacteria were incubated in Mueller-Hinton broth without antibiotics. The tolerance demonstrated by adherent cells is likely to play a role in the difficulties encountered in the antimicrobial chemotherapy of biofilm infections. Moreover, our assay system was considered to be useful in the therapeutic selection of antibiotics for these infections.


In vitro adherence of Streptococcus constellatus to dense hydroxyapatite and titanium

Bacterial adherence to dense hydroxyapatite (HA) and titanium (Ti) rods was examined in vitro using Streptococcus constellatus ATCC27832. Streptoccus constellatus is associated with dental abscess and was isolated from the peri-implantitis. The dense HA was etched with 5% hydrofluoric acid for 0, 10, 20, 30, 40, and 50 s Adherence to the untreated dense HA was twice as high as the adherence to the Ti. The adherence to saliva-treated Ti was 13% higher than that to untreated Ti. The adherence was increased by acid-treatment of the dense HA, following a sigmoid curve from 20 to 50 s of treatment time. The dense HA has the capacity to receive more bacterial adherence than Ti and furthermore, the etched dense HA becomes more adherent.


A long-term survey of methicillin-resistant Staphylococcus aureus in the oral cavity of children

Methicillin-resistant Staphylococcus aureus (MRSA), an indigenous bacteria in healthy people, often causes nosocomial infections. If the host human becomes compromised, MRSA can cause a serious infection. The long-term colonization of MRSA increases this risk. The purpose of this study was to demonstrate the incidence of S. aureus and MRSA colonization in the oral cavities of healthy children, and to examine the stability of identical strains of MRSA over a long-term period. Fourteen children were examined in two stages (first stage: 1987-88, second stage: 1992-93). Five of the 14 children were negative for S. aureus in both stages, seven children were positive in both stages and two children were positive in only the second stage. The children who were colonized with S. aureus in the first stage always harbored the bacteria in the second stage. Of the seven children that were positive for S. aureus in both stages, three persisted in carrying MRSA. We compared two MRSA strains isolated from the same children in both stages by coagulase typing, antibiogram typing and DNA fingerprinting. In two children, the strains showed the same coagulase types, similar antibiograms and similar DNA fragment profiles. These data strongly suggest that identical strains of MRSA persisted in the oral cavities for more than five years, and that the oral cavity can serve as a reservoir for MRSA with the potential to cause nosocomial infections.


Pharyngeal bacteria and professional oral health care in elderly people

プロフェッショナル·オーラル·ヘルス·ケアを受けた高齢者の咽頭細菌数の変動


Treatment of gingivitis in elderly patients in a nursing home

特別養護老人ホーム入所者における歯肉炎の改善に関する研究


Rapid bone resorption adjacent to hydroxyapatite-coated implants

This paper describes rapid bone resorption in the peri-implantitis of HA implants based on both our clinical observations of and histological research on extracted dense hydroxyapatite (HA) implants. The surfaces of extracted HA implants were rough, although they were smooth at fixture placement. Plaque formed on the necks of the implants, whereas little plaque was seen on the bottoms. The plaque consisted of cocci and rods, including filamentous bacteria. Few spirochetes were observed. Although surrounding bone was formed rapidly around the HA implant, bone thickness gradually decreased compared with the titanium implant. These facts suggest that the rigid biointegration of HA with the thin surrounding bone--that is, the overstressing of the bone--causes rapid bone resorption rather than plaque accumulation on HA.


High incidence of Staphylococcus aureus] from dentures and tongues of maxillary resection patients


老人病院入院患者の口腔状態とデンチャープラークの細菌構成について --- 第1報 予備調査結果

The Journal of the Japan Prosthodontic Society


電解水の義歯洗浄効果 --- -細菌学的検討-

電解水の義歯洗浄効果 --- -細菌学的検討-


Isolation and characterization of teichoic acid-like substance as an adhesin of Staphylococcus aureus to HeLa cells

A cell wall component that bound to HeLa cells (HeLa cell-binding CWC) was isolated from a clinical isolate of Staphylococcus aureus. The HeLa cell-binding CWC was resistant to heat (100 C, 1 hr) and proteases, did not stain with Coomassie Brilliant Blue R-250 on SDS-PAGE but stained as a broad band with antiserum against the strain on Western blots. These data suggest that the HeLa cell-binding CWC is not a protein, and may be teichoic acid. Purified teichoic acid bound to HeLa cells, whereas fractions without teichoic acid did not. In Western blots, HeLa cell-binding CWC appeared as a broad band of less than 35 kDa, similar to that of purified teichoic acid. These data suggest that the HeLa cell-binding CWC obtained in this study is teichoic acid. Teichoic acid inhibited S. aureus adherence to HeLa cells and bound to the cells time and dose dependently, in a saturable and reversible manner, and therefore appears to be an adhesin of S. aureus to HeLa cells.


Highly expressed human sialyl Lewisx antigen on cell surface of Streptococcus gallolyticus


アパタイトインプラントの細菌付着に関する研究 --- -摘出インプラントの観察と細菌付着試験-


In vitro antibacterial activity of pazufloxacin against bacteria adhering to solid phase


An investigation of gram-negative tannin-protein complex degrading bacteria in fecal flora of various mammals

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.

The Journal of Veterinary Medical Science


Identification of endo-β-N-acetylglucosaminidase and N-acetylmuramyl-L-alanine amidase as cluster-dispersing enzymes in Staphylococcus aureus

Two proteins which are capable of dispersing cell clusters of Staphylococcus aureus have been purified from a S. aureus FDA209P culture supernatant. Both of them were found to have bacteriolytic activity. From the elution profile of column chromatography and Western blot (immunoblot) analysis, one of them was identified as a 51-kDa endo-beta-N-acetylglucosaminidase (GL). The other was a 62-kDa protein on the basis of sodium dodecyl sulfate gel electrophoresis. Analysis of the peptidoglycan fragments following treatment with the 62-kDa protein indicated that this protein is an N-acetylmuramyl-L-alanine amidase (AM). In vitro studies of cluster dispersion activities using S. aureus mutant strains Lyt66 or S. aureus Wood46 grown as clusters demonstrated that these two enzymes act synergistically to disperse clusters into single cells. Antiserum against the 51-kDa GL cross-reacted with the 62-kDa AM, and S. aureus FDA209P grown in the presence of anti-51-kDa-GL immunoglobulin G induced giant clusters. Clusters induced by anti-51-kDa GL and by Cibacron blue F3G-A were dispersed by coincubation with the 51-kDa GL and the 62-kDa AM. Western blot analysis demonstrated that the 51-kDa GL and the 62-kDa AM were missing in culture supernatants of S. aureus Lyt66, Wood46, and RUSAL2 (Tn551 autolysin-defective mutant), which grow in clusters. These results strongly suggest that the 51-kDa GL and 62-kDa AM are involved in cell separation of daughter cells after cell division.


Cross-reactivity between human sialyl Lewisx oligosaccharide and common causative oral bacteria of infective endocarditis

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.

FEMS Immunology and Medical Microbiology


In vitro activity of tetracyclines, macrolides, quinolones, clindamycin and metronidazole against periodontopathic bacteria

We re-evaluated several antibiotics including newer ones, for their in vitro killing activity, as well as their inhibitory activity, against clinical isolates of periodontopathic bacteria. Tetracyclines were active against Porphyromonas gingivalis, and were highly active against Prevotella intermedia, but demonstrated only a low killing activity against Actinobacillus actinomycetemcomitans. Rokitamycin, a new macrolide, and clindamycin were highly active against P. gingivalis and P. intermedia, but showed very weak killing activity against A. actinomycetemcomitans. Quinolones demonstrated excellent bactericidal activity against A. actinomycetemcomitans, and good inhibitory and bactericidal activity against P. gingivalis and P. intermedia. Metronidazole had an activity almost equivalent to quinolones against P. gingivalis and P. intermedia; but it was the least active against A. actinomycetemcomitans.


Mirobiological features of gingivitis in pubertal children

The subgingival microflora of 42 pubertal children (aged 12-15 years) and 18 young adults (aged 21-25 years) was investigated by anaerobic culture and phase contrast microscopy. Motile rods, spirochetes and Prevotella intermedia were elevated proportionately in pubertal children with gingivitis (median GI > or = 1); however, no statistically significant differences in enumerated organisms on selective media were observed between pubertal children and young adults. These organisms were positively correlated with the index of bleeding on probing and the gingival index of pubertal children. Pubertal children were divided into 3 distinct subject clusters according to the similarity of subgingival microbial features, and the subjects who were then selected for the gingivitis group were distributed into clusters 1 and 2. The proportions of motile rods, P. intermedia and Eikenella corrodens were significantly higher in cluster 2 than in cluster 1. These results suggest that these 2 species and motile rods which differentiated features of clusters, are useful for screening of high-risk subjects for worsening of inflammation.


Isolation and characterization Staphylococcus aureus mutans which form altered cell clusters

Staphylococcus aureus FDA 209P produces two extracellular bacteriolytic enzymes, 51-kDa endo-beta-N-acetylglucosaminidase (GL) and 62-kDa N-acetylmuramyl-L-alanine amidase (AM), both of which can disperse cell clusters. To characterize the physiological roles of these enzymes in vivo, mutants with altered autolysin activity were isolated, and their degree of cluster formation in broth culture was assessed. Bacteriolytic activities of GL and AM, produced and secreted from these mutants into the culture fluid and detected with activity gels, coincided well with the degree of cluster formation of the mutants. The mutants with little or no enzyme activity grew in clusters, whereas those with high activity grew as well-separated cocci, suggesting that these enzymes are involved in cell separation of S. aureus in vivo.


In vitro antibacterial activity of biapenem against adherent bactera

固層に付着した細菌に対するbiapenemのIn vitro 抗菌力


緑膿菌の抗菌薬抵抗性に対する新規化合物の作用機序

緑膿菌の抗菌薬抵抗性に対する新規化合物の作用機序


新規化合物によるレボフロキサシン及びトブラマイシン殺菌作用の増強効果

新規化合物によるレボフロキサシン及びトブラマイシン殺菌作用の増強効果


Streptococcus intermedius biofilm componentsの糖鎖結合能と炎症反応誘導能に関する解析


新規化合物によるビアペネム殺菌作用の増強効果について

新規化合物によるビアペネム殺菌作用の増強効果について


クラリスロマイシンによる緑膿菌c-di-GMPの誘導

クラリスロマイシンによる緑膿菌c-di-GMPの誘導

第50回緑膿菌感染症研究会講演記録 /緑膿菌感染症研究会事務局編

第50回緑膿菌感染症研究会講演記録 /緑膿菌感染症研究会事務局編


水溶性MPCポリマーの口腔細菌付着抑制効果

水溶性MPCポリマーの口腔細菌付着抑制効果

口腔細菌によるbiofilm，すなわちdental plaqueの形成にはいくつかのkey stepがあり，dental plaque形成の抑制のためにはそれらのkey stepに効果的に働く方法を選択する必要が有る．我々は，超親水性表面を得ることができる2-methacryloloxethyl phosphorylcholine(MPC)co-polymerを用いてプラスチック表面をコートした界面は微生物の付着抑制に関与することを報告してきた．本実験では5%水溶性MPCポリマーで処理したHydroxyapatite(HY)プレートと培養歯肉上皮細胞(RT-7)へのStreptococcus mutansの付着状況を顕微鏡下にて観察した．その結果，5%水溶性MPCポリマー処理でHYプレート及び培養歯肉上皮細胞上へのS.mutansの付着は抑制されることが示された．以上よりHYプレートや培養歯肉上皮細胞に吸着した水溶性MPCポリマーは，プラスチックへのコーティングと同様にS.mutansの付着抑制効果を発揮することが示唆された．


緑膿菌臨床分離株におけるRSCVsの分離とその性状について

緑膿菌臨床分離株におけるRSCVsの分離とその性状について

緑膿菌感染症において，寒天培地上で小さな皺上のコロニー性状を示す，rugose small colony variants(RSCVs)の存在が報告されている．このRSCVsは，細胞内のセカンドメッセンジャーであるc-di-GMPの細胞内濃度が上昇しているためであると考えられている．今回我々は，徳島大学病院で分離された緑膿菌30株についてRSCVsの有無を検討したところ，2株が小さな皺状のコロニー性状を示し，バイオフィルム形性能が高く，運動性も低下していたことから，RSCVsであると考えられる．そのうちの1株は，c-di-GMP合成に関与する遺伝子オペロンの1つであるwspF遺伝子の変異の結果である事が明らかとなった．もう1株では，wspF遺伝子には大きな変異は見られなかった．これらの結果から，臨床分離株30株中2株のRSCVsが確認されたものの，その原因となる遺伝子は異なっていることが明らかとなった．


The effect of culture supernatants of intestinal flora bacteria on the biofilm formation

付着状態における緑膿菌の抗菌薬抵抗性とQuorum sensing機構について


付着緑膿菌の抗菌薬抵抗性とQuorum sensing機構について

付着緑膿菌の抗菌薬抵抗性とQuorum sensing機構について


デンタルデバイス感染症 --- デンチャープラークから高齢者の健康を考える

デンタルデバイス感染症 --- デンチャープラークから高齢者の健康を考える


デンチャープラークの最前線

デンチャープラークの最前線


要介護高齢者における咽頭微生物叢について --- 特に義歯装着の有無による影響

要介護高齢者における咽頭微生物叢について --- 特に義歯装着の有無による影響


含嗽の咽頭ぬぐい液細菌数に対する影響

含嗽の咽頭ぬぐい液細菌数に対する影響

Bulletin of School of Medical Sciences, the University of Tokushima

徳島大学医療技術短期大学部紀要


The effect of Triton X-100 on the in-vitro susceptivility of mehicillin-resistant Staphylococcus aureus ot oxacillin

The effect of the non-ionic detergent, Triton X-100, on the in-vitro activity of oxacillin against methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) strains of Staphylococcus aureus was investigated. In the presence of Triton X-100, the MICs of oxacillin for both MRSA and MSSA isolates were reduced; this enhancing effect was particularly marked for the MRSA strains. Triton X-100 therefore counteracted the resistance to methicillin encoded by mecA. In the presence of oxacillin at subinhibitory concentrations, Triton X-100 induced the bacteriolysis of MRSA and potentiated the autolysis of these organisms. However, the detergent had no effect on the bacteriolytic enzyme profile or the susceptibility of the bacterial cell wall to bacteriolytic enzymes, nor did it promote the binding of oxacillin to the penicillin-binding protein (PBP) 2A. On the other hand, it stimulated the release from the bacteria of acylated lipoteichoic acid (LTA), a putative endogenous regulator of autolysins. Autolytic enzyme-deficient MRSA mutants were equally as sensitive as the parent strain to the effect of Triton X-100 on susceptibility to oxacillin. These results indicate that the enhanced in-vitro activity of oxacillin against MRSA in the presence of Triton X-100 cannot be accounted for simply by the induction of bacteriolysis following activation of autolytic enzymes by the detergent-stimulated release of LTA.

The Journal of Antimicrobial Chemotherapy


細菌学的に見た細菌カウンタの有用性

細菌学的に見た細菌カウンタの有用性


Biological Roles of Extracellular DNA in Bacterial Infection

Asean Plus and Tokushima Joint International Conference, Program and Proceeding Book


High prevalence of Pseudomonas aeruginosa from oropharyngeal biofilm in patients with cerebrovascular infarction and dysphagia.


透析液中の細菌検出における遺伝学的検討

透析液中の細菌検出における遺伝学的検討

腎と透析65巻別冊 ハイパフォーマンスメンブレン'08

腎と透析65巻別冊 ハイパフォーマンスメンブレン'08


Intermedilysin plays a key role in the invasion of human cells by Streptococcus intermedius

Streptococci New Insights into an Old Enemy: International Congress Series