Factors influencing compliance with infection control practice in Japanese dentists.

BACKGROUND: In recent years, dentists have more opportunity of treating patients infected with blood-borne pathogens. Although compliance with infection control practice (ICP) in dental practice is required, it is not still sufficiently spread in Japan. OBJECTIVE: To identify factors associated with compliance with ICPs in the population of Japanese dentists. METHODS: In a questionnaire-based cross-sectional study in 2009, 2134 dentists in Aichi prefecture, Japan, were surveyed. They were asked for their demographic characteristics, willingness to treat HIV/AIDS patients, and knowledge about universal/standard precautions and ICP. RESULTS: Many ICP items had significant association with age, specialty for oral surgery, number of patients treated per day, willingness to treat HIV/AIDS patients and knowledge about the universal/standard precautions. In logistic regression model, knowledge about the precautions had significant associations with all ICP items. Among participants with disadvantageous characteristic group for ICP (ie, age ≥50 years, being general dentist, and treating ≤35 patients/day), knowledge about the universal/standard precautions had greater impact on exchanging handpiece for each patient and installing extra-oral vacuum in those with age of ≥50 years than in those who visited ≤35 patient per day. CONCLUSION: Knowledge about the meaning of universal/standard precautions is the most significant predictor of compliance with ICPs among Japanese dentists.

Effects of short-chain fatty acids on Actinomyces naeslundii biofilm formation.

Actinomyces naeslundii is an early colonizer and has important roles in the development of the oral biofilm. Short-chain fatty acids (SCFA) are secreted extracellularly as a product of metabolism by gram-negative anaerobes, e.g. Porphyromonas gingivalis and Fusobacterium nucleatum; and the SCFA may affect biofilm development with interaction between A. naeslundii and gram-negative bacteria. Our aim was to investigate the effects of SCFA on biofilm formation by A. naeslundii and to determine the mechanism. We used the biofilm formation assay in 96-well microtiter plates in tryptic soy broth without dextrose and with 0.25% sucrose using safranin stain of the biofilm monitoring 492 nm absorbance. To determine the mechanism by SCFA, the production of chaperones and stress-response proteins (GrpE and GroEL) in biofilm formation was examined using Western blot fluorescence activity with GrpE and GroEL antibodies. Adding butyric acid (6.25 mm) 0, 6 and 10 h after beginning culture significantly increased biofilm formation by A. naeslundii, and upregulation was observed at 16 h. Upregulation was also observed using appropriate concentrations of other SCFA. In the upregulated biofilm, production of GrpE and GroEL was higher where membrane-damaged or dead cells were also observed. The upregulated biofilm was significantly reduced by addition of anti-GroEL antibody. The data suggest biofilm formation by A. naeslundii was upregulated dependent on the production of stress proteins, and addition of SCFA increased membrane-damaged or dead cells. Production of GroEL may physically play an important role in biofilm development.

Inhibiting effects of Streptococcus salivarius on competence-stimulating peptide-dependent biofilm formation by Streptococcus mutans.

INTRODUCTION: The effects of Streptococcus salivarius on the competence-stimulating peptide (CSP)-dependent biofilm formation by Streptococcus mutans were investigated. METHODS: Biofilms were grown on 96-well microtiter plates coated with salivary components in tryptic soy broth without dextrose supplemented with 0.25% sucrose. Biofilm formations were stained using safranin and quantification of stained biofilms was performed by measuring absorbance at 492 nm. RESULTS: S. mutans formed substantial biofilms, whereas biofilms of S. salivarius were formed poorly in the medium conditions used. Furthermore, in combination cultures, S. salivarius strongly inhibited biofilm formation when cultured with S. mutans. This inhibition occurred in the early phase of biofilm formation and was dependent on inactivation of the CSP of S. mutans, which is associated with competence, biofilm formation, and antimicrobial activity of the bacterium, and is induced by expression of the comC gene. Comparisons between the S. mutans clinical strains FSC-3 and FSC-3DeltaglrA in separate dual-species cultures with S. salivarius indicated that the presence of the bacitracin transport ATP-binding protein gene glrA caused susceptibility to inhibition of S. mutans biofilm formation by S. salivarius, and was also associated with the regulation of CSP production by com gene-dependent quorum sensing systems. CONCLUSION: It is considered that regulation of CSP by glrA in S. mutans and CSP inactivation by S. salivarius are important functions for cell-to-cell communication between biofilm bacteria and oral streptococci such as S. salivarius. Our results provide useful information for understanding the ecosystem of oral streptococcal biofilms, as well as the competition between and coexistence of multiple species in the oral cavity.

Role of lysine in interaction between surface protein peptides of Streptococcus gordonii and agglutinin peptide.

INTRODUCTION: Streptococcus gordonii interacts with the salivary pellicle on the tooth surface and plays an important role in dental biofilm formation. Reports show that the analog Ssp peptide (A11K; alanine to lysine at position 11 in the arranged sequence, (1)DYQAKLAAYQAEL(13)) of SspA and SspB of S. gordonii increased binding to the salivary agglutinin (gp-340/DMBT1) peptide (scavenger receptor cysteine-rich domain 2: SRCRP2). To determine the role of lysine in the binding of the Ssp(A11K) peptide to SRCRP2, we investigated whether an additional substitution by lysine influenced the binding of Ssp(A11K) peptide to SRCRP2 using a BIAcore biosensor assay. METHODS: Six analogs of the Ssp peptide with positive charges in surface positions on the structure were synthesized using substitution at various positions. RESULTS: The binding activity of analog Ssp(A4K-A11K) peptide was significantly higher than the other Ssp analogs. The binding activity rose under low ionic strength conditions. The distance between positively charged amino acids in the Ssp(A4K-A11K) peptide between 4K and 11K was 1.24 +/- 0.02 nm and was close to the distance (1.19 +/- 0.00 nm) between Q and E, presenting a negative charged area, on SRCRP2 using chemical computing graphic analysis. The molecular angle connecting 1D-11K-4K in the Ssp(A4K-A11K) peptide secondary structure was smaller than the other peptide angles (1D-11K-XK). The Ssp(A4K-A11K) peptide showed higher inhibiting activity for Streptococcus mutans binding to saliva-coated hydroxyapatite than the (A11K) peptide. CONCLUSION: The positioning of lysine is important for binding between Ssp peptide and SRCRP2, and the inhibiting effect on S. mutans binding to the tooth surface.

Association between commensal bacteria and opportunistic pathogens in the dental plaque of elderly individuals.

Opportunistic infections in the oral cavity of the elderly may increase the incidence of systemic disease. The objective of this study was to investigate the differences in the oral bacterial flora between dependent elderly (inpatients) and independent elderly (community-dwelling residents). After multiple variables were taken into account, inpatients had significantly lower detection rates than community-dwelling residents for alpha-streptococci (p < 0.001) and Neisseria (p 0.004), and higher detection rates for Pseudomonas aeruginosa (p 0.024), methicillin-resistant Staphylococcus aureus (MRSA) (p 0.011) and Actinomyces spp. (p 0.005). Among inpatients, the requirement for a high degree of care was related negatively to detection of alpha-streptococci, but was related significantly to detection of P. aeruginosa (p 0.018) or MRSA (p 0.004). Tube-fed inpatients had a significantly lower detection rate for alpha-streptococci (p 0.041) and a higher detection rate for P. aeruginosa (p 0.004) than those who did not require tube feeding. Inpatients with a history of antibiotic use had a significantly lower detection rate for alpha-streptococci (p 0.049) and a higher detection rate for MRSA (p 0.007) than those without a history of antibiotic use. The detection rates for P. aeruginosa or MRSA in inpatients without alpha-streptococci were higher than in inpatients with alpha-streptococci after controlling for age and gender (P. aeruginosa, p 0.006; MRSA, p 0.001). Overall, detection of alpha-streptococci had an inverse correlation with the detection of P. aeruginosa and MRSA in the oral cavity and is likely to be an indicator of pathogenic bacterial infection.

Post-operative infection by pathogenic micro-organisms in the oral cavity of patients with prostatic carcinoma.

The aim of this study was to analyse the change in the oral cavity microflora of 14 patients who had undergone a radical prostatectomy for prostatic carcinoma. The detection of micro-organisms in the oral cavity was compared before and after the surgical procedure. Post-operative infection, defined as those patients who had increased Candida species counts and/or pathogenic bacteria only at the post-operative examination, was observed in 10 patients. Six patients showed increased Candida species counts at the post-operative examination compared with the pre-operative examination. In five patients, pathogenic bacterial species were detected at the post-operative examination but not at the pre-operative examination. One patient had detectable pathogenic bacterial species only at the post-operative examination along with increased Candida species counts. Our findings suggest that pre-operative oral hygiene to remove bacterial and Candida species from patients who are scheduled for surgical procedures is important for satisfactory clinical outcomes.

Human T-cell responses to oral streptococci in human PBMC-NOD/SCID mice.

We investigated cellular and humoral immune responses to oral biofilm bacteria, including Streptococcus mutans, Streptococcus anginosus, Streptococcus sobrinus, and Streptococcus sanguinis, in NOD/SCID mice immunized with human peripheral blood mononuclear cells (hu-PBMC-NOD/SCID mice) to explore the pathogenicity of each of those organisms in dental and oral inflammatory diseases. hu-PBMC-NOD/SCID mice were immunized by intraperitoneal injections with the whole cells of the streptococci once a week for 3 weeks. FACS analyses were used to determine the percentages of various hu-T cell types, as well as intracellular cytokine production of interleukin-4 and interferon-gamma. Serum IgG and IgM antibody levels in response to the streptococci were also determined by enzyme-linked immunosorbent assay. S. anginosus induced a significant amount of the proinflammatory cytokine interferon-gamma in CD4(+) and CD8(+) T cells in comparison with the other streptococci. However, there was no significant differences between the streptococci in interleukin-4 production by CD4(+) and CD8(+) T cells after inoculation. Further, S. mutans significantly induced human anti-S. mutans IgG, IgG(1), IgG(2), and IgM antibodies in comparison with the other organisms. In conclusion, S. anginosus up-regulated Th1 and Tc1 cells, and S. mutans led to increasing levels of their antibodies, which was associated with the induction of Th2 cells. These results may contribute to a better understanding of human lymphocyte interactions to biofilm bacteria, along with their impact on dental and mucosal inflammatory diseases, as well as endocarditis.

Telomere size and telomerase activity in Epstein-Barr virus (EBV)-positive and EBV-negative Burkitt's lymphoma cell lines.

The telomere repeat lengths of BL cell lines were quantified by measuring terminal restriction fragment (TRF). Epstein-Barr virus (EBV)-positive Namalwa, Raji, and EB-3 cell lines have long telomeres, i.e. TRFs 10-19 kbp, whereas the Daudi cell line, producing a transformation-defective EBV mutant, has TRFs approximately 2.2 kbp. EBV-negative BJAB and DG75 cell lines have short TRFs 3.9-5.4 kbp, shorter than the approximately 12 kbp TRFs in PBLs. Telomerase activities of these BL cell lines are similar. TRFs of non-BL lymphoma cell lines are 2.3-5.5 kbp. Fluorescent in situ hybridization (FISH) studies of these cell lines showed remarkable heterogeneity of telomere size in chromosomes in the same BL cell. These results suggest that EBV-positive and EBV-negative BL cell lines have experienced various telomere dynamics.

Role of peptide antigen for induction of inhibitory antibodies to Streptococcus mutans in human oral cavity.

The alanine-rich repeating region (A-region) in the surface protein antigen (PAc) of Streptococcus mutans has received much attention as an antigenic component for vaccines against dental caries. The PAc (residue 361-386) peptide in the A-region possesses a multiple binding motif (L- -V-K- -A) to various HLA-DR molecules and a B-cell core epitope (- Y- - -L- -Y- - - -) that recognizes the inhibiting antibody to S. mutans. In the present study, we investigated the immunogenicity of the PAc (361-386) peptide in humans and regulators of induction of the anti-PAc (361-386) peptide IgA antibody (aPPA) in saliva. The PAc (361-386) peptide was confirmed as an ideal peptide antigen for induction of the inhibiting antibody to S. mutans in 151 healthy human subjects (36.6 +/- 12.6 years old) by quantitative analyses of oral bacteria and ELISA, as the aPPA titre in human saliva decreased significantly in an age-dependent manner. Homozygous DRB1*0405 and 1502, and heterozygous DRB1*0405/1502 showed a negative association with production of aPPA and tended to reduce the number of total streptococci in saliva. In contrast, the DRB1*1501 allele was significantly correlated with a high level of induction of the antibodies, and also tended to reduce lactobacilli and mutans streptococci. Further, peptide immunogenicity was confirmed in NOD-SCID mice grafted with human peripheral blood mononuclear cells. Our results indicate that the interplay between regulators such as age, DRB1 genotype, cytokines, and peptide immunogenicity may provide a potential means for developing a vaccine useful for the prevention of dental caries as well as their diagnosis.

Role of gene E2f1 in susceptibility to bacterial adherence of oral streptococci to tooth surfaces in mice.

Dental plaque is composed of a biofilm community of microorganisms on teeth that coats the oral cavity, including attaching to the teeth, and provides a protective reservoir for oral microbial pathogens, which are the primary cause of persistent and chronic infectious diseases. Oral streptococci are pioneering organisms that play an important role in biofilm formation on tooth surfaces as well as being primary causative agents of dental caries. The purpose of this study was to clarify the role of the E2f1 gene in susceptibility to dry mouth and bacterial adherence of oral streptococci to tooth surfaces in animal model experiments. A mutation of the E2f1 gene in mice is known to cause enhanced T-lymphocyte proliferation, leading to testicular atrophy, splenomegaly, salivary gland dysplasia, and other systemic and organ-specific autoimmunity. We found a decreased volume of saliva production and protein production rate, along with increased amylase activity, IgA concentration, and mucin 1 concentration in E2F-1(-/-) mice as compared with the control C57BL/6 mice. Further, we quantified the recolonization of oral streptococci in E2F-1(-/-) mice and found that a higher number of some oral streptococci were colonized on the teeth of these mice. In particular, following oral ingestion of 1% sucrose in water, the colonization of Streptococcus mutans increased in comparison with other streptococci. Our results suggest that the E2f1 gene may affect susceptibility for oral biofilm formation by streptococci in humans with dry mouth.

Systemic diseases in association with microbial species in oral biofilm from elderly requiring care.

BACKGROUND: The oral cavity is a reservoir for colonization and infection of systemic organs by pathogenic bacteria. It is understood that aging, tooth eruption, hormonal changes, active disease, oral hygiene, and other factors have an influence on biofilm formation and bacterial accumulation in the oral cavity. OBJECTIVE: To understand the influence of systemic health care on microfloral changes, we conducted epidemiological studies of nursing home residents in an attempt to elucidate the relationship between underlying systemic diseases and the isolation frequency of oral opportunistic pathogens. METHODS: The prevalence of bacteria and fungi causing pneumonia in association with oral biofilm bacteria were determined using detection culture plates. The influences of gender, age, denture-wearing status, number of teeth, and bedridden status in the patients residing in nursing homes were then analyzed. RESULTS: The isolation frequency rates of Candida albicans, Pseudomonadaceae, Staphylococcus spp., and some strains of Enterobacteriaceae in plaque samples, as well as C. albicans and Xanthomonas maltophilia in samples from the pharynx, were significantly higher in those requiring systemic care (mean age 83.9 years) than in those who did not require such care (mean 71.0 years). In particular, the frequencies of Pseudomonas spp., C. albicans, and Serratia marcescens in plaque were significantly higher in those who were bedridden. Furthermore, the isolation of Pseudomonas spp. and Klebsiella pneumoniae, and/or C. albicans in plaque was significantly associated with heart disease. CONCLUSION: The coexistence of Pseudomonas spp. and C. albicans in elderly with 10-19 teeth is a potential indicator of high risk for pneumonia and heart disease. Therefore, attention to oral hygiene and professional care for removing the indicators may diminish the occurrence of systemic disease in the elderly requiring systemic care.

Real-time interaction of oral streptococci with human salivary components.

Oral streptococci are present in large numbers in dental plaque and several types interact with the enamel salivary pellicle to form a biofilm on tooth surfaces. The respective affinity of individual streptococci for salivary components has an influence on the etiologic properties of oral biofilm in the development of dental caries. We studied real-time biospecific interactions between oral streptococci and salivary components utilizing biosensor technology to analyze surface plasmon resonance. Streptococcus sanguis and Streptococcus mutans showed significant responses for binding to salivary components, in comparison with other bacteria. Further, the association rates (4.1 x 10-11/bacterium) and dissociation rate (5.7 +/- 0.9 x 10-3 Second(s)-1) were higher for S. sanguis than for S. mutans (2.4 x 10-11 and 2.9 +/- 0.8 x 10-3) and Streptococcus mitis (1.3 x 10-11 and 3.5 +/- 1.3 x 10-3). However, the association equilibrium constants (8.2 S/bacterium) for S. mutans was 2 times higher in than S. mitis (3.8) and slightly higher than S. sanguis (7.2). These findings may provide useful information regarding the mechanism of early biofilm formation by streptococci on the tooth surface.

Identification of the peptide motifs that interact with HLA-DR8 (DRB1*0802) in Streptococcus mutans proteins.

A glucosyltransferase (GTF) and a surface protein antigen (PAc) of Streptococcus mutans have been suggested as possible components of an effective dental caries vaccine. To identify antigenic peptides in GTF and PAc that bind to MHC class II (HLA-DR8, DRB1*0802) molecules, we investigated binding activities to DR8 molecules of overlapping synthetic peptides at several sites in GTF and in the alanine-rich repeating region of PAc using an ELISA-inhibition competitive binding assay for the interaction between the HLA-DR molecule and the PAc (316-334) peptide. Six GTF peptides and 10 PAc peptides strongly bound to the HLA-DR8 molecule. In a homology analysis of the amino acid sequences of the six GTF peptides, two binding motifs were found in L/Y--Y/L-A/N and Y/L--N/G/E--Y-V/L/P. Moreover, a new binding motif in PAc was found in L--Y-A. It is suggested that these binding motifs could be useful in designing a dental caries vaccine in humans.

Streptococcus mutans binding to solid phase dextran mediated by the glucan-binding protein C.

Streptococcus mutans GbpC is a wall-anchored surface protein which is involved in dextran-dependent aggregation. The GbpC phenotype is observed only in cells grown under stress conditions. In order to detect the GbpC protein of S. mutans, we isolated the wall fraction following digestion of the cell wall of this organism by N-acetylmuramidase, and detected the GbpC protein from S. mutans cells by western analysis with anti-GbpC serum. Interestingly, S. mutans cells exhibiting the negative dextran(alpha-1,6 glucan)-dependent aggregation (ddag) phenotype expressed the protein and could bind to immobilized dextran.

Epstein-Barr virus nuclear antigen-1-dependent and -independent oriP-binding cellular proteins.

OBJECTIVE: Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA-1) and the replication origin, oriP, are essential for the replication and maintenance of latent EBV DNA in cells, but no enzymatic activity has been associated with EBNA-1 protein alone. In this study, we have searched for host cellular proteins that interact with EBNA-1 protein in various B cell lines latently infected with EBV, including a recently EBV growth-transformed cell line. METHODS: By using gel shift analysis, we investigated the interactions of an oligonucleotide containing a single EBNA-1 recognition site, derived from the family of repeats (FR) element of oriP, with protein from cell extracts. RESULTS: The FR oligonucleotide bound a (72-kD) cellular protein in the absence of EBNA-1 and without induction of the previously reported 'anti-EBNA-1 proteins'. The FR oligonucleotide formed complexes with additional proteins from EBNA-1-synthesizing cell lines; these complexes were abolished or supershifted by anti-EBNA-1 monoclonal antibodies. SDS-PAGE analyses of 35S-Met-labeled proteins that bound to a biotin- conjugated FR oligonucleotide, fractionated by a glycerol gradient centrifugation and affinity-purified with streptavidin, showed three major bands, a 72-kD protein, the FR binding of which seemed to be independent of EBNA-1, a 64-kD protein in both EBNA-1-transfected and latently EBV-infected cell lines, and a 45-kD protein in EBV-infected cell lines, which was most prominent in a recently EBV growth-transformed cell line. CONCLUSIONS: The FR element forms complexes with cellular proteins in the absence and presence of EBNA-1. These 72-, 64- and 45-kD cellular proteins might be involved in the function of the oriP and EBNA-1 system.

Identification of an antigenic epitope in Helicobacter pylori urease that induces neutralizing antibody production.

We previously reported a mouse monoclonal antibody (MAb), termed L2, specific for Helicobacter pylori urease strongly inhibited its enzymatic activity. Here, to gain insight into how this antibody affects urease activity, the epitope that was recognized by the antibody was determined. By screening a panel of overlapping synthetic peptides covering the entire sequence of the two subunits (UreA and UreB), we identified a stretch of UreB-derived 19 amino acid (aa) residues (UB-33; aa 321 to 339, CHHLDKSIKEDVQFADSRI) that was specifically recognized by the L2 antibody. Further sequential amino acid deletion of the 19-mer peptide from either end allowed us to determine the minimal epitope as 8 amino acid residues (F8; SIKEDVQF) for L2 reactivity. This epitope appears to lie exactly on a short sequence which formed a flap over the active site of urease, suggesting that binding of the L2 antibody sterically inhibits access of urea, the substrate of urease. Finally, immunization of rabbits with either the 19-mer peptide or the 8-mer minimal epitope resulted in generation of antiurease antibodies that were capable of inhibiting the enzymatic activity. Since urease is critical for virulence of H. pylori, antigenic peptides that induce production of antibodies to inhibit its enzymatic activity may potentially be a useful tool as a vaccine for prevention and treatment of H. pylori infection.

Inhibitory effects of MoAbs against a surface protein antigen in real-time adherence in vitro and recolonization in vivo of Streptococcus mutans.

A surface protein antigen (PAc) of Streptococcus mutans, particularly the A-region of the molecule, has been reported to interact with salivary components on the tooth surface. It might be a candidate antigen inducing the production of antibodies against the adherence of S. mutans to the tooth surface. We investigated the effects of monoclonal antibodies (MoAbs) obtained by immunization of synthetic PAc peptides that completely correspond to the amino acid sequence of part of the A-region. These MoAbs recognize several core B-cell epitopes in the sequence. Two (KH5 and SH2) of these antibodies reacted with both S. mutans and Streptococcus sobrinus, but not with Streptococcus sanguis, Streptococcus salivarius, Porphyromonas gingivalis or Lactobacillus casei. They clearly inhibited the real-time adherence of S. mutans to salivary components in a biosensor. KH5, which showed a real-time inhibition (71%), also significantly prevented the recolonization of S. mutans on the tooth surface in rats. These results suggested that the core B-cell epitope (-Y---L--Y----) recognized by KH5 was the essential sequence in the antigenic epitopes of PAc protein recognized specifically by the inhibitory antibody. Therefore, the amino acid residues were found to be important in the initial attachment of S. mutans to the tooth surface. These results provide for the mechanism of PAc molecule in the initial attachment of S. mutans on the tooth surface and more effective designs for the removal of S. mutans and S. sobrinus from the oral cavity.

Periodontitis and serum interleukin-6 levels in the elderly.

The elderly lose teeth as a result of dental caries and periodontitis caused by pathogenic oral bacteria. Periodontitis produces inflammatory cytokines due to the presence of lipopolysaccharides from oral gram-negative bacteria. Although the number of circulating inflammatory cytokines is related to the severity of the periodontitis, it is unclear whether the concentrations also correlate with periodontitis in the elderly. We investigated the relationship between periodontitis status and the concentrations of serum interleukin-6 (IL-6) in the serum from 276 subjects of 70- and 80-year-olds. Of the 276 subjects, 227 (82%) were dentate, 149 (54%) were found to be positive for serum IL-6, and 29 (13%) of the dentate subjects had severe periodontitis. However, there were no significant differences between the severity of periodontitis or the number of teeth and the mean serum IL-6 concentrations. These results provided no evidence to support an association between circulating IL-6 and periodontitis in the elderly.