Effects of xylitol wipes on cariogenic bacteria and caries in young children.

The aim of the study was to investigate the efficacy of the use of xylitol-containing tooth-wipes in preventing dental caries in young children. In a double-blinded randomized controlled clinical trial, 44 mothers with active caries and their 6- to 35-month-old children were randomized to xylitol-wipe or placebo-wipe groups. The children's caries scores were recorded at baseline and 1 year. Salivary levels of mutans streptococci and lactobacilli were enumerated at baseline, 3, 6, and 12 months. Data were analyzed by intent-to-treat modeling with imputation for caries lesions and a linear mixed-effect model for bacterial levels. Significantly fewer children in the xylitol-wipe group had new caries lesions at 1 year compared with those in the placebo-wipe group (P < 0.05). No significant differences between the two groups were observed in levels of mutans streptococci and lactobacilli at all time-points. Daily xylitol-wipe application significantly reduced the caries incidence in young children as compared with wipes without xylitol, suggesting that the use of xylitol wipes may be a useful adjunct for caries control in infants (Clinicaltrials.gov registration number CT01468727).

A randomized clinical trial of anticaries therapies targeted according to risk assessment (caries management by risk assessment).

This randomized parallel group clinical trial assessed whether combined antibacterial and fluoride therapy benefits the balance between caries pathological and protective factors. Eligible, enrolled adults (n = 231), with 1-7 baseline cavitated teeth, attending a dental school clinic were randomly assigned to a control or intervention group. Salivary mutans streptococci (MS), lactobacilli (LB), fluoride (F) level, and resulting caries risk status (low or high) assays were determined at baseline and every 6 months. After baseline, all cavitated teeth were restored. An examiner masked to group conducted caries exams at baseline and 2 years after completing restorations. The intervention group used fluoride dentifrice (1,100 ppm F as NaF), 0.12% chlorhexidine gluconate rinse based upon bacterial challenge (MS and LB), and 0.05% NaF rinse based upon salivary F. For the primary outcome, mean caries increment, no statistically significant difference was observed (24% difference between control and intervention groups, p = 0.101). However, the supplemental adjusted zero-inflated Poisson caries increment (change in DMFS) model showed the intervention group had a statistically significantly 24% lower mean than the control group (p = 0.020). Overall, caries risk reduced significantly in intervention versus control over 2 years (baseline adjusted generalized linear mixed models odds ratio, aOR = 3.45; 95% CI: 1.67, 7.13). Change in MS bacterial challenge differed significantly between groups (aOR = 6.70; 95% CI: 2.96, 15.13) but not for LB or F. Targeted antibacterial and fluoride therapy based on salivary microbial and fluoride levels favorably altered the balance between pathological and protective caries risk factors.

Fluoride varnish efficacy in preventing early childhood caries.

To determine the efficacy of fluoride varnish (5% NaF, Duraphat, Colgate) added to caregiver counseling to prevent early childhood caries, we conducted a two-year randomized, dental-examiner-masked clinical trial. Initially, 376 caries-free children, from low-income Chinese or Hispanic San Francisco families, were enrolled (mean age +/- standard deviation, 1.8 +/- 0.6 yrs). All families received counseling, and children were randomized to the following groups: no fluoride varnish, fluoride varnish once/year, or fluoride varnish twice/year. An unexpected protocol deviation resulted in some children receiving less active fluoride varnish than assigned. Intent-to-treat analyses showed a fluoride varnish protective effect in caries incidence, p < 0.01. Analyzing the number of actual, active fluoride varnish applications received resulted in a dose-response effect, p < 0.01. Caries incidence was higher for 'counseling only' vs. 'counseling + fluoride varnish assigned once/year' (OR = 2.20, 95% CI 1.19-4.08) and 'twice/year' (OR = 3.77, 95% CI 1.88-7.58). No related adverse events were reported. Fluoride varnish added to caregiver counseling is efficacious in reducing early childhood caries incidence.

Clinical trial of a plant-derived antibody on recolonization of mutans streptococci.

OBJECTIVE: This double-blinded, placebo-controlled clinical trial tested the safety and efficacy of a topical secretory IgA antibody manufactured in tobacco plants (plantibody) in preventing recolonization of mutans streptococci (MS) in human plaque as measured by whole stimulated saliva samples. METHODS: Following a 9-day antimicrobial treatment with chlorhexidine (CHX), 56 eligible adults (enrollment salivary MS > or = 10(4) CFU/ml; no current caries) were randomized equally to a group receiving 0, 2, 4, or 6 topical applications of plantibody followed by 6, 4, 2, or 0 applications of placebo, respectively, over a 3-week period. RESULTS: Among the 54 subjects who completed the trial, the CHX regimen eliminated salivary MS in 69%. After 6 months, there were no significant differences in MS levels by number of applications, relative to placebo (p > 0.43). No adverse effects were observed. CONCLUSION: Plantibody is safe but not effective at the frequency, concentration, and number of applications used in this study.

Separate oligosaccharide determinants mediate interactions of the low-molecular-weight salivary mucin with neutrophils and bacteria.

The low-molecular-weight human salivary mucin (MG2) coats oral surfaces, where it is in a prime location for governing cell adhesion. Since oligosaccharides form many of the interactive facets on mucin molecules, we examined MG2 glycosylation as it relates to the molecule's adhesive functions. Our previous study of MG2 oligosaccharide structures showed that the termini predominantly carry T, sialyl-T, Lewisx (Lex), sialyl Lex (sLex), lactosamine, and sialyl lactosamine determinants [Prakobphol, A., et al. (1998) Biochemistry 37, 4916-4927]. In addition, we showed that sLex determinants confer L-selectin ligand activity to this molecule. Here we studied adhesive interactions between MG2 and cells that traffic in the oral cavity: neutrophils and bacteria. Under flow conditions, neutrophils tethered to MG2-coated surfaces at forces between 1.25 and 2 dyn/cm2, i.e., comparable to the shear stress generated at the tooth surface by salivary flow ( approximately 0.8 dyn/cm2). MG2 was also found in association with neutrophils isolated from the oral cavity, evidence that the cells interact with this mucin in vivo. Since MG2 serves as an adhesion receptor for bacteria, the MG2 saccharides that serve this function were also identified. Seven of 18 oral bacteria strains that were tested adhered to MG2. Importantly, six of these seven strains adhered via T antigen, sialyl-T antigen, and/or lactosamine sequences. No adherence to Lex and sLex epitopes was detected in all the strains that were tested. Together, these results suggest that distinct subsets of MG2 saccharides function as ligands for neutrophil L-selectin and receptors for bacterial adhesion, a finding with interesting implications for both oral health and mucin function.

Cloning and regulated expression of the Candida albicans phospholipase B (PLB1) gene.

Degenerate oligonucleotides (derived from conserved regions of PLB1 genes from S. cerevisiae and other fungi) were used to amplify PLB1 homolog fragments from C. albicans and C. tropicalis by using the polymerase chain reaction. The C. albicans PLB1 fragment was then used as a probe to clone the full-length gene and to monitor PLB1 mRNA expression. The C. albicans PLB1 gene consists of a 1815-bp open reading frame encoding a putative protein of 605 amino acids. It contains the highly conserved Gly-X-Ser-X-Gly catalytic motif, found in all lipolytic enzymes, and exhibits significant homology with other fungal PLB1 gene products (approximately 63% similarity, approximately 45% identity). Blastospores and pseudohyphae expressed higher levels of PLB1 mRNA than germ-tube-forming cells. TUP1, a general transcriptional repressor, may regulate PLB1 expression in C. albicans, since PLB1 expression was the highest in tup1 delta mutants and did not vary in response to environmental stimuli. Together, these results suggest that expression of the C. albicans PLB1 gene is regulated as a function of morphogenic transition.

Isolation and characterization of transposon-induced mutants of Porphyromonas gingivalis deficient in fimbriation.

Fimbriae are considered to be an important virulence factor of Porphyromonas gingivalis. In order to identify genes essential for fimbriation, other than fimA which encodes the major subunit protein of fimbriae, transposon mutagenesis and immunological screening techniques were used to isolate fimbria-deficient mutants. R751::*Omega4, a suicide vector that carries Tn4351, was transferred from Escherichia coli to P. gingivalis by conjugation. Twenty-two independent fimbria-deficient mutants were identified among the resulting transformants. Southern hybridization analysis with pBlue 4351, a transposon-specific probe, and R751 indicated that 45% of the mutants resulted from single transposon insertions and that the remaining 55% of the mutants resulted from cointegration of R751 sequences. Southern hybridization analysis with pUCBg12.1, a probe for the fimA region, indicated that nine of the mutants contained insertions within the 2.5 kb SacI DNA fragment of P. gingivalis that contains fimA, ORF1 (which encodes a 15 kDa protein), and the C-terminal portion of ORF5 (which encodes a 63 kDa protein). Polymerase chain reaction (PCR) analysis and further Southern hybridization analysis indicated that the insertion site(s) for all nine of these mutants was within the fimA gene. Southern hybridization analysis also indicated that the remaining thirteen mutants contained insertions somewhere outside the 10 kb fimA region. Analysis by pulsed field gel electrophoresis (PFGE) revealed that insertions for most of the thirteen mutants mapped to a 300 kb NotI fragment and are located at least approximately 200 kb away from fimA. These results identify genetic loci other than fimA, that are required for fimbriation of P. gingivalis. Future cloning and characterization of these genetic loci should be straightforward since they are now marked by antibiotic resistance genes carried by the transposon.

Palmitoyl carnitine, a lysophospholipase-transacylase inhibitor, prevents Candida adherence in vitro.

Candida adherence is poorly understood. The results of this study indicate that interactions of Candida with the lyso-forms of phospholipids may be one important attachment mechanism. C. tropicalis and C. albicans adhered to purified lysophospholipids immobilized on microtiter wells, as well as to a human laryngeal epidermoid carcinoma (HEp-2) cell line. Adherence to both lysophospholipids and HEp-2 cells was significantly reduced by palmitoyl carnitine, a lysophospholipase-transacylase inhibitor. Over time there was a positive correlation between Candida adherence and its transacylase activity. The data suggest that palmitoyl carnitine interferes with Candida adherence to lysophospholipids and the HEp-2 cell line by blocking the interaction between the Candida-associated transacylase enzyme receptor site and its lysophospholipid substrate ligand.

Identification of Porphyromonas gingivalis prefimbrilin possessing a long leader peptide: possible involvement of trypsin-like protease in fimbrilin maturation.

Fimbriae of Porphyromonas gingivalis have been shown to be important as one of the virulence factors for colonization on mucosal surfaces. The gene (fimA) encoding the fimbrial subunit (fimbrilin) was overexpressed in Escherichia coli by using a bacteriophage T7 promoter-polymerase expression vector system. Analysis of the resulting fimA gene product revealed that the prefimbrilin had a 46 amino acid leader peptide. This extremely long leader peptide was cleaved from the prefimbrilin by treatment with trypsin or P. gingivalis extracts containing trypsin-like protease activity, resulting in production of a mature fimbrilin. We also found that some transposon-induced trypsin-like protease deficient mutants of P. gingivalis exhibited deficiency in fimbriation and that one of the mutants accumulated a fimbrilin precursor possessing a 25 amino acid leader peptide in the cell. The presence of an extremely long leader peptide and the requirement for a leader peptidase with a substrate specificity similar to that of P. gingivalis trypsin-like protease for fimbrilin maturation indicate that P. gingivalis fimbrilin is a novel type that is different from fimbrilins of type I and IV families.

Transposon-induced pigment-deficient mutants of Porphyromonas gingivalis.

Trypsin-like protease activity, hemagglutination activity, and accumulation of heme-containing compounds (black pigment) are considered to be virulence factors of Porphyromonas gingivalis. Transposon-mutagenesis was used for the first time to isolate pigment-deficient mutants. These mutants exhibited simultaneous deficiency in trypsin-like protease activity and hemagglutination activity. Two major membrane-associated proteins, observed by SDS-PAGE with the parent strain, were essentially absent from the mutant strains. Immunoblot analysis indicated that these two proteins correspond to putative hemagglutinin and hemagglutinin/protease products of P. gingivalis. Each mutant contained only one transposon insertion, thus the pleiotropic phenotype resulted from single site-specific mutations. The results indicate that trypsin-like protease activity is required for accumulation of protoheme from hemoglobin by P. gingivalis and that genetic and/or physiological linkage exists between trypsin-like protease activity and hemagglutination activity.

Transposition of Tn4351 in Porphyromonas gingivalis.

Genetic analysis of Porphyromonas gingivalis, an obligately anaerobic gram-negative bacterium, has been hindered by the apparent lack of naturally occurring bacteriophages, transposable elements, and plasmids. Plasmid R751::*omega 4 has previously been used as a suicide vector to demonstrate transposition of Tn4351 in B. uniformis. The erythromycin resistance gene on Tn4351 functions in Bacteroides and Porphyromonas. Erythromycin-resistant transconjugants were obtained at a mean frequency of 1.6 x 10(-7) from matings between Escherichia coli HB101 containing R751::*omega 4 and P. gingivalis 33277. Southern blot hybridization analysis indicated that about half of the erythromycin-resistant P. gingivalis transconjugants contained simple insertions of Tn4351 and half contained both Tn4351 and R751 sequences. The presence of R751 sequences in some P. gingivalis transconjugants most likely occurred from Tn4351-mediated cointegration of R751, since we were unable to detect autonomous plasmid in these P. gingivalis transconjugants. The P. gingivalis-Tn4351 DNA junction fragments from different transconjugants varied in size. These results are consistent with transposition of Tn4351 and with insertion at several different locations in the P. gingivalis chromosome. Tn4351 may be useful as a mutagen to isolate well-defined mutants of P. gingivalis.

Correlation of haemagglutination activity with trypsin-like protease activity of Porphyromonas gingivalis.

Porphyromonas gingivalis is a Gram-negative anaerobic bacterium associated with various forms of periodontal disease. Several characteristics of P. gingivalis are thought to contribute to its pathogenicity; these include haemagglutination and trypsin-like protease activity. Previous studies suggest an association between haemagglutination and trypsin-like protease activity of P. gingivalis. To investigate this, two complementary quantitative experimental approaches were taken. Five independent mutants of P. gingivalis deficient in trypsin-like protease activity were shown to exhibit reduced haemagglutination activity. In addition, enhancers (cysteine and dithiothreitol) and inhibitors (N-ethylmaleimide, N-p-tosyl-L-lysine-chloromethyl ketone, and phenylmethylsulphonyl fluoride) of trypsin-like protease activity were shown, respectively, to significantly enhance and inhibit haemagglutination activity of washed, wild-type P. gingivalis cells (p less than 0.05, paired t-test). Statistical analysis indicated a strong correlation between haemagglutination and trypsin-like protease activity (r = 0.85, p less than 0.001, Spearman rank correlation). The effect of the protease enhancers and inhibitors on haemagglutination activity was specific for P. gingivalis, as they did not significantly change the haemagglutination activity of Fusobacterium nucleatum. These results suggest that the proteolytic site of the trypsin-like protease participates in haemagglutination activity of P. gingivalis.

Adherence of oral streptococci to salivary glycoproteins.

We used an overlay method to study the ability of human salivary glycoproteins to serve as receptors for several strains of streptococci that colonize the oral cavity. Parotid and submandibular-sublingual salivas were collected as ductal secretions, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. The resulting blots were overlaid with [35S]methionine-labeled bacteria, and salivary components to which the bacteria bound were detected by autoradiography. Potential glycoprotein receptors were identified for 8 of the 16 strains tested. In three cases (Streptococcus sanguis 72-40 and 804 and Streptococcus sobrinus OMZ176), highly specific interactions with a single salivary component were detected. Removal of sialic acid residues from the low-molecular-weight salivary mucin prevented adherence of one of these strains (S. sanguis 72-40), suggesting that this saccharide either mediates binding or is a critical component of the receptor site. In the remaining five strains (Streptococcus gordonii G9B and 10558, S. sanguis 10556, and Streptococcus oralis 10557 and 72-41), interactions with multiple salivary components, including the low-molecular-weight salivary mucin, highly glycosylated proline-rich glycoproteins, and alpha-amylase, were detected. These results suggest that some oral streptococci can bind specifically to certain of the salivary glycoproteins. The interactions identified may play an important role in governing bacterial adherence and clearance within the oral cavity.

Identification of genomic clonal types of Actinobacillus actinomycetemcomitans by restriction endonuclease analysis.

To evaluate its utility in discriminating different strains, restriction endonuclease analysis was applied to 12 strains of Actinobacillus actinomycetemcomitans (3 serotype a, 5 serotype b, and 4 serotype c strains). DNA isolated from each strain was digested by 12 different restriction endonucleases, and the electrophoretic banding patterns of the resulting DNA fragments were compared. The DNA fragment patterns produced by SalI, XhoI, and XbaI for the 12 A. actinomycetemcomitans strains were simple (less than 30 bands) and allowed us to recognize easily 10 distinct genomic clonal types. The three serotype a strains exhibited distinctly different clonal types from one another, the five serotype b strains exhibited an additional four distinct clonal types, and the four serotype c strains showed another three different clonal types. The other endonucleases tested were less useful in typing A. actinomycetemcomitans. We conclude that restriction endonuclease analysis is a powerful tool for typing and discerning genetic heterogeneity and homogeneity among A. actinomycetemcomitans strains. It should, therefore, be very useful for epidemiologic studies.

Association of proteases of Porphyromonas (Bacteroides) gingivalis with its adhesion to Actinomyces viscosus.

P. gingivalis adheres to A. viscosus on mineral surfaces mimicking teeth. To study whether P. gingivalis proteases contribute to its binding, mutants of P. gingivalis deficient in proteases were compared with their parent strain and a P. gingivalis-type strain for their adherence to A. viscosus on saliva-coated hydroxyapatite by manipulating a radio-isotope binding assay. Adherence of P. gingivalis 2561 to A. viscosus was studied by tests of the effects of incubation temperature and known inhibitors or promoters of proteases. Controls were handled by the assay run in PBS buffer at 22 degrees C. Two mutants deficient in trypsin-like protease were found to be deficient in adherence (% attachment relative to control: 3.2 +/- 0.1% and 11.2 +/- 0.4%), while a collagenase-deficient mutant had an adherence score (51.6 +/- 8.4) closer to that of the parent strain (75.6 +/- 7.2%). Heating P. gingivalis at 70 degrees C decreased its subsequent adherence at 22 degrees C by 80%. Adherence decreased by 60% when the assay was run at 4 degrees C, but increased by 70% at 37 degrees C. Reducing agents (dithiothreitol, cysteine, and mercaptoethanol) enhanced P. gingivalis adherence by 50 to 60%. Protease inhibitors (BZMD, SBTI, TPCK, TLCK, CMPS, PMSF) decreased adherence by 10 to 50%. Also, Hg2+ and Zn2+ decreased adherence by 30 to 50%, and arginine decreased it by 50%. Most of these effects on P. gingivalis adherence were statistically significant (p less than 0.05). Analysis of these data suggests that P. gingivalis proteases may contribute to the cohesion of P. gingivalis and A. viscosus.

Electrophoretic heterogeneity of lipopolysaccharides of Actinobacillus actinomycetemcomitans.

Electrophoretic banding patterns of lipopolysaccharides (LPS), as observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), have proved to be useful in studies relating LPS structure to virulence and as epidemiological markers. In this report, LPS of actinobacillus actinomycetemcomitans from outer membrane fractions and hot phenol-water extracts were analyzed by SDS-PAGE and LPS-specific silver-staining techniques. Both intra- and inter-strain heterogeneity of A. actinomycetemcomitans LPS was observed. Twelve strains of A. actinomycetemcomitans, representative of the three described serotypes, were assigned to LPS subtypes based on the relative mobility of their most rapidly migrating LPS band. All three serotype a strains (29523, aB75, and GA3), two (29524 and SAC11A) of five serotype b strains, and two (aB67 and SAC5A) of four serotype c strains were assigned to LPS subtype I. The three remaining serotype b strains (29522, Y4, and JP2) were assigned to LPS subtype II, and the remaining two serotype c strains (SAC6A and SAC12A) were assigned to LPS subtype III. LPS subtyping may serve as an adjunct or alternative to serotyping in epidemiological studies.

An in vitro model to study bacterial invasion of periodontal tissues.

In periodontal disease, the abilities of bacteria to adhere to and degrade in vivo basement membranes should be considered as two of the rate-limiting steps for the potential active or passive invasion of gingival connective tissues. To study these mechanisms in greater detail, we used the PF HR-9 basement-membrane-like matrix to establish an in vitro model of bacterial invasion and degradation. Three gram-negative anaerobic periodontopathic organisms, Bacteroides gingivalis, Fusobacterium nucleatum, and Actinobacillus actinomycetemcomitans, bound in considerably higher numbers to the HR-9 matrix than did 6 strains of gram-positive facultative organisms typically associated with periodontal health. In a further experiment with B. gingivalis, the organism rapidly degraded Type IV collagen, the major macromolecular component constituting the HR-9 matrix. Streptococcus mitis, the nonperiodontopathic bacterium tested, did not degrade this model matrix. This study provides evidence that B. gingivalis, a periodontopathic bacterium, is able to adhere to and degrade basement membranes, whereas nonperiodontopathic organisms appear not to share in these abilities.

Attachment of oral bacteria to a basement-membrane-like matrix and to purified matrix proteins.

The purpose of this study was to investigate the adherence of oral bacteria to an in vitro basement-membrane-like matrix and to selected individual macromolecular constituents of this matrix. Radiolabeled bacteria were incubated with basement-membrane-like matrices isolated from PF HR-9 cells. Bacteroides gingivalis 33277, Fusobacterium nucleatum FN-2, and Actinobacillus actinomycetemcomitans GA3(A) bound to the matrix in the range of 44 to 70%, considerably higher than the ranges of A. actinomycetemcomitans GA3(NA) and SUNY AB67 (range, 20 to 25%). The attachment of selected strains of gram-positive bacteria such as Streptococcus and Actinomyces spp. was much less frequent (range, 6 to 25%). Competitive inhibition studies demonstrated that preincubating the bacteria with fibronectin significantly decreased the binding of B. gingivalis by 51% but increased the binding of other gram-negative and gram-positive organisms tested. Similarly, preincubating the matrices with antifibronectin antibodies decreased the binding of B. gingivalis by 31%, whereas the other bacteria tested were either unaffected or binding was increased. The adherence of bacteria to purified basement membrane proteins was also investigated. Strain and species differences were seen in binding, but no clear relationship emerged between binding to an intact matrix and binding to isolated matrix proteins. The results of this study suggest that some gram-negative oral bacteria commonly associated with periodontal disease, such as B. gingivalis, A. actinomycetemcomitans, and F. nucleatum, bound in high numbers to basement-membrane-like matrices in vitro. On the other hand, the gram-positive strains tested bound in much fewer numbers. The results suggest that further studies with this in vitro model may aid in understanding the mechanisms by which oral bacteria adhere to basement membranes.