bluuebunny/crossref_metadata_2025 huggingface dataset:
Survival in transport media of Actinobacillus actinomycetemcomitans, Porphymmonas gingivalis and Prevotella intermedia in human subgingival samples
Authors: van Steenbergen T. J. M. , Petit M. D. A. , Tijhof C. J. , van Winkelhoff A. J. , van der Velden U. , de Graaff J.
Published: December 2007

The purpose of this study was to evaluate the survival in 3 transport media of 3 suspected periodontal pathogens, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia. Subgingival samples were taken from 10 patients with severe periodontitis, all harboring at least two of the above‐mentioned species. The material was dispersed and aliquots were added to vials containing reduced transport fluid, reduced transport fluid containing 10% Fildes extract, or viability‐maintaining microbioslatic medium, anaerobically prepared (VMGA III). Viable counts were determined after 1, 2, 4, 24 and 48 h of storage at 4°C or at room temperature. The results showed that, for up to 4 h of storage, no significant differences existed for all parameters tested. A large increase of the total viable counts was found in VMGA III at room temperature after 24 and 48 h. This was due to an outgrowth of mainly streptococci. Incubation at 4°C yielded often a significantly higher recovery compared to room temperature. After storage at room temperature, the tested bacteria were below detection level in some samples.
Participation of an arginyl residue of insulin chain B in the inhibition of hemagglutination by Porphyromonas gingivalis
Authors: Hayashi H. , Morioka M. , Ichimiya S. , Yamato K. , Hinode D. , Nagata A. , Nakamura R.
Published: December 2007

Insulin chain B, containing each one arginyl and lysyl residue in its peptide chain, inhibited hemagglutination by Porphyromonas gingivalis. To determine the further inhibitory profile, chain B was digested into 4 fragments by protease, which was contained in the preparation of hemagglutinin from P. gingivalis. Identification of each fragment by the amino acid analysis revealed that the chain was cleaved at the carboxyl site of arginyl and/or lysyl residues, but one fragment contained citrulline instead of arginine at its carboxyl terminal. This citrulline might have originated from arginine by an arginine deaminase‐like enzyme of P. gingivalis. Only one fragment that contained the arginyl residue exhibited inhibitory activity on hemagglutination, but it was considerably weakened compared with that of the intact chain B. The difference in the inhibitory activity seemed to depend on the position of an arginyl residue in the peptide; this was also confirmed using several derivatives of bradykinin. The present result suggests that the internal arginyl residue in a peptide chain may be critical for the inhibition of the hemagglutination by P. gingivalis.
Branched‐chain amino acid transport in Streptococcus mutans Ingbritt
Authors: Dashper S. G. , Reynolds E. C.
Published: December 2007

Leucine transport in glucose‐energized cells of Streptococcus mutans exhibited Michaelis‐Menten‐type kinetics at low extracellular concentrations, with a Kt, of 15.3 μM and a Vmax of 6.1 nmol/mg dry weight/min. At high extracellular leucine concentrations, the transmembrane diffusion of leucine was not saturable, indicating that passive diffusion becomes a significant mechanism of leucine transmembrane movement under these conditions. The proton motive force (PMF) was measured in glucose‐energized cells of S. mutans and was found to have a maximum value of 126 mV at an extracellular pH (pH0) of 5.0; this decreased to 45 mV at pH0 8.0. The intracellular accumulation of leucine was significantly correlated with the magnitude of the PMF. The addition of excess isoleucine or valine caused a marked decrease in the leucine transport rate. Maximal rates of leucine transport occurred at pH0 6.0, and the rate of leucine transport was independent of the growth medium. The results suggest that there is a PMF‐driven, branched‐chain amino acid carrier in S. mutans with a proton:substrate stoichiometry of 1.
The in vitro Iysozyme susceptibility of Candida albicans cultured in carbohydratesupplemented media
Authors: Samaranayake Y. H. , MacFarlane T. W. , Aitchison T. C. , Samaranayake L. P.
Published: December 2007

The in vitro lysozyme susceptibility of three oral isolates of Candida albicans cultured in carbohydrate‐supplemented media was studied. Lysozyme was shown to have a dose‐ and time‐dependent killing effect on C. albicans isolates. Fungieidal activity persisted to varying degrees when yeast isolates were cultured in a variety of carbohydrates (glucose, galascrose. sucrose, maltose. xylitol and laelose) before exposure to 20 μg/ml lysozyme. Sucrose and galactose grown yeasts exhibited increased resistance to iysozyne conipared with (in decreasing order) those grown in glucose, maltose, xylnol or laelose. Further, the C albicans isolates tested demonstrated strain variations in their susceptibility to lysozyme. These results suggesl that dietary carbohydrate may play a role in modulating the yeast cell populations in the oral —– by altering the fungal susceptibility to salivary lysozyme.
Sensitization of periodontopathogenic bacteria to killing by light from a low‐power laser
Authors: Wilson M. , Dobson J. , Sarkar S.
Published: December 2007

Cultures of Porphyromonas gingivalis, Fusobacterium nucleatum and Actinobacillus actinomycetemcomitans were treated with a range of photosensitizers and then exposed to light from a 7.3 m W helium/neon laser for up to 80 s. Toluidine blue O (25 μg/ml) and methylene blue (25 μg/ml) were effective lethal photosensitizers of all 3 target organisms, enabling substantial light dose‐related reductions in viable counts. Dihaematoporphyrin ester and aluminium disulphonated phthalocyanine were lethal photosensitizers only of P. gingivalis. In the absence of a photosensitizer, exposure to laser light had no significant effect on the viability of the cultures. If such low doses of light (22 J/cm2) are effective at killing bacteria in vivo, the technique may be useful as a means of eliminating periodontopathogenic bacteria from diseased sites.
In vitro adherence of rat lymphocytes to salivary gland epithelia
Authors: O'Sullivan N. L. , Montgomery P. C.
Published: December 2007

Glandular mucosal tissues contain lymphocyte populations that contribute to expression of IgA antibodies in external secretions. Interaction of circulating lymphocytes with glandular structures may regulate lymphocyte accumulation. An in vitro assay was used to investigate adhesive interactions between lymphocytes and salivary gland tissues. Thoracic duct lymphocytes (TDL) bound to the serous acinar epithelia of parotid salivary glands and to the mucous tubulo‐acinar epithelium of submandibular salivary glands. Lymph node cells and splenocytes adhered to these tissues in lesser numbers and thymocytes bound in negligible numbers. TDL adherence was an active process, being time‐ and cell dose‐dependent and requiring intact membrane as well as cytoskeletal and metabolic function. Calcium was required in each case and binding was mediated by a trypsinsensitive lymphocyte surface determinant. These findings suggest that the lymphocyte composition of salivary gland tissues is regulated by active lymphocyte interaction with the glandular epithelium.
Bactericidal action of tachyplesin I against oral streptococci
Authors: Masuda K. , Ohta M. , Ito M. , Ohsuka S. , Kaneda T. , Kato N.
Published: December 2007

Tachyplesin I, a polycationic antimicrobial peptide isolated from hemocytes of horseshoe crabs, kills bacteria by disrupting the membrane potential of the cytoplasmic membrane. The present study shows that, among 36 oral streptococcal strains, 12 of 21 Streptococcus sanguis, 3 Streptococcus mutans, 9 Streptococcus salivarius and 3 Streptococcus milleri strains were susceptible to tachyplesin I, whereas 9 S. sanguis strains were resistant. Interestingly, these resistant strains include the clinical isolates from both Kawasaki disease and Behget patients. According to the time‐kill study, tachyplesin I inhibited irreversibly the growth of S. sanguis. S. tuutans and S. salivarius strains within 20 min and an S. milleri strain within 20 min. Although it has been suggested that Escherichia coli cultured in rich media were more susceptible to tachyplesin I, the present results show thai only 3 S. milleri strains were more sensitized to tachyplesin I in a glucose‐supplemented medium, and other tested strains were not. Similarly, only 4 strains were more resistant to tachyplesin I in saline than these were in a rich medium.
Molecular detection of Bacteroides forsythus in human periodontitis
Authors: Lotufo R. F. M. , Flynn J. , Chen C. , Slots J.
Published: December 2007

The usefulness of a digoxigenin‐labeled genomic DNA probe for the detection of subgingival Bacteroides forsythus was examined. In addition, the arbitrarily primed polymerase chain reaction (AP‐PCR) was used to delineate the genetic diversity of B. forsythus periodontal isolates. The DNA probe detected 103B. forsvthus cells and yielded a strong signal at 104 cells. It reacted with B. forsythus ATCC 43037T and 44 clinical isolates and showed no detectable reactivity with 75 strains of 24 other oral microbial species. In comparison to culture, the DNA probe in a dot‐blot method demonstrated a sensitivity of 88.8% and a specificity of 38.4% (accuracy, 72.5%). By colony‐blotting on primary plates, a sensitivity of 98.1% and a specificity of 53.8% (accuracy, 82.5%i) were obtained. B. forsythus was detected in 449 (73.1%) of 614 periodontitis patients. The occurrence of the organism was closely associated with Porphyromonas gingivalis, both species being present in 54.8%. and absent in 22.2%, of 270 study samples. AP‐PCR identified 24 B. forsythus genotypes among 27 test strains. This study demonstrated the utility of a non‐radioactive genomic probe for direct detection of B. forsvthus in subgingival specimens. The species showed a considerable degree of genetic diversity. DNA analysis may help to determine the role of B. forsythus in periodontal disease and its mode of transmission among exposed individuals.
Effect of lactoferrin on interaction of Prevotella intermedia with plasma and subepithelial matrix proteins
Authors: Alugupalli K. R. , Kalfas S. , Edwardsson S. , Forsgren A. , Arnold R. R. , Naidu A. S.
Published: December 2007

A lactoferrin‐binding protein with an estimated molecular mass of 57 kDa was identified in the cell envelope of Prevotella intermedia by gel electrophoresis and Western‐blol analysis. Peroxidase‐labeled bovine lactoferrin and human lactoferrin showed similar specific binding to this protein. Whole cells of P. intermedia were also examined for interactions with 5 125I‐labeled plasma and subepithelial matrix proteins. A high degree of binding was found with fibronectin, collagen type I and type IV and laminin, whereas a moderate interaction was detected with fibrinogen. The ability of bovine lactoferrin to affect the interactions of the above proteins with P. intermedia was examined. In the presence of unlabeled bovine lactoferrin, a dose‐dependent inhibition of binding was observed with all 5 proteins tested. Unlabeled bovine lactoferrin also dissociated the bacterial complexes with these proteins. The complexes with laminin or collagen type I were more effectively dissociated than fibronectin or fibrinogen, whereas the interaction with collagen type IV was affected to a lesser extent. A strain‐dependent variation in the effect of bovine lactoferrin was observed. These data establish the presence of a specific lactoferrin‐binding protein in the cell envelope of P. intermedia. The ability of lactoferrin to inhibit the binding of some plasma and subepithelial matrix proteins to P. intermedia could be a protective mechanism against the establishment of this pathogen in the periodontal pocket.
Transposon Tn5 mutagenesis of Actinobacillus actinomycetemcomitans via conjugation
Authors: Kolodrubetz D. , Kraig E.
Published: December 2007

To facilitate molecular genetic studies on the virulence of the periodontopathogen Actinobacillus actinomycetemcomitans, we have extended the use of Tn5 transposon mutagenesis to this organism. A suicide plasmid was used to deliver a spectinomycin‐resistant Tn5 derivative [mini‐Tn5(Sp)] via conjugation from Escherichia coli to A. actinomycetemcomitans. Spectinomycin‐resistant exconjugants were found at a frequency of about 1 × 10‐7 per recipient cell. Southern blot analysis showed that the transposon had moved into the A. actinomycetemcomitans genome, and each exconjugant had a transposon at a single location. Importantly, the mini‐Tn5(Sp) appeared to transpose into relatively random sites: there were 32 different sites of integration among the 33 exconjugants examined by Southern blot analysis. Finally, the mini‐Tn5(Sp) was stably integrated, since all of the exconjugants were still spectinomycin‐resistant after 7 passages on non‐selective media. Thus, the mini‐Tn5(Sp) system is a useful tool for the mutagenesis of A. actinomycetemcomitans in studies of its virulence factors and their regulation.
Absence of Helicobacter pylori in subgingival samples determined by polymerase chain reaction
Authors: Asikainen S. , Chen C. , Slots J.
Published: December 2007

The polymerase chain reaction was used for the detection of Helicobacter pylori from subgingival plaque in 336 periodontitis patients. A pair of primers derived from the H. pylori urease gene A served to amplify a targeted 411‐bp fragment of genomic DNA. This technique permitted the detection of as few as 60 H. pylori cells. Paper point samples from 3 deep periodontal pockets per patient were immersed in 1 ml of phosphate‐buffered saline or distilled water, DNA was solubilized by detergent/protease method, 3.7 μl or 37 μl of lysate supernatant was used as template, and the amplification product was analyzed in 1% agarose gel containing ethidium bromide. Each experiment included purified DNA and cell lysate of H. pylori as positive controls. The presence of bacteria in the sample was verified by a primer pair common to prokaryote 16S rRNA. The present study did not reveal the specific polymerase chain reaction amplification product characteristic of H. pylori. We conclude that periodontal pockets do not constitute a natural reservoir for H. pylori.
Characteristics of accumulation of oral gram‐positive bacteria on mucin‐conditioned glass surfaces in a model system
Authors: Li Y. H. , Bowden G. H.
Published: December 2007

Strains of Streptococcus, Actinomyces and Lactobacillus were grown on glass surfaces in semi‐defined medium (pH 7.0) with mucin. at a dilution rate of D = 0.1 h−1, in a modified chemostat. The accumulation of cells followed four phases. In phase 1 (0‐1 h). cells did not divide on the surfaces and adhesion accounted for rapid accumulation. Phase 2 (1‐4 h) comprised adhesion and cell division, and accumulation slowed, cell number doubling times (Cdt) Streptococcus, 2.7 h to 8.6 h, Actinomyces, 2.3 h to 7.5 h and Lactobacillus, 3.6 h to 3.8 h. Cell division on surfaces accounted for accumulation in phase 3 (4 h to 12 h): Cdt Streptococcus, 1.7 h to 5.2 h. Actinomyces, 2.4 h to 7.5 h and Lactobacillus, 2.2 h to 7.2 h. The biofilm stabilized in Phase 4, Cd1 18.5 h to 90.2 h. The numbers (106 colony‐forming units per cm2) of cells in stable biofilms were Streptococcus, 4.02 to 5.12, Actinomyces, 12.5 and 34.0 and Lactobacillus, 2.77. Accumulation increased (Cd1 0.9 h‐2.7 h) when cells were exposed to glucose excess or high dilution rates and phase 2 of accumulation did not occur.
The influence of saliva on interbacterial adherence
Authors: Skopek R. J. , Liljemark W. F.
Published: December 2007

The mechanism of bacterial adherence to the mat of bacteria in preformed dental plaque is not well defined. This study measured the influence of saliva on the adherence of bacteria in suspension to a continuous bacterial surface in vitro. Twenty different pairs of bacteria were tested, consisting of Streptococcus spp., Huenwphilus spp. and Actinomyces spp. The species were chosen based on the parameters of coaggregation. and salivary agglutination. The results were expressed as bacteria that adhered per mm2 of bacterial surface. When both the surface bacteria and the bacteria in suspension agglutinated in saliva, interbacterial adherence was increased 2.5‐fold when a salivary coating was placed on the surface. When one or both of the bacteria did not agglutinate in saliva, interbacterial adherence was increased only slightly when the surface was saliva‐coated. The results suggested that salivary‐mediated adherence is significant to plaque formation once the tooth surface becomes covered with bacteria. Thus bacteria that are capable of agglutinating in saliva may have a distinct advantage in colonization of the plaque surface.
Oral microflora of the ferret at the gingival sulcus and mucosa membrane in relation to ligature‐induced periodontitis
Authors: Fischer R. G. , Edwardsson S. , Klinge B.
Published: December 2007

The aim of this study was to describe, in the domestic ferret, the composition of the normal gingival sulcus and the near related mucosa microflora and the shift in the composition during an acute periodontal breakdown induced by the placement of ligatures. Five animals were used. At the start of the experiment (day 0) and before the placement of the ligatures, 2 microbiological samples were obtained from the gingival sulcus and the related mucosa at the mid‐buccal surface of the right upper P4. The same sites were sampled on the left side, serving as control sites. On day 28, new microbiological samples were taken from the same 4 sites. The samples were analyzed with darkfield microscopy and with viable counts. In the darkfield analysis, on day 0, the predominant bacterial morphotype was cocci, ranging from 88–92%. The viable counts showed that nearly 100% of the total cultivable flora was composed of facultative anaerobic gram‐negative and gram‐positive rods. Pasteurella spp., Corynebacterium spp. and Rothia spp. were the major components of these bacterial groups. No anaerobic bacteria were detected. On day 28, cocci decreased to approximately 75% in the experimental gingival sulcus sample, and filaments and rods increased significantly. Spirochetes remained largely unchanged during the experiment. The composition of the experimental site samples changed. There was a decrease in the mean numbers of facultative anerobic gram‐positive rods, and approximately one third of the microflora comprised anaerobic gram‐negative rods, mainly Porphyromonas gingivalis and Fusobacterium spp. Although the composition of the microflora of the ferret presented few similarities to that described in dogs and monkeys, the anaerobic portion of the gingival sulcus and oral mucosa microflora related to ligature‐induced periodontitis presented microorganisms observed in experimental periodontitis both in dogs and monkeys as well as in human periodontal diseases.
Use of the Syrian golden hamster for the induction of intraoral abscesses by sutures contaminated with human subgingival plaque
Authors: Wasfy M. O. , McMahon K. T. , Santos A. C. , Minah G. E. , Falkler W. A. , Lloyd D. R.
Published: December 2007

Analysis of normal oral flora in 150 cheek pouches of hamsters (Mesocricetus auratus) defined the microbial working environment and demonstrated the absence of human oral black‐pigmented bacteria. Silk sutures saturated with Porphyromonas gingivalis, Prevotella intermedia or subgingival plaque were used to close wounds made in hamster's cheek pouches. Abscesses were formed when sutures had solitary P. gingivalis or other bacteria mixed with P. gingivalis or when P. intermedia was mixed with other bacteria besides P. gingivalis. A concentration of black‐pigmented bacteria emanating from 3×105 colony‐forming units/inoculum was required for abscess formation. Six abscesses (14.3%) were developed in association with the presence of other odontopathic bacteria, primarily Fusobacterium nucleatum and Actinomyces viscosus. The hamster cheek pouch with iatrogenic wounds closed with plaque‐impregnated sutures is a novel and effective model to study the pathology of wound infections and virulence of human subgingival organisms.
Degradation of immunoglobulin G by periodontal bacteria
Authors: Jansen H.‐J. , van der Hoeven J. S. , van den Kieboom C. W. A. , Göertz J. H. C. , Camp P. J. M. , Bakkeren J. A. J. M.
Published: December 2007

Several subgingival microorganisms were tested for their ability to utilize human immunoglobulin G (IgG) as a substrate for growth. This was done using a protein‐free chemically defined medium, supplemented with IgG. Stimulation of growth was observed for Capnocytophaga ochracea, Porphyromonas asaccharolytica, Porphyromonas endodontalis, Porphyromonas gingivalis, Prevotella intermedia, Prevotella oralis, Lactobacillus catenaforme and Streptococcus intermedius. Immunoelectrophoresis, sodium dodecyl sulfatepolyacrylamide gel electrophoresis and a protein assay demonstrated that P. intermedia and P. endodontalis completely degraded the protein chains of IgG. Partial breakdown of IgG was observed for P. asaccharolytica and C. ochracea, whereas P. oralis cleaved the IgG heavy chain, yielding Fc and Fab fragments. All these bacteria utilized IgG as a substrate for growth. Binding studies using an enzyme‐linked immunosorbent assay, revealed complete loss of in vitro antigen‐antibody binding capacity after incubation of specific IgG with P. endodontalis and partial loss of binding with P. intermedia, P. gingivalis, C. ochracea or Fusobacterium nucleatum. Degradation or inactivation of IgG by oral bacteria is thought to be important in the causation of polymicrobial infections.
Effect of binding of fibrinogen to each bacterium on coaggregation between Porphymmonas gingivalis and Streptococcus oralis
Authors: Nagata H. , Amano A. , Ojima M. , Tanaka M. , Kataoka K. , Shizukuishi S.
Published: December 2007

Fibrinogen inhibits the coaggregation between Porphyromonas gingivalis and Streptococcus oralis. In this study, we determined which bacterium interacts with fibrinogen in this inhibitory process. Although preincubation of each bacterium with fibrinogen did not inhibit coaggregation, its activity was completely eliminated by the addition of protease inhibitors such as N‐ethylmaleimide (NEM), p‐chloromercuriphenyl sulfonate and Nα‐p‐tosyl‐L‐lysine chloromethyl ketone to the preincubation mixture with fibrinogen and P. gingivalis. However, the inhibition of coaggregation was not found after preincubation of S. oralis with fibrinogen in the presence or absence of the protease inhibitors. Labelled materials were recovered from the extract of P. gingivalis cells incubated with radioiodinated fibrinogen in the presence of NEM but not in the absence of NEM. In the binding experiment, P. gingivalis showed a much higher binding activity to fibrinogen than S. oralis. These findings suggest that fibrinogen and its fragment(s) may mask directly or indirectly the aggregation site with S. oralis on the P. gingivalis cells in its inhibitory process of coaggregation.
Leukocyte adhesion molecules in oral lichen planus: a T cell‐mediated immunopathologic process
Authors: Eversole L. R. , Dam J. , Ficarra G. , Hwang C.‐Y.
Published: December 2007

Oral lichen planus exhibits features of a mucosal type IV immunopathologic process. Adhesion molecules involved in the trafficking and homing of T lymphocytes to the subepithelial compartment were assessed by immunohistochemical methods. Laminin, type IV collagen and type VII collagen extracellular matrix components at the epithelial‐connective tissue junction are significantly increased and serve as ligands for β1 integrins on lymphocyte membranes. Endothelial‐associated intercellular adhesion molecule 1 and extracellular matrix basement membrane components are also significantly increased in the submucosa. Keratinocyte expression of major histocompatibility complex class II molecules and intercellular adhesion molecule 1 may serve as ligands for lymphocyte T cell receptor complex and β2 integrins, respectively. These adhesion molecules are probably involved in the trafficking of lymphocytes to the epithelial connective tissue interface in response to as of yet undefined antigens.
Restriction fragment length polymorphism analysis of two hemagglutinin loci, serotyping and agglutinating activity of Porphyromonas gingivalis isolates
Authors: Lépine G. , Savett D. A. , Progulske‐Fox A.
Published: December 2007

Restriction fragment length polymorphisms (RFLPs) of two hemagglutinin loci were analyzed in 36 Porphyromonas gingivalis isolates from human and monkey origins using portions of hag A and hagB as probes. The P. gingivalis strains were differentiated into 9 RFLP groups based on the heterogeneity of the hag A locus and 10 different groups based on hybridization with hagB. Homology to hagA was detected in all human derived and all but three monkey derived strains. All P. gingivalis isolates exhibited DNA homologous to hagB. Multiple alleles of the hemagglutinin genes were detected for most P. gingivalis strains. No DNA homologous to either hemagglutinin gene could be detected in 6 other bacterial species tested. Serotyping and hemagglutination liters of each P. gingivalis isolate were obtained in an attempt to establish a correlation between these pheno‐typic parameters and RFLP group. Although no correlations were found with these parameters, a correlation between RFLP group and invasiveness in the mouse abscess model was noted.
Ribotyping on small‐sized spirochetes isolated from subgingival plaque
Authors: Fiehn N.‐E. , Bangsborg J. M. , Colding H.
Published: December 2007

In the present study DNA restriction patterns and corresponding ribotypes of 17 subgingival small‐sized spirochetes (1:2:1 and 2:4:2 isolates), 2 Treponema socranskii strains and two Treponema denticola strains were examined. Purified chromosomal DNA was digested by BamHI, HindIII, PstI and ClaI. The DNA fragments were separated in a horizontal slab of 0.7% agarose containing ethidium bromide and transferred by nylon membranes. Hybridization was carried out with digoxigenin‐labelled copy DNA of 16S and 23S ribosomal RNA from Escherichia coli. Depending on the restriction endonuclease used, up to 4 distinct bands were observed for the 2:4:2 isolates and the T. denticola strains. For each of the endonucleases used, identical band patterns were always observed for this group of isolates, and these patterns differed persistently from the T. denticola strains. For the 1:2:1 strains, up to 11 distinct bands were observed after digestion with Hindlll, whereas a maximum of 6 bands were observed when PstI or ClaI was used. By using Clal, the examined 1:2:1 isolates were separated into 8 groups, whereas PstI and HindIII separated these isolates into 5 groups. The ribotyping showed that the tested 1:2:1 spirochetes were more heterogeneous than the 2:4:2 spirochetes examined.
Actinobacillus actinomycetemcomitans genotypes in relation to serotypes and periodontal status
Authors: Asikainen S. , Chen C. , Slots J.
Published: December 2007

Actinobacillus actinomycetemcomitans is prevalent in periodontitis but is found in some periodontally healthy individuals as well. The arbitrarily primed polymerase chain reaction (AP‐PCR) was used to fingerprint clinical A. actinomycetemcomitans isolates of different serotypes to determine the association between individual clonal types and periodontal conditions. Fifteen different AP‐PCR genotypes were distinguished among 93 A. actinomycetemcomitans isolates from 86 uncohabiting individuals with adult periodontitis, localized juvenile periodontitis or no periodontal destruction. The 3 most common AP‐PCR genotypes accounted for 68% of the isolates. Seven of the remaining AP‐PCR genotypes were found only in periodontitis. The isolates of a given AP‐PCR genotype usually belonged to the same serotype. The distribution of the AP‐PCR genotypes among serotype b isolates seemed to differ among the subject groups. The results revealed a major genetic dissimilarity between A. actinomycetemcomitans serotypes and suggested a relationship between some A. actinomycetemcomitans clones and periodontal disease.
Role of prostaglandin in the formation of osteoclasts induced by capsular‐like polysaccharide antigen of Actinobacillus actinomycetemcomitans strain Y4
Authors: Ueda N. , Nishihara T. , Ishihara Y. , Amano K. , Kuroyanagi T. , Noguchi T.
Published: December 2007

We found no reports that capsular‐like polysaccharide antigen purified from Actinobacillus actinomycetemcomitans either induces osteoclastic bone resorp‐tion in mouse organ cultures or promotes osteoclast formation in mouse marrow cultures. In contrast, capsular‐like polysaccharide antigen purified from A. actinomycetemcomitans strain Y4 induced bone resorption in mouse organ culture. To examine the mechanism of bone resorption induced by A. actinomycetemcomitans, mouse bone marrow cells were cultured with A. actinomycetemcomitans strain Y4 capsular‐like polysaccharide antigen. A. actinomycetemcomitans strain Y4 capsular‐like polysaccharide antigen stimulated osteoclast‐like cell formation in mouse bone marrow cultures. However, the polysaccharide of A. actinomycetemcomitans lipopolysaccharide did not induce the formation of osteoclast‐like cells. Indomethacin inhibited osteoclast‐like cell formation mediated by A. actinomycetemcomitans strain Y4 capsular‐like polysaccharide antigen in a dose‐dependent manner. There was a good correlation between the number of osteoclast‐like cells formed in the marrow culture and the amount of prostaglandin E2 released into the culture media. When mouse bone marrow cells were cultured with prostaglandin E2 during the culture periods, many osteoclast‐like cells were formed. These results indicate that prostaglandin E2 is involved in the mechanism of the formation of osteoclast‐like cells mediated by A. actinomycetemcomitans strain Y4 capsular‐like polysaccharide antigen. A. actinomycetemcomitans strain Y4 capsular‐like polysaccharide antigen may play an important role in inflammatory bone resorption by promoting osteoclast formation in periodontal disease.
Demonstration of adherence properties of Porphyromonas gingivalis outer membrane vesicles using a new microassay
Authors: Duchesne P. , Grenier D. , Mayrand D.
Published: December 2007

Vesicles made by Porphyromonas gingivalis possess several biological activities, including the ability to adhere to oral surfaces and to bacteria. In this study, a new and simple method was developed to measure the adherence capability of outer membrane vesicles from P. gingivalis. Vesicles were conjugated to fluorescent microspheres (0.7 μ) and added to wells of a Teflon‐coated microscope slide previously covered with a variety of soluble ligands. After incubation and washes, the number of fluorescent microspheres per microscopic field were counted. Vesicle‐coated microspheres attached best to gelatin (<200 per field), whereas other compounds (such as fibronectin, fibrinogen, collagen and laminin) provided moderate attachment, and no attachment was observed to bovine serum albumin. Adherence to any of the tested ligands was not observed when fluorescent micro‐spheres were conjugated to bovine serum albumin or lipopolysaccharides from P. gingivalis. The adherence of vesicle‐coated microspheres to ligands was not significantly affected when the pH of the reaction mixture was between 4 and 10. None of the tested carbohydrates lowered the attachment capability of vesicle‐coated microspheres to substrates. When vesicle‐coated microspheres were treated with trypsin and chymotrypsin or heated, this resulted in a significant loss of attachment, suggesting a possible involvement of proteinaceous molecules in the process. The present study confirms that vesicles of P. gingivalis are capable of attachment to various molecules and indicate their potential role in colonization.
A novel method to study the hyphal phase of Candida Albicans and to evaluate its hydrophobicity
Authors: Nikawa H. , Nishimura H. , Yamamoto T. , Samaranayake L. P.
Published: December 2007

As little is known about the surface properties of the hyphal phase of the dimorphic fungus Candida albicans due to the difficulty in examining the latter phase in isolation, a novel method was designed to compartmentalize the two phases of the yeast using a commercially available filter‐type device – Chemotaxicell. When yeast cells in the blastospore phase were incubated in filter chambers of Chemotaxicell submerged in hyphal‐induction media, hyphae traversed through the pores of the filter into the exterior of the chamber enmeshing the entire outer filter surface, after 72 h of incubation. However, the inner or the chamber surface of the filter comprised mainly blastospores. The hydrophobicity of the two morphologic phases of the yeast was then compared using this method, by contact angle measurement. When the 72‐h specimens with matted hyphal elements were evaluated, the contact angles varied depending on the incubation medium (such as TC 199 or serum), and in each case hydrophobicity of the hyphal phase was significantly higher than the blastospore phase yeasts. This simple, reproducible method should help not only in evaluating the properties of the hyphal elements of C. albicans but also in studying parameters such as saliva and serum, which are known to affect hyphal formation in vivo.
Evidence for two distinct locomotory phenotypes of Treponema denticola ATCC 35405
Authors: Chan E. C. S. , Qiu Y.‐S. , Siboo R. , Noble P.
Published: December 2007

Using a two‐layer system, a bottom layer containing treponema cells suspended in NOS (New Oral Spirochete)‐Noble agar medium or NOS‐Bacto agar medium and overlaid with cell‐free NOS‐agarose medium resulted in the spirochete cells migrating into the top layer. However, if the positions of the medium layers were reversed with the cells inoculated into the bottom layer containing NOS‐agarose, there was no migration into the upper layer. This suggests migration of the spirochetes away from Bacto and Noble agars. Using a 3‐layer system in which cells were inoculated into a middle layer consisting of NOS‐agarose medium and sandwiched between cell‐free NOS‐agarose medium layers, cells remained within the middle layer. If the cells were inoculated into a middle layer consisting of NOS‐Bacto agar medium while the upper and lower layers remained unchanged, cells migrated into both upper and lower layers. If cells that had migrated into the upper layer were transferred into a middle layer, they virtually all migrated into the upper layer repeatedly. Cells that had migrated into the lower layer and transferred to the middle layer migrated repeatedly into the lower layer. These results suggest the possible existence of two distinct locomotory phenotypes within this strain of treponeme.
Characterization of glutamine transport in Streptococcus mutans
Authors: Dashper S. G. , Riley P. F. , Reynolds E. C.
Published: December 2007

Glutamine transport in glucose‐energized cells of Streptococcus mutans Ingbritt exhibited Michaelis‐Menten‐type kinetics with a Vmax of 13.4 nmol/mg dry weight/min and a K1 of 4.1 μM. Diffusion of glutamine into de‐energized cells of S. mutans displayed similar type kinetics, with a Kt of 6.8 μM but with a markedly reduced Vmax of 53.9 pmol/mg dry weight/min. Glutamine transport in S. mutans is not proton motive force‐driven, as the intracellular accumulation of glutamine by energized cells far exceeded the thermodynamic limits of the proton motive force, and the dissipation of this proton motive force by gramicidin in a high K+ medium did not decrease the intracellular glutamine concentration. Glutamine transport is therefore likely to be energized by ATP hydrolysis. The activity of the transporter was maximal between pH 6.0 and 7.0 and decreased rapidly above pH 7.0. The transport of glutamine was not competitively inhibited by asparagine, glutamate or aspartate, indicating a specific glutamine transport system. Reversed‐phase high‐pressure liquid chromatography of cell extracts revealed that approximately 26% of the glutamine taken into the cell was converted to glutamate within 10 min. The results are consistent with transported glutamine being converted to glutamate and ammonia by the action of an intracellular glutaminase. Glutamine therefore may be an important source of nitrogen for the cell.
Age‐related changes in immunoglobulin isotypes in whole and parotid saliva and serum in healthy individuals
Authors: Challacombe S. J. , Percival R. S. , Marsh P. D.
Published: December 2007

Mucosal Infections account for the majority of infections seen in elderly people, but little is known of whether mucosal immunity decrease with age. The objective of this study was to investigate the effect of age on the levels of salivary and serum immunoglobulins and the salivary immunoglobulin secretion rates in a healthy adult population. Healthy subjects (116 total) were divided into the following age groups: 20–39; 40–59; 60–79 and >80 years. Unstimulated (resting) whole and stimulated parotid saliva and serum were collected from all participants. Salivary and serum immunoglobulins were quantified by enzyme‐linked immunosorbent capture assays. The levels of serum immunoglobulin G (IgG) and IgM were significantly reduced in the oldest age group, whereas no significant reduction in the level of IgA with age was observed. The IgG and IgA levels in whole saliva increased significantly in the oldest age group D, but no changes were detected in IgM levels. No significant changes in any immunoglobulin levels with age were found in parotid saliva. However, significant reductions in the secretion rates of IgA and IgM, but not IgG, in whole saliva were detected in the oldest age group. No significant changes in the secretion rates in parotid saliva were found with age. Our results demonstrate a decline in immunoglobulin secretion rates in saliva and in circulating immunoglobulin concentrations with increased age, which may contribute to the increased susceptibility of elderly individuals to infectious diseases.
Population genetic analysis of oral treponemes by multilocus enzyme electrophoresis
Authors: Dahle U. R. , Olsen I. , Tronstad L. , Caugant D. A.
Published: December 2007

Seventeen treponemes recently isolated from necrotic pulps, periodontal and periapical infections and 17 previously well characterized oral treponemal strains were analyzed by multilocus enzyme electrophoresis. Ten genetic loci were characterized on the basis of the electrophoretic mobilities of their enzymatic products. All loci were polymorphic. The average number of alleles per locus was 7.8. The genetic diversity among the electrophoretic types at each locus ranged from 0.624 to 0.836 with a mean genetic diversity per locus of 0.751. The 34 strains represented 34 electrophoretic types, constituting 6 main divisions (I‐VI) separated at genetic distances greater than 0.75. Several of the previously characterized treponemes revealed multiple bands of enzyme activity at several loci, indicating that they were not pure. The characterized strains usually clustered within established species, whereas fresh clinical isolates overlapped species borders. There was a large genetic difference between some reference and clinical strains, indicating that the latter may contain undescribed species. Treponema socranskii and Treponema denticola strains clustered in distinct divisions (IV and V, respectively), with the exception of T. denticola strain FDC 51B2 and T. socranskii subsp. paredis strain VPI D46CPE1, both previously well described. This indicated that the taxonomic assignment of these 2 strains should be reconsidered.
Characterization of oral treponemes isolated from human periodontal pockets
Authors: Koseki T. , Ishikawa I. , Umeda M. , Benno Y.
Published: December 2007

A total of 90 clinical strains of oral treponemes was isolated from subgingival plaque in patients with periodontal disease. They were characterized by biochemical means as well as cell enzyme, protein analysis using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and DNA dot blot hybridization. Sixty strains were isolated on Medium 10 (M10), which was fundamentally serum‐free. The remainder were isolated on serum‐containing media. Isolates were divided into 6 groups according to their biochemical characteristics. Three of the 6 groups were asaccharolytic, and 2 of these 3 groups were Treponema denticola and “Treponema vincenti”. The other 3 groups were saccharolytic and further divided into 9 subgroups. The analyses of cell enzyme, cell protein and dot blot hybridization with the DNA probe of Treponema socranskii indicated that all the saccharolytic groups were T. socranskii or closely related species. This study indicated that the newly characterized saccharolytic oral treponemes could be identified using M10 from the human periodontal pocket.
The occurrence of treponemes and their spherical bodies on polytetrafluoroethylene membranes
Authors: Wecke J. , Wolf V. , Path S. , Bernimoulin J.‐P.
Published: December 2007

The occurrence of small (diameter 0.125 μm) and large (diameter 0.27 μm) treponemal species on expanded polytetrafluoroethylene membranes were demonstrated in vivo. In the apical part of the expanded polytetrafluoroethylene membrane only the smaller treponemes were colonizing, while in other parts a mixed population of different bacterial species including large treponemes was seen. Only the smaller treponemes were able to form spherical bodies in the occlusive part of the membrane. All morphological characteristics of spherical bodies such as common outer sheath, randomly distributed axial flagella, sheathless protoplasmic cylinders and central bodies could be differentiated. Possible causes for the formation of spherical bodies are discussed.
Molecular genetic detection of Bacteroides heparinolyticus in adult periodontitis
Authors: Ashimoto A. , Flynn M. J. , Slots J.
Published: December 2007

Bacteroides heparinolyticus in subgingival plaque was identified using a digoxigen‐in‐labeled whole genomic DNA probe and a polymerase chain reaction (PCR) assay based on 16S rRNA species‐specific primers (5′‐ATG GTG ATT CCG CAT GGT TTC TCC‐3’(base position, 188‐212) and 5′‐CAA ACT TTC ACA GCT GAC TTA AGC‐3’(592‐615)). Subgingival specimens obtained by paper points from 3 deep periodontal pockets in each of 113 adults were examined. The DNA probe reacted with all pure isolates tested of B. heparinolyticus and did not react with other oral species tested; the probe showed positive reactions in 74.3% of the patient samples examined. The PCR primers produced the 428 bp species specific amplification product in all B. heparinolyticus test strains and did not reveal detectable amplicons with strains of other subgingival species. The PCR method detected 50 B. heparinolyticus cells dispersed in subgingival plaque. PCR only revealed B. heparinolyticus in 6.2% of the patient samples studied. The higher level of positive specimens with the DNA probe was probably due to false‐positive reactions from cross‐hybridization with unknown subgingival species. This study suggests that the PCR method amplifying specific 16S rRNA sequences represents an easy and valuable means to detect B. heparinolyticus in subgingival plaque. The low prevalence of subgingival B. heparinolyticus does not incriminate the organism in the etiology of adult periodontitis.
Colorimetric microtiter plate based assay for detection and quantification of amplified Actinobacillus actinomycetemcomitans DNA
Authors: Fujise O. , Hamachi T. , Hirofuji T. , Maeda K.
Published: December 2007

We developed a Colorimetric microtiter plate‐based assay for the detection and quantification of polymerase chain reaction‐amplified DNA fragment specific for Actinobacillus actinomycetemcomitans. We amplified the 396‐bp leukotoxin‐specific DNA fragment by using two oligonucleotide primers, one carrying a biotin group at the 5’end and another one with a digoxigenin at the 5’end. Following amplification, the biotinylated polymerase chain reaction products were applied to a microtiter well precoated with avidin. The Colorimetric detection and quantification were achieved by an enzyme‐linked immunosorbent assay using alkaline phosphatase‐conjugated anti‐digoxigenin antibody. The detection limit of the Colorimetric assay was found to be as little as 500 fg of purified A. actinomycetemcomitans DNA and as few as 50 A. actinomycetemcomitans cells. The absorbance values in the Colorimetric assays were log‐linear between 500 fg and 1 ng of purified DNA and between 50 and 105 cells of A. actinomycetemcomitans. Therefore, this Colorimetric assay was able to estimate the amount of A. actinomycetemcomitans in subgingival plaque samples. We concluded that the colorimetric assay of the PCR product is a very useful method not only to detect the presence of A. actinomycetemcomitans but also to quantify the amount of A. actinomycetemcomitans in large numbers of subgingival plaque samples.
Identity of 1:2:1 and 2:4:2 subgingival spirochetes by DNA hybridization
Authors: Olsen I. , Fiehn N.‐E. , Johnson J. L.
Published: December 2007

A group of 1:2:1 and 2:4:2 subgingival spirochetes, well characterized by transmission electron microscopy, biochemical tests, cellular fatty acid and carbohydrate analyses, and ribotyping, was recently suggested to represent new treponemal species. The present study used DNA hybridization to examine this possibility. When DNA of a representative strain (no. 16) of the 8 1:2:1 spirochetes examined was labeled by iodination, it showed, after SI nuclease treatment, from 58 to 104% (average 76%) homology with DNA from the 1:2:1 spirochetes. 94% homology with DNA from the type strain of Treponema socranskii and of T. socranskii subsp. socranskii, i.e., ATCC 35536T, and 62% homology with DNA from T. socranskii subsp. buccale, strain ATCC 35534T. Similarly treated DNA from a representative strain (no. 3) of 8 2:4:2 spirochetes exhibited from 90 to 105% (average 97%) homology with DNA from the 2:4:2 spirochetes, and 85% and 87% homology, respectively, with DNA from Treponema denticola strains ATCC 33520 and FDC T1. There was a negligible degree of homology between the 1:2:1 and 2:4:2 spirochetes. Thus, all the 2:4:2 spirochetes belonged to T. denticola. 1:2:1 strains with DNA homology levels >70% (5 strains) belonged to T. socranskii or T. socranskii subsp. socranskii, while those with homology levels from 58 to 63% (3 strains) most likely belonged to other subspecies of T. socranskii.
Immunogenicity of Actinobacillus actinomycetemcomitans serotype b‐specific polysaccharide‐protein conjugate
Authors: Takamatsu‐Matsushita N. , Yamaguchi N. , Kawasaki M. , Yamashita Y. , Takehara T. , Koga T.
Published: December 2007

To enhance the Immunogenicity of capsular‐like serotype b‐specific polysaccharide antigen (SPA) of Actinobacillus actinomycetemcomitans, the purified antigen was coupled with bovine serum albumin via an adipic acid hydrazide functional group. The conjugate (SPA‐bovine serum albumin) or the native SPA was administered subcutaneously or intranasally to BALB/c mice. Neither subcutaneous immunization with SPA emulsified in Freund adjuvant nor intranasal immunization with SPA and cholera toxin B subunit elicited any antibody responses to the polysaccharide antigen. High serum immunoglobulin M (IgM) and IgG responses to SPA were induced by subcutaneous immunization with SPA‐bovine serum albumin in Freund adjuvant. Serum and salivary IgA responses to SPA in addition to IgM and IgG responses were induced by intranasal immunization with the conjugate and cholera toxin B subunit. To investigate the functional activity of SPA‐specific antibodies, the opsonic activity of sera from BALB/c mice immunized with the conjugate was assessed by chemiluminescence assay using human polymorphonuclear leukocytes. Murine antisera efficiently opsonized A. actinomycetemcomitans serotype b in the assay, suggesting that antibodies to SPA of the organism might serve as a protective role.