bluuebunny/crossref_metadata_2025 huggingface dataset:
Spirochetes in early lesions of necrotizing ulcerative gingivitis experimentally induced in beagles
Authors: Mikx F. H. M. , Maltha J. C. , van Campen G. J.
Published: December 2007

The presence of Spirochetes and other bacteria during the induction of necrotizing ulcerative gingivitis (NUG) in beagle dogs was studied by transmission electron microscopy. NUG was induced by application of dental debris from diseased dogs to 4 beagles treated with corticosteroid. Biopsies of interdental papillae were collected during a period of 7 weeks. Spirochetes and other bacteria were observed in enlarged intercellular spaces between non‐keratinized epithelial cells. Intracellular bacteria were found with and without surrounding vesicular spaces. The invasive Spirochetes were mainly intermediately sized and of flagellar “4‐8‐4” configuration or higher. Bacterial invasion of the sulcular epithelium occurred before clinical evidence of NUG could be observed. It is concluded that invasion by intermediate spirochetes and other bacteria plays a role in initiating ulcerative gingivitis in beagles.
An in vivo study of neutrophil locomotion in relation to periodontal disease status and local chlorhexidine
Authors: Seymour Kevin G. , Watts Trevor L. P. , Addison Ian E. , Johnson Beryl
Published: December 2007

Neutrophil migration from skin window abrasions was studied in 10 patients with no history of periodontitis, 10 with localised juvenile or post‐juvenile periodontitis, and 10 with chronic adult periodontitis. Filters contained either saline or chlorhexidine (0.002% or 0.02%). The leading front was measured in filters placed for 30 min after cell migration had been established for 2 h. Subjects in the juvenile/post juvenile group showed a reduced range of migration distances, but were still within the normal range when compared with the other 2 groups. Chlorhexidine at 0.002% tended to increase leading front distances, and 0.02% to decrease them. We conclude that: 1) migrating neutrophils in vivo may move less far in patients with a history of juvenile periodontitis; 2) chlorhexidine may inhibit cell migration, possibly decreasing the host response in vivo if applied at current therapeutic concentrations.
Binding of streptococci of the “mutans” group to type 1 collagen associated with apatitic surfaces
Authors: Liu Tianjia , Gibbons R. J.
Published: December 2007

The present study surveyed the ability of 21 strains of cariogenic streptococci of the “mutans” group to bind to human type 1 collagen adsorbed on hydroxyapatite (HA) surfaces. All strains of Streptococcus cricetus (serotype a) and Streptococcus rattus (serotype b) tested attached strongly to collagen‐treated HA (C‐HA). Streptococcus mutans strains of serotype c and Streptococcus sobrinus strains of serotype d or g attached poorly, or not at all, to C‐HA. S. mutans strains OMZ‐175 (serotype f) and B14 (serotype e) were exceptions which exhibited significant binding to C‐HA. Binding of S. cricetus AHT and S. rattus LB‐1 to C‐HA occurred in a dose‐dependent manner, and was specific since it was inhibited by the presence of soluble collagen, but not by egg albumin, or by human fibrinogen or fibronectin. Binding occurred maximally between pH 6 and 8 and was not affected by any of several sugars tested. Trypsin treatment the streptococci had little affect, but heating at 100°C reduced their ability to bind to C‐HA.
Amino acid utilization by Fusobacterium nucleatum grown in a chemically defined medium
Authors: Dzink JoAnn L. , Socransky Sigmund S.
Published: December 2007

The present investigation evaluated amino acid utilization by 120 strains of Fusobacterium nucleatum in a chemically defined medium and attempted to relate the patterns to 3 proposed subspecies of F. nucleatum. Strains were inoculated into a chemically defined medium, with and without 2 g/1 glucose, consisting of 14 inorganic salts, 21 amino acids, 23 vitamins and cofactors, and 7 purities and pyrimidines. After 7 days of anaerobic incubation, the spent culture medium, as well as the uninoculated control medium, were analyzed for amino acid content by ion chromatography. Amino acid utilization was determined by the differences in concentrations of amino acids found in inoculated and uninoculated samples. If > 34% of the amino acid was removed from the medium, the amino acid was considered to be utilized. Of the 21 amino acids present in the chemically defined medium, 8 amino acids, lysine, glutamine, asparagine, histidine, threonine, serine, glutamate and cysteine were consistently utilized. Four amino acids, tyrosine, tryptophan, methionine and aspartate were utilized by some strains but not others. Nine amino acids, alanine, leucine, isoleucine, glycine, valine, phenyl‐alanine, proline, ornithine, and arginine were not utilized by any of the strains. The utilization patterns did not relate to subspecies formed on the basis of SDS‐PAGE and DNA hybridization.
Control of sugar utilization in oral streptococci. Properties of phenotypically distinct 2‐deoxyglucose‐resistant mutants of Streptococcus salivarius
Authors: Gauthier Lucie , Bourassa Sylvie , Brochu Denis , Vadeboncoeur Christian
Published: December 2007

The physiological and biochemical characterization of Streptococcus salivarius mutants isolated by positive selection for resistance to 0.5 mM 2‐deoxyglucose in the presence of lactose are reported. We found 2 classes of mutants following a series of experiments that included: growth rate determinations, uptake studies, measurement of phosphotransferase system (PTS) activities and detection of the IIIman proteins by Western blotting and analysis of [32P]PEP‐phosphorylated proteins. Class 1 mutants did not possess the low‐molecular‐weight form of IIIman. They did not grow on mannose and were unable to transport 2‐deoxyglucose. On the other hand, class 2 mutants possessed the 2 forms of IIIman, grew readily on mannose and transported 2‐deoxyglucose, albeit at a lower rate than the parental strain. Both classes of mutants exhibited abnormal growth in media containing mixtures of sugars. Moreover, derepression of genes coding for catabolic enzymes was observed in all the mutant strains. Our data suggested that the role of the mannose PTS in the control of sugar utilization in S. salivarius is complex and may involve the participation of several components.
Collagenase activity in gingival crevicular fluid of patients with juvenile periodontitis
Authors: Suomalainen Kimmo , Sorsa Timo , Saxén Leena , Vauhkonen Matti , Uitto Veli‐Jukka
Published: December 2007

The nature and origin of collagenases in gingival crevicular fluid of juvenile periodontitis patients was investigated. Gingival crevicular fluid collected from deep untreated periodontal pockets of juvenile periodontitis patients was found to contain only vertebrate collagenase (EC 3.4.24.7) activity that cleaved soluble type‐I and ‐III collagens into 3/4 and 1/4 length fragments, as analyzed by SDS‐polyacrylamide gel electrophoresis. Type II collagen was degraded at a markedly slower rate. This substrate specificity is indicative of collagenases produced by fibroblasts, epithelial cells and macrophages. We have previously found that collagenase in gingival crevicular fluid of adult periodontics patients appears to be mainly derived from polymorphonuclear leukocytes (PMN). The reasons for the apparent difference in collagenase source between the groups were investigated. We examined whether the pathogen characteristic for juvenile periodontitis, Actinobacillus actinomycetemcomitans, can release collagenase from normal human PMNs. All 10 A. actinomycetemcomitans strains tested, freshly isolated from the subgingival plaque of juvenile periodontitis patients, caused release of collagenase from PMNs in vitro. These results suggest that the lack of normally functioning PMNs in the periodontium of juvenile periodontitis patients may result in a colonization of bacteria that activate the resident periodontal cells to produce increased amounts of collagenase.
The effect of sanguinarine on human peripheral blood neutrophil viability and functions
Authors: Agarwal S. , Reynolds M. A. , Pou S. , Peterson D. E. , Charon J. A. , Suzuki J. B.
Published: December 2007

Human polymorphonuclear cell (PMN) viability, morphology, adherence, chemotaxis, oxidative metabolism, degranulation and phagocytosis were evaluated following treatment with sanguinarine (SANG). SANG was noncytotoxic to PMNs at all concentrations tested (0.31–200 μM). SANG entered the PMNs rapidly without altering the membrane fluidity and localized in the nuclear matrix. SANG (1.56–6.21 μM) inhibited chemotaxis, chemokinesis and adhesion in a dose‐dependent manner, with a complete inhibition at 6.2 μM concentration. Concentrations of SANG up to 1.56 μM did not affect PMN oxidative burst; however, higher concentrations were found to inhibit basal as well as PMA‐induced superoxide anion generation. The effect of SANG was time‐ and dose‐dependent, and could be reversed if the PMNs were exposed to 12.5 μM or lower concentrations of SANG for less than 5 min. Autologous serum increased the tolerance of PMNs to SANG. Exogenous Ca2+ or Mg2+ did not alter the SANG‐mediated inhibition of PMN functions. Treatment of PMNs with 3.12 μM or higher concentrations of SANG also resulted in inhibition of PMN degranulation and phagocytosis. The results suggest that SANG‐mediated inhibition of PMN functions, without cytolysis or resultant release of inflammatory mediators, may have clinical implications.
Use of randomly cloned DMA fragments for the identification of oral spirochetes
Authors: DiRienzo Josheph M. , Cornll Susan , Boehrnger Hansruedi
Published: December 2007

DNA probes were produced for the detection and identification of 4 cultivable species of oral spirochetes, Treponema denticola, Treponema socranskii, Treponema vincentii and Treponema pectinovorum. To obtain probe sequences, chromosomal DNA, isolated from representative strains within each species, was cloned in Escherichia coli K‐12. Cloned DNA fragments were screened for the ability to hybridize to DNA only from homologous strains. Several such fragments were identified and shown to be specific when tested against a series of DNAs from gram‐negative and gram‐positive oral bacteria. The selected probe sequences were semi‐conserved within strains of T. denticola and T. socranskii such that restriction fragment length polymorphism (RFLP) was observed. In the case of T. socranskii, RFLP was useful in distinguishing between the 3 known subspecies. Chromosomal DNA fragments from 2 strains of T. vincentii failed to cross‐hybridize, under stringent conditions, to genomic DNA from each of these strains. The hybridization probes were suitable for the identification of clinical isolates of T. denticola and could be used to detect the presence of individual Treponema species in mixed cultures. On this basis, the probes were used successfully to detect T. denticola in uncultured plaque samples.
Use of monoclonal antibodies to enumerate spirochetes and identify Treponema denticola in dental plaque of children, adolescents and young adults
Authors: Barren S. L. , Riviere G. R. , Simonson L. G. , Lukehart S. A. , Tira D. E. , O'Neil D. W.
Published: December 2007

Identification of spirochetes in dental plaque is difficult. Not all spirochetes can be cultured and microscopic analysis based on darkfield or phase optics cannot determine the genus and species of individual bacterial cells. The purpose of this study was to use monoclonal antibodies in an immuno enzyme technique to stain spirochetes in dental plaque. Separate mAb were used to estimate total spirochetes and relative numbers of 2 distinct types of Treponema denticola. Plaque samples were collected from 40 subjects grouped by age. Results showed that older subjects are more likely to have spirochetes, to have more spirochetes and to have more diverse populations or spirochetes than younger subjects. Our studies suggest that T. denticola may be the first treponeme to colonize the primary dentition, that T. denticola appears to comprise a major proportion of all spirochetes at all ages and that two distinct serotypes of T. denticola are found to coexist in Plaque from most children.
Associations of microbiological factors and plaque index with caries prevalence and water fluoridation status
Authors: Eisenberg A. D. , Mundorff S. A. , Featherstone J. D. B. , Leverett D. H. , Adair S. M. , Billings R. J. , Proskin H. M.
Published: December 2007

Plaque indices, numbers of mutans streptococci, lactobacilli and total viable flora in plaque and saliva were assessed as part of a large‐scale cross‐sectional study designed to determine the factors that are associated with high caries activity in children. Subjects, 12 to 15 years old, residing in areas where the water supplies were either fluoridated (n= 140) or fluoride‐deficient (n= 173) were studied. Mutans streptococci and lactobacilli numbers in saliva were positively correlated with plaque index. Plaque indices were significantly higher in the high‐caries group than in the zero‐caries group of the fluoride‐deficient community. In the fluoridated community, there was no difference in the plaque index between the zero‐ or high‐caries groups. In each community, pooled dental plaque and saliva harbored fewer mutans streptococci and lactobacilli in the zero‐caries than the high‐caries group. Greater numbers of mutans streptococci were found in the dental plaque and saliva of the zero‐caries subjects in the fluoridated community than in the fluoride‐deficient community, suggesting that a greater caries challenge can coexist with zero‐caries status in the fluoridated community.
Sorbitol inhibition of glucose metabolism by Streptococcus sanguis 160
Authors: Hamilton I. R. , Svensater G.
Published: December 2007

Hamilton IR, Svensater G. Sorbitol inhibition of glucose metabolism by Streptococcus sanguis 160.

Clinical studies in Sweden have shown that the proportion of sorbitol‐utilizing strains of Streptococcus sanguis increases in dental plaque from individuals using sorbitol‐containing products for prolonged periods. We have undertaken to study the metabolism of glucose and sorbitol by S. sanguis 160, isolated from a subject consuming sorbitol‐containing chewing‐gum 4 times a day for 4 years. Growth on glucose was inhibited by the presence of sorbitol in the growth medium and sorbitol was utilized in the presence of glucose, albeit, at a slower rate than glucose. In addition, pulses of glucose added to cultures growing on sorbitol resulted in the expulsion of sorbitol from the cell. In order to examine further the relationship of sorbitol and glucose, uptake assays were carried out with S. sanguis 160 grown in continuous culture (pH 7.0, dilution rate = 0.1 h−1) with glucose, sorbitol or nitrogen (sorbitol excess) limitations. The uptake of [14C]‐glucose by sorbitol‐limited cells, but not by glucose‐limited cells, was inhibited by sorbitol, as was glycolysis. Kinetic experiments with glucose‐limited cells showed 2 transport systems for glucose with Ks values of 5.2 and 40 μM, and glucose phosphorylation activity by decryptified cells indicated transport by the P‐enolpyruvate (PEP) phosphotransferase system (PTS) with lesser activity for an ATP‐dependent transport process. Transition from glucose‐limited growth to sorbitol‐limited growth revealed repression of total [14C]‐glucose uptake by intact cells and activity for Enzyme II for glucose (Ellglc) of the PTS measured in membrane preparations in the presence of an excess of the soluble PTS proteins in crude cell‐free supernatant fractions. In a ‘crossover’ experiment, Ellglc activity in membranes of glucose‐limited cells was repressed when the glucose‐limited soluble fraction was replaced by the soluble supernatant derived from sorbitol‐limited and sorbitol‐excess cells. This suggests that the transition from a glucose limitation to a sorbitol limitation resulted in the repression of a soluble PTS component, possibly Enzyme III for glucose (IIIglc), necessary for glucose transport. In addition, the 8‐fold lower Ellglc activity in sorbitol‐limited membranes when combined with the glucose‐limited soluble components revealed repression of Ellglc by sorbitol. Higher repression (103‐fold) of Ellglc activity was observed with sorbitol‐excess membranes. This evidence indicates that sorbitol is an effective catabolite repressor of the glucose‐PTS, exhibiting negative regulation over synthesis of both Ellglc and a soluble component, presumably IIIglc.
Identification of oral yeasts by polyacrylamide gel electrophoresis
Authors: Maiden M. F. J. , Tanner A.
Published: December 2007

Protein profiles of sonicated cells for 9 species of yeasts isolated from oral samples were obtained by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), and visualized with a silver stain. The profiles obtained on 12% acrylamide gels were distinct, and characteristic for 6 species of Candida, and Torulopsis glabrata, Yarrowia lipolytica, and Saccharomyces cerevisiae. Using this method, 79 fresh isolates of yeasts from saliva samples were identified; 58 as Candida albicans, 9 as Candida parapsilosis, 1 as Candida tropicalis, and 11 as T. glabrata. SDS‐PAGE offers a rapid, convenient alternative or adjunct to yeast identification systems based on carbohydrate assimilation tests.
Extracellular vesicle‐associated and soluble trypsin‐like enzyme fractions of Porphyromonas gingivalis W50
Authors: Smalley John W. , Birss Andrew J.
Published: December 2007

Soluble vesicle‐associated trypsin‐like enzyme fractions (VSF) were prepared by‐sonication from extracellular vesicles (ECV) from strains W50 and W50/BE1. High‐(H), intermediate‐(I) and low‐(L) molecular‐weight VSF enzyme subfractions were identified by non‐dissociative gel filtration chromatography with Mr 160, 95 and 60 kDa respectively. The chromatographic profiles of W50 VSF from 48‐h and 72‐h cultures were identical. W50/BE1 VSF displayed a higher ratio of the 160 to 60 kDa components. This ratio was reduced in VSF from 72‐h cultures. Extracellular soluble protein (EP) trypsin profiles were similar to their respective VSF, but the 60 kDa component predominated. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) revealed a loss of soluble extracellular polypeptides with culture age. A polyclonal antiserum to EP subfraction L reacted in immunoblots with a 50 kDa peptide of subfraction L of W50. Whole EP and its subfraction H displayed a 50 kDa immunoreactive peptide but no peptides of higher molecular weight. This antiserum reacted with a similar sized peptide, and with lower‐molecular‐weight components in whole ECV. Gelatin substrate zymography of whole EP following non‐reducing SDS‐PAGE revealed a major 80 kDa protease that increased with culture age. Minor protease bands of 70 and 50 kDa were also detected.
Cell surface hydrophobicity and electrokinetic potential of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus
Authors: Holm Anders , Kalfas Sotirios
Published: December 2007

Laboratory strains and fresh isolates of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus were examined for cell surface hydrophobicity and for electrokinetic properties under different experimental conditions. Fresh isolates of A. actinomycetemcomitans and H. aphrophilus were hydrophobia Laboratory strains of A. actinomycetemcomitans were 20–30% less hydrophobic than fresh isolates. No difference was observed between laboratory and fresh isolates of H. aphrophilus. The pH of the suspending buffer, growth phase or incubation atmosphere did not significantly affect the hydrophobicity of the 2 species, whereas agar cultures of H. aphrophilus were less hydrophobic than broth cultures. All A. actinomycetemcomitans strains treated with sterile filtered saliva showed a concentration‐dependent decrease in hydrophobicity of at most 30%. H. aphrophilus strains were not affected by the same treatment. Laboratory strains of H. aphrophilus were more negatively charged than A. actinomycetemcomitans, whereas fresh isolates of the 2 species exhibited similar surface charge. In the presence of saliva the mean cell surface charge of laboratory strains decreased by 56% for A. actinomycetemcomitans and by 73% for H. aphrophilus. The results indicate that the 2 species differ in expression of cell structures accounting for hydrophobicity and surface charge and that environmental factors might differently influence the physical properties of the two species analyzed.
Adhesive properties of strains of Fusobacterium nucleatum of the subspecies nucleatum, vincentii and polymorphum
Authors: Xie H. , Gibbons R. J. , Hay D. I.
Published: December 2007

This study surveyed some adhesive properties of strains of Fusobacterium nucleatum representative of the 3 recently defined groups or subspecies that could relate to their colonization and virulence. With one exception, F. nucleatum strains agglutinated sheep erythrocytes, but the quantity of bacteria required and the sensitivity of the hemagglutination reactions to inhibition by 0.05 M galactose or arginine varied between strains, and did not exhibit clear‐cut correlations with subspecies. Neuraminidase treatment of erythrocytes generally enhanced the hem‐agglutinating activity of most strains, but trypsin treatment had no effect. Strains of F. nucleatum also attached in moderate numbers to buccal epithelial cells. Treatment of the epithelial cells with neuraminidase or with trypsin increased the numbers of all Fusobacterium strains that attached. Treatment of hydroxyapatite (HA) beads with submandibular or parotid saliva also promoted the adhesion of all strains of F. nucleatum studied. Treatment of HA with human serum or albumin produced a selective effect. Adhesion of some strains was promoted by serum and albumin treatment, and that of other strains was unaffected. Adhesion of all strains of F. nucleatum was enhanced to statherin‐treated HA, whereas HA treated with salivary pro line‐rich protein‐1 did not foster F. nucleatum attachment. Three of 4 strains of the subspecies vincentii, and each of 2 polymorphum strains studied exhibited strong adhesion to HA treated with either human type I or type IV collagen. However, only 1 of 5 strains of the subspecies nucleatum bound well to collagen‐treated HA. The data obtained indicate that strains of F. nucleatum are heterogeneous in their adhesive properties, and possess different collections of adhesins that mediate their attachment to surfaces. The enhanced attachment of F. nucleatum cells to neuraminidase or trypsin‐treated epithelial cells likely contributes to their increased proportions being associated with poor oral hygiene and gingivitis. In addition, the binding of some F. nucleatum strains to serum or albumin‐treated apatitic surfaces may contribute to their tropism for colonizing subgingival sites, and the ability of some strains to bind to collagen may contribute to their invasive potential.
Comparison of the amino acid uptake profile of reference and clinical isolates of Fusobacterium nucleatum subspecies
Authors: Gharbia Saheer E. , Shah Haroun N.
Published: December 2007

Human isolates of Fusobacterium nucleatum subspecies appear to colonize different niches in the oral cavity, which may be reflected in their nutritional properties. Consequently the utilization of nitrogenous substrates, their sources of energy (supplied here as amino acids) were compared between the 3 subspecies using the reference strain and fresh clinical isolates of each subspecies. All strains incorporated mainly acidic and basic amino acids but significant differences occurred between subspecies. Both reference and clinical isolates of F. nucleatum subspecies polymorphum utilized all amino acids in the medium but the levels of glutamate, arginine and cysteine were noticeably higher in the reference strain. By contrast, F. nucleatum subspecies fusiform used a very restricted range of amino acids, of which only glutamate, arginine, histidine and cysteine were taken up at greater than 0.5 mM. F. nucleatum subspecies nucleatum utilized fewer amino acids than F. nucleatum subspecies polymorphum but higher concentrations were taken up by the former. Clinical isolates of F. nucleatum subspecies nucleatum incorporated polar and nonpolar neutral amino acids poorly but their levels increased steadily as a clinical isolate was subcultured over a period of 4 months, and was eventually similar to the reference strain. The effect of adding the key catabolic substrate, glutamate (10 mM), on the amino acid uptake profile of F. nucleatum subspecies nucleatum resulted in the complete suppression of the dibasic amino acids arginine, ornithine and histidine. Strains of this subspecies could grow on glutamate as a major source of carbon and energy but, morphologically, the cells appeared somewhat distended and had a tendency to clump.
Cloning and expression of protease genes from Treponema denticola in Escherichia coli
Authors: MacDougall J. H. , Beighton D. , Russell R. R. B.
Published: December 2007

A gene bank of random fragments of chromosomal DNA from Treponema denticola ATCC 33520 was constructed in the bacteriophage vector λL47.1. The gene bank was plated on Escherichia coli C600 and screened for the presence of plaques in which enzyme activity was expressed, using overlays of fluorogenic synthetic substrates and a two‐step procedure in which immunological screening was followed by enzyme assays of immunopositive lysates. Recombinants expressing trypsin‐like, chymotrypsin‐like and proline iminopeptidase activities were found. The gene for the trypsin‐like activity was subcloned into plasmid pJDC9 and maintained in E. coli.
Acidogenesis in relation to fluoride resistance of Streptococcus mutans
Authors: Van Loveren C. , Van de Plassche‐Simons Y. M. , De Soet J. J. , De Graaff J. , Ten Gate J. M.
Published: December 2007

The velocity of acid production (Vap) of Streptococcus mutans C180‐2 and of 2 fluoride‐resistant mutant strains S. mutans C180‐2FR and S. mutans C180‐2MFR was examined in vitro at pH values between 7.0 and 4.5. The Vap of the fluoride‐resistant mutants was lower than the Vap of the parent strain at pH ≥6.0. At pH 5.5 and 5.0, the Vap of the mutant strains was higher than the Vap of the parent strain, whereas at pH 4.5 no significant differences were observed between the Vap of the 3 strains. The fluoride sensitivity of all 3 strains was amplified by a low pH environment. The fluoride concentration necessary to inhibit the acid production of the parent strain completely was 27 mM at pH 7 and 0.1 mM at pH 4.5. For the 2 mutants, the corresponding concentrations were 65 mM at pH 7 and 0.5–0.7 mM at pH 4.5. The results suggest that, if S. mutans acquires fluoride resistance in vivo, the rate of acid production in dental plaque may be decreased at pH ≥6, but increased at lower pH levels. Low concentrations of fluoride inhibit acid production less effectively.
Human leukocyte antigens in relation to colonization by mutans streptococci in the oral cavity
Authors: Wallengren Marie Louise , Ericson Dan , Forsberg Björn , Johnson Ulf
Published: December 2007

Mutans streptococci are well established as caries‐inducing microorganisms in man. Most humans carry the bacteria, but in highly different numbers. This cannot be explained by environmental factors only. The aim of this study was to investigate a possible association between levels of colonization by mutans streptococci and the presence of certain B and DR human leukocyte antigens (HLA). Altogether, 170 subjects who had their HLA antigens determined (76 renal' transplant patients and 94 healthy blood donors) were selected for the investigation. Paraffin‐stimulated saliva samples were taken using the wooden spatula method with subsequent cultivation of mutans streptococci on mitis salivarius bacitracin agar plates. An association between the absence of HLA‐DR 4 antigens and low, or undetectable, levels of mutans streptococci was found. This was statistically significant for the immunosuppressed renal transplant subjects. The same trend was observed among the healthy blood donors.
Metabolism of intracellular polysaccharide in the cells of Streptococcus mutatis under strictly anaerobic conditions
Authors: Takahashi Nobuhiro , Iwami Yoshimichi , Yamada Tadashi
Published: December 2007

Streptococcus mutans, which had accumulated glycogen‐like iodophilic intracellular polysaccharide (IPS), produced large amounts of formate, acetate and ethanol from the IPS by pyruvate formate‐lyase (PFL) under strictly anaerobic conditions without exogenous sugar. Under aerobic conditions, the same S. mutans produced exclusively lactate and pyruvate from the IPS because of the inactivation of PFL by oxygen. The total amount of acid produced under anaerobic conditions was larger than that under aerobic conditions. The analysis of intracellular glycolytic intermediates revealed that levels of fructose 1,6‐bisphos‐phate (lactate dehydrogenase (LDH) activator) and glyceraldehyde 3‐phosphate and dihydroxyacetone phosphate (PFL inhibitors) were low when IPS was used as a glycolytic substrate, implying that PFL functions more efficiently than LDH in IPS metabolism. These findings suggest that the PFL pathway contributes to the acid production from IPS, and may explain partially why the acids in starved dental plaque are mainly acetate and formate.
Purification and properties of neuraminidase from the culture medium of Bacteroides loescheii
Authors: Takeshita Tetsuo , Takagaki Masaru , Amano Atsuo , Murakami Yukitaka , Nagata Hideki , Shizukuishi Satoshi
Published: December 2007

Neuraminidase was purified from the culture medium of Bacteroides loescheii ATCC 15930 by ultrafiltration followed by a DEAE‐Sephacel anion exchange chromatography, fast‐protein liquid chromatography using Mono P column and high‐performance liquid chromatography using a Shim‐pack Diol‐300 column. The molecular weight of the purified enzyme was measured to be approximately 87 kDa and the optimal pH was at 4.8. Although the enzyme was able to hydrolyze the substrates with α, 2–3, α,2–6, and α,2–8 linkages of N‐acetylneuraminic acid, the rate of hydrolysis of N‐acetylneuraminosyl‐α,2–3‐lactose was greater than that of the α,2–6‐isomer.
Identification of the major surface protein antigens of Porphyromonas gingivalis using IgG antibody reactivity of periodontal case‐control serum
Authors: Curtis M.A. , Slaney J.M. , Carman R.J. , Johnson N.W.
Published: December 2007

The identity of the major surface antigens of Porphyromonas gingivalis was investigated. Outer membranes of P. gingivalis strains W83, W50, 381 and NCTC 11843 were prepared following inactivation of the trypsin‐like enzyme activity. Three proteins, molecular weight 115, 55 and 40 kDa, were major components of the outer membranes of strains W83 and W50 and were also present in strains 381 and NCTC 11834. Two proteins, 55 and 47 kDa, were released from the cells during the sonication step of the outer membrane preparation procedure. Immunoblots using preparations of P. gingivalis W83 and serum from a case‐control study of adult periodontitis demonstrated higher mean antibody reactivity in the case population to all the major proteins except for the 115 kDa outer membrane protein, which was recognized equally well by both populations. We conclude that the 55, 47 and 40 kDa proteins are important surface antigens of P. gingivalis. Characterization of the structure and function of these components should lead to an improved understanding of the host‐parasite interactions in adult periodontitis.
Phenotypic analysis of B‐cells extracted from human periodontal disease tissue
Authors: Gemmell E. , Seymour G. J.
Published: December 2007

B‐cells extracted from periodontal disease tissue were analyzed for the presence of activation markers using a range of monoclonal antibodies. In adult periodontitis (AP), 6% of B‐cells expressed the IL‐2 receptor (CD25) compared with 1–2% in peripheral blood and healthy or marginal gingivitis (H/MG) gingival B‐cells. There was also an increase in the mean percentage of IgD‐positive B‐cells and a decrease in CD21 and CD22 expression. In both AP and H/MG lesions, 20–22% of the B‐cells expressed CD23 compared with < 5% in peripheral blood. As B‐cells are activated by day 3 in culture and start differentiating into immunoglobulin‐secreting cells by day 6, B‐cell phenotypes were assayed at these times in this study. Following stimulation with the periodontopathic bacterium Porphyromonas gingivalis, the expression of CD23, CD21 and CD22 on B‐cells extracted from AP lesions remained relatively constant over the 6‐d culture period. However, with Fusobacterium nucleatum stimulation, there was a significant decrease in CD23, CD21 and CD22 expression after 3 d in culture, which corresponds to the activation time for B‐cells. These results show that B‐cells extracted from periodontal disease tissue display a range of activation markers and on stimulation, demonstrate differing responses to individual periodontopathic bacteria.
Effect of lipopolysaccharide from Porphyromonas gingivalis on prostaglandin E2 and interleukin‐1β release from rat periosteal and human gingival fibroblasts in vitro
Authors: Sismey‐Durrant Helen J. , Hopps Rosamund M.
Published: December 2007

Lipopolysaccharide (LPS) was extracted from Porphyromonas gingivalis (W83) by the Westphal procedure, nuclease‐digested and ultracentrifuged. Fibroblasts were obtained from human gingival tissue and rat periosteum, grown to confluence then stimulated in serum‐free medium with 0.1, 1.0 and 10.0μg/ml LPS. The levels of prostaglandin E2 (PGE2) and interleukin‐1β (IL‐1β) released were measured after 2, 4 and 6 d by specific radioimmunoassays. Unstimulated gingival fibroblasts produced low levels of PGE2 (24.5 ± 1.5 (SD) ng/ml) and IL‐1β (0.34 ± 0.29 ng/ml). LPS stimulated statistically significant dose‐related increases hi PGE2 and IL‐1β at the concentrations of LPS tested. At 10.0μg/ml, LPS‐stimulated fibroblasts produced 363.5 ± 40.3 ng/ml PGE, and 1.81±0.1 ng/ml IL‐1β in 6 d. These results demonstrate that LPS from P. gingivalis is capable of stimulating PGE, and IL‐1β release from fibroblasts. This would appear to be an additional mechanism by which LPS can induce tissue breakdown in periodontal disease.
Peptostreptococcus micros in human periodontitis
Authors: Rams Thomas E. , Feik Diane , Listgarten Max A. , Slots Jørgen
Published: December 2007

Peptostreptococcus micros is a recognized pathogen in medical infections, and its association with progressive periodontitis was examined in this study. P. micros was isolated from paper‐point subgingival samples on anaerobic enriched blood agar plates and identified on the basis of cellular and colonial morphology and selected biochemical tests. In a cross‐sectional study involving 907 people with advanced adult periodontitis, 127 with early‐onset periodontitis, and 12 with localized juvenile periodontitis, P. micros in these patient groups occurred with a prevalence of 58‐63%. In culture‐positive patients, P. micros averaged 12‐15% of total viable counts. P. micros demonstrated similar occurrence and proportional recovery in all age groups. In a longitudinal study of 91 adult periodontitis patients on maintenance therapy, P. micros demonstrated a significantly higher prevalence in disease‐active than in disease‐inactive patients (47% vs 14%). Mechanical subgingival debridement and 0.12% chlorhexidine pocket irrigation was unable to eradicate subgingival P. micros from 18 of 22 adult periodontitis patients. In vitro antimicrobial susceptibility testing showed P. micros to be sensitive to therapeutic levels of penicillin, clindamycin and metronidazole. Our findings indicate that P. micros is a potential pathogen in adult periodontitis. The methods for its eradication from subgingival sites remain to be determined.
Dot blot hybridization in detection of human papillomavirus (HPV) infections in the oral cavity of women with genital HPV infections
Authors: Kellokoski Jari , Syrjänen Stina , Yliskoski Merja , Syrjänen Kari
Published: December 2007

The presence of human papillomavirus (HPV) types 2, 6, 7, 11, 13 and 16 DNA in cytologic scrapings of oral mucosa was studied in 309 women with genital HPV infections. The objective was to test the usefulness of oral mucosal scrapings (3 sequential swabs) in HPV DNA detection by dot blot hybridization. Based on hybridization with the 32P‐Labeled Alu‐repeat probe, most samples contained more than 105 cells, which is an adequate number of cells for dot blot hybridization. Hybridization with 32P‐lapelled HPV DNA probes showed that 3.8% of the 309 women had an oral HPV infection. Of these, only 2 had clinical lesions indicative of HPV. All other oral HPV positive subjects had clinically healthy mucosa. HPV 6 was the most common (3.1%) type, followed by HPV 11 and 16 (1.1%). In 3 cases the genital mucosa harboured the same HPV type as found in the oral cavity. The results indicate that oral mucosal scraping results in adequate number of cells for dot blot hybridization with HPV DNA. Although the method is likely to result in an underestimation of latent and subclinical HPV infections, it is useful for studying the clinical HPV infections as well as other viral infections known to be present in exfoliated cells.
The early colonization of gram‐negative anaerobic bacteria in edentulous infants
Authors: Könönen E. , Asikainen S. , Jousimies‐Somer H.
Published: December 2007

The occurrence of oral gram‐negative anaerobes was examined in 30 edentulous infants (mean age 3 months, range 1‐7 months). One pooled swab sample from mucosal surfaces (cheeks, palate, tongue) and one saliva sample was taken from each infant. The samples were cultured aerobically and anaerobically using non‐selective and selective media. Prevotella (Bacteroides) melaninogenica was the most frequently isolated anaerobe, found in 70% of the infants. The other common anaerobes were Fusobacterium nucleatum, Veillonella spp. and nonpigmented Prevotella (Bacteroides) spp., found in 60%, 57% and 57% of the infants, respectively. Of corroding bacilli, Bacteroides gracilis was detected in 23% of the infants, Wolinella spp. and microaerophilic Eikenella corrodens in one infant (3% each). Leptotrichia spp., microaerophilic Capnocytophaga spp., Prevotella (Bacteroides) loescheii and Prevotella (Bacteroides) intermedia were found in 17%, 13%, 13% and 7% of the infants, respectively. In addition to these 30 infants, 21 edentulous infants were investigated for the presence of Actinobacillus actinomycetemcomitans only. A. actinomycetemcomitans was not detected in any of the 51 edentulous infants. The number of different anaerobic bacterial species in the same mouth varied from 0 to 7. No anaerobic bacteria were detected in 3 of 30 children (10%). These data suggest that various anaerobic bacterial species readily colonize the edentulous mouth in infants.
Use of a replica‐plate assay for the rapid assessment of salivary protein‐bacteria interactions
Authors: Tseng Ching‐Chung , Scannapieco Frank A. , Levine Michael J.
Published: December 2007

A replica‐plate assay was used to screen for the interaction of salivary molecules with dental plaque bacteria. Bacterial colonies cultured from supragingival plaque on sheep‐blood (SB) agar were replica‐plated onto nitrocellulose membranes overlaying SB or mitis‐salivarius agar. Membranes with attached colonies were removed and incubated with 125I‐amylase or 125I ‐proline‐rich glycoprotein (PRG). Positive interactions were detected by autoradiography. Only strains of Streptococcus gordonii and Actinomyces viscosus bound amylase, and strains of A. viscosus bound PRG. The results suggest that amylase and PRG bind to selected species of aerobic dental plaque bacteria.
Characterization of the adherence of Porphyromonas gingivalis to oral streptococci
Authors: Lamont R. J. , Hersey S. G. , Rosan B.
Published: December 2007

Adherence of Porphyromonas gingivalis to strains of Streptococcus sanguis and Streptococcus mitis deposited on nitrocellulose paper was investigated. A variety of laboratory strains and clinical isolates of P. gingivalis bound to both S. sanguis and S. mitis. Binding of P. gingivalis to all but one of the streptococci was not inhibited by salivary molecules. Pretreatment of P. gingivalis with periodate and pretreatment of S. sanguis and S. mitis with pronase decreased binding, suggesting that adherence may be mediated by a protein on the streptococci interacting with a carbohydrate on P. gingivalis. Binding was not inhibited by a selection of simple sugars. The ability to adhere to early plaque bacteria such as S. sanguis and S. mitis may be important in the colonization of the mouth by P. gingivalis.
Survey of a receptor protein in human erythrocytes for hemagglutinin of Porphyromonas gingivalis
Authors: Hayashi H. , Nagata A. , Hinode D. , Sato M. , Nakamura R.
Published: December 2007

The purpose of this study is to survey a receptor protein in human erythrocyte membrane for the hemagglutinin (HA) of Porphyromonas gingivalis. Human erythrocytes were modified by either chymotrypsin or P. gingivalis HA along with the disappearance of their hemagglutinating ability and the removal of the band 3 protein. By preparative electrophoresis, this protein was isolated and purified from human erythrocytes. The purified protein showed strong inhibitory activity for hemagglutination and the binding to P. gingivalis cells, whose binding sites were calculated to be approximately 9000, suggesting its binding to the active site of HA. Hemagglutinin purified from P. gingivalis by affinity absorption to sheep erythrocyte ghosts possessed strong trypsin‐like activity, and both the HA and the enzyme activities were inhibited by arginine. Specific modification of arginyl residues in human erythrocytes by phenylglyoxal diminished the hemagglutinating ability. From the similarity of the inhibition profile and possible active sites between HA and the trypsin‐like protease, it is suggested that hemagglutination may occur as a result of the primary reaction of the enzyme (protease) and the substrate. These results suggest that band 3 may be a key protein in human erythrocyte membrane for HA from P. gingivalis and its binding sites may be arginyl residues of the protein.
Suppressive effect of soluble factor(s) derived from Prevotella loescheii ATCC 15930 on proliferation of human lymphocytes
Authors: Terao N. , Saito S. , Hayakawa M. , Abiko Y.
Published: December 2007

Soluble sonic extracts of Prevotella loescheii caused a dose‐dependent inhibition of human peripheral blood lymphocyte proliferation by mitogen and of the proliferation of a leukemic cell line, BALL‐1, when assessed by DNA synthesis (3H‐thymidine incorporation). RNA (3H‐uridine incorporation) and protein (3H‐leucine incorporation) synthesis were similarly altered after exposure to the extract. There was no effect on cell viability as measured by either trypan blue exclusion or extracellular release of the cytoplasmic enzyme lactate dehydrogenase. Preliminary characterization indicates the suppressive factor(s) derived from P. loescheii to be a protein since it is heat‐labile and trypsin‐sensitive. The factor eluted in a peak on a high‐pressure liquid chromatography gel filtration corresponding to a molecular weight of approximately 32,000. Since black‐pigmented anaerobic rods have been implicated in the pathogenesis of periodontal disease, the data suggest that P. loescheii contributes to the disease process by suppressing lymphocyte function.
CD45RA and CD45RO positive CD4 cells in human peripheral blood and periodontal disease tissue before and after stimulation with periodontopathic bacteria
Authors: Gemmell E. , Feldner B. , Seymour G. J.
Published: December 2007

Flow cytometric analysis was used to examine naive and primed or memory CD4 cells extracted from periodontal lesions compared with cells from peripheral blood of healthy subjects before and after stimulation with the periodontopathic bacteria, Porphyromonas gingivalis and Fusobacterium nucleatum. In peripheral blood, approximately 60% and 40% of CD4 cells were CD45RO+ and CD45RA+ respectively at day 0. Phytohaemagglutinin (PHA) induced CD45RO expression on almost 100% of CD4 cells. However, P. gingivalis and F. nucleatum stimulation did not cause any significant change in percentage of CD45RO+ CD4 cells except for a loss of antigen at day 6 together with re‐expression at day 7, which also occurred on cells cultured in medium only. CD45RA expression on PHA and bacterial‐stimulated peripheral blood CD4 cells remained fairly stable for the 10‐d culture period. Greater than 90% CD4 cells extracted from healthy or marginal gingivitis (H/MG) and adult periodontitis (AP) lesions were CD45RO+ and this was maintained on AP cells throughout the 6‐d culture period, except for a small decrease in the percentage of positive cells induced by P. gingivalis at day 3. Approximately 9% CD4 cells from H/MG tissue were CD45RA+, but about 22% AP cells expressed this antigen, and this increased again in P. gingivalis‐ and F. nucleatum‐stimulated cultures after 3 d. Therefore, in peripheral blood P. gingivalis and F. nucleatum do not act as nonspecific T‐cell mitogens and, in AP cells, these bacteria induce changes in phenotype, supporting previous data that although they may be polyclonal B‐cell activators, they activate antigen specific T‐cells.
Bacterial synergy of Treponema denticola and Porphyromonas gingivalis in a multinational population
Authors: Simonson L. G. , McMahon K. T. , Childers D. W. , Morton H. E.
Published: December 2007

Treponema denticola and Porphyomonas gingivalis have been associated with human adult severe periodontitis. In this study, we quantified these putative pathogens in subgingival plaque samples collected from 74 Fijians, 74 Colombians and 73 U.S. Americans stationed at the Multinational Force and Observers encampment in the Sinai Desert, Egypt. A contingency table of T. denticola and P. gingivalis frequency revealed a highly significant synergistic relationship. We discovered that the occurrence of T. denticola apparently requires the presence of P. gingivalis. This represents the first observation of a synergistic relationship between these putative oral pathogens associated with adult severe periodontal disease.
Antibiotic resistance of the subgingival microbiota following local tetracycline therapy
Authors: Goodson J. M. , Tanner A.
Published: December 2007

The antibiotic resistance of the subgingival microbiota was studied by 3 approaches. First, we assessed the ability of subgingival isolates taken following therapy to grow on media containing tetracycline (TC). Higher percentages of TC‐resistant organisms appeared at TC fiber‐treated periodontal sites and within the saliva 1 week after treatment as compared with pre‐treatment levels. By 1 month, the percentage of TC‐resistant organisms had returned to levels comparable to those seen before treatment. In the second approach, subgingival isolates taken following therapy were grown on media without antibiotics, and isolates were selected for Gram‐stain and cell morphology determination. This study indicated that subgingival sites became colonized with gram‐positive cocci in the same time period that an increase of TC‐resistant isolates was observed in the first study. This may account for the transient increase in TC resistance, because many gram‐positive cocci are intrinsically resistant to TC. In the third approach, the antibiotic resistance of subgingival gram‐negative species was determined. The predominant cultivable microbiota of 9 sites from 3 subjects were isolated immediately before and 6 months after TC fiber treatment. Gram‐negative rods were characterized and tested for sensitivity to TC (minimum inhibitory concentration [MIC] 1‐128 μg/ml), penicillin at 80 μg/ml, and erythromycin at 8 μg/ml. None of the gram‐negative rods were resistant to TC (MIC 16 μg/ml), either before or after treatment. Before treatment 98% of the gram‐negative rods were susceptible to TC at 1–2 μg/ml and after therapy 88% were susceptible. The percentage of gram‐negative rods exhibiting intermediate TC susceptibility (MIC 4–8 μg/ml) changed from 2.0% before therapy to 5.2% after therapy. In no case was either penicillin or erythromycin resistance associated with increased resistance to TC.