bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA fingerprinting of Eikenella cormdens by pulsed‐field gel electrophoresis
Authors: Steffens L. , Franke S. , Nickel S. , Schwarzkopf A. , Flemmig T. F. , Karch H.
Published: December 2007

In order to conduct molecular typing of Eikenella corrodens strains by macrorestriction fingerprinting, we evaluated different restriction enzymes for digestion of genomic DNA and determined the optimal parameters for separating E. corrodens DNA by pulsed‐field gel electrophoresis. Ten E. corrodens strains isolated from oral and extraoral infection sites in different individuals were analyzed. The rare‐cutting restriction endonucleases DraI, SmaI and XbaI usually used for pulsed‐field gel electrophoresis analyses were not suitable for digestion of E. corrodens genomic DNA because they either did not digest the DNA or produced bands of similar molecular weights that could not be separated. Accordingly, among additional enzymes including BamHI, BglII, EcoRI and Hind III, we found BamHI and BglII to be the most suitable rare‐cutting enzymes for pulsed‐field gel electrophoresis analysis. They cleaved the genomes of all the above strains into 15–20 fragment bands that were clearly separated by the following pulsed‐field gel electrophoresis conditions: 140 V with a running time of 40 h, pulse times of 5 to 50 s with linear ramping and an electrical field angle of 120. These conditions enabled us to distinguish 8 individual pulsed‐field gel electrophoresis patterns from the 10 strains analyzed. However, only 4 identical outer membrane protein profiles were differentiated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The data obtained in this analysis showed clonal divergence among members of the E. corrodens species, at the same lime revealing this pulsed‐field gel electrophoresis as being a highly attractive procedure for epidemiological investigation of this organism, including its acquisition and transmission.
The most frequently isolated gram‐negative anaerobes in saliva and subgingival samples taken from young women
Authors: Könönen E. , Jousimies‐Somer H. , Asikainen S.
Published: December 2007

Stimulated saliva and a pooled subgingival sample from the first molars were collected from 30 Caucasian women (mean age 30 years) for the presence of gram‐negative anaerobic bacteria. The samples were inoculated on nonselective and selective media. The gram‐negative anaerobic species occurring in the oral cavity of more than 50% of the subjects were selected for further analysis. Fusobacterium nucleatum and Prevotella melaninogenica were not recovered from subgingival sites without a simultaneous recovery in saliva. When positive subgingivally, Leplotrichia spp., nonpigmented Prevotella spp., Prevotella intermedia, Capnocytopha‐ga spp., Bacteroides gracilis and Prevotella loescheii were simultaneously detected from saliva in 76%, 72%), 64%>, 64%, 61% and 55% of the subjects, respectively. Intraindividually, the proportions of F. nucleatum, nonpigmented Prevotella spp., Leptotrichia spp., B. gracilis and P. loescheii of the anaerobic flora were quite similar in the salivary and subgingival samples. Capnocytophaga spp. and P. intermedia were present in higher proportions in subgingival samples than in saliva, whereas P. melaninogenica was present in higher proportions in saliva. Our data indicate that many oral gram‐negative anaerobes are frequently recovered from stimulated saliva. Furthermore, when subgingivally positive, most subjects harbored these anaerobes simultaneously in saliva.
A comparison of salivary immunoglobulin A (IgA) and IgA subclass concentrations in predentate and dentate children and adults
Authors: Tappuni A. R. , Challacombe S. J.
Published: December 2007

Local immune factors may play a role in the protection against oral disease and defences against microflora may be related to immunoglobulin A (IgA) subclass responses. The objectives of this study were to determine the concentrations and ratios of salivary IgA and IgA subclasses in predentate and dentate children and adults and to compare the levels in breastfed and bottle‐fed infants. Whole saliva samples were collected from: a) 44 predentate children (mean age 4 months), b) 29 dentate children (1.5 years) and c) 28 adults (29.5 years). Immunoglobulin levels were assayed by enzyne‐linked immunosorbent assay using colostral IgA standard. In the predentate children, the mean IgA, IgA1 and IgA2 levels were lower than in the dentate group but the difference reached statistical significance with IgA2 levels only. The comparative concentrations in the adult group were significantly higher than that in both children's groups. The IgA1:IgA2 ratio was similar in all 3 groups, at approximately 58:42. No significant differences were found in the concentration of salivary IgA and IgA subclasses in the breastfed infants compared with the bottle‐fed ones. The results of this investigation confirm other studies suggesting that the concentration of salivary IgA subclasses are not significantly different between predentate and dentate children and that they do not reach adult levels until well after 2 years of age.
Polymerase chain reaction for the identification of Porphyromonas gingivalis collagenase genes
Authors: Bodinka A. , Schmidt H. , Henkel B. , Flemmig T. F. , Klaiber B. , Karch H.
Published: December 2007

Porphyromonas gingivalis has been shown to exhibit genetic diversity possibly resulting in variation of virulence. In the present study a potential virulence factor was targeted for the detection of P. gingivalis. A 548 bp fragment of the collagenase gene (prtC) from Porphyromonas gingivalis ATCC 33277 was amplified by polymerase chain reaction (PCR) using oligonucleotides derived from the middle portion of prtC. From 16 of 21 clinical P. gingivalis strains, a PCR product of similar size to the prtC could be obtained. These 16 P. gingivalis strains were confirmed as positive for prtC using DNA hybridization with a digoxigenin‐labeled prtC PCR product as a probe. In 12 of the 16 prtC positive strains, the restriction analysis of the PCR products revealed fragment patterns identical to the known sequence. In the other 4 prtC positive strains, 4 distinct patterns were found. Of these strains, nucleotide sequence analysis of a 400 bp PCR product stretch revealed 79.1%, 83.0%, 84.8 and 89.5% homology with the known nucleotide sequence for this specific region. Sequence analysis of the PCR products from the ATCC 33277 strain demonstrated 93.7% homology. The limit of detection for the PCR was about 100 organisms. None of the other 48 tested strains of 16 bacterial species derived from oral and extraoral infections yielded a PCR product. The PCR was also used for the detection of prtC sequences in dental plaque. Our data indicate that not all P. gingivalis strains have prtC. Nucleotide heterogeneity exists among P. gingivalis with prtC. Using a potential virulence factor for the detection of putative periodontal pathogens such as P. gingivalis may be valuable for the epidemiology of infection and clinical diagnosis of periodontal diseases.
The in vitro effect of human polymorphonuclear leukocyte azurophil granule components on natural killer cell cytotoxicity
Authors: Lindemann R. A. , Lala A. , Miyasaki K. T.
Published: December 2007

We previously demonstrated that human polymorphonuclear leukocyte (PMN) secretions are capable of activating and inhibiting natural killer cell (NK) cytotoxicity depending on the eliciting PMN stimulus. Serum‐opsonized zymosan induced PMN to secrete substances that enhanced NK activity in vitro. Serum‐opsonized zymosan stimulates the release of PMN azurophil granules, which contain both human neutrophil peptides (HNPs) and neutral serine proteases (NSPs). When HNPs and NSPs were tested for their ability to activate NK cells in peripheral blood lymphocytes, all but cathepsin G consistently enhanced cytotoxicity above control values. HNP‐induced cytotoxicity was significantly enhanced within 12 h, peaking at approximately 24 h. Of the HNPs, HNP‐1 was the most potent activator, enhancing NK activity at 1.25 μ/ml. Interleukin‐2 and inter‐feron‐1 were not involved in this activational process, as antibodies to interleukin‐2 and interferon‐y did not block activation by HNPs and NSPs, and interleukin‐2 receptor expression was unaltered after 24 h of incubation. Enzymatically inactivated elastase and cathepsin G produced equivalent activational effects to their active counterparts. Antisera to elastase and cathepsin G decreased but did not eliminate NK activation over untreated peripheral blood lymphocytes. These data suggest that certain PMN azurophil granule components, including HNPs and NSPs directly increase the cytotoxic activity of NK cells.
Effect of proteolytic enzymes on the lysis and growth of oral bacteria
Authors: Grenier D.
Published: December 2007

Previous studies have shown increased levels of proteolytic enzymes in affected periodontal sites. The aim of the present investigation was to evaluate the effect of proteolytic environments on the lysis and growth of selected oral bacteria associated with either healthy or diseased periodontal sites. The effect of trypsin, chymotrypsin and proleinase K on cell lysis was determined following incubation with bacteria, whereas the effect of the same proteolytic enzymes on bacterial growth was tested using a disc‐plate technique. Overall, gram‐positive bacteria appeared to be more resistant to lysis than gram‐negative bacteria. The most susceptible bacteria were Actinomyces spp., Eubacterium saburreum, Prerotella intermedia, Capnocytophaga ochracea, Fitsobacterium nucleatum, Prevotella loescheii, Treponema clenticola and Actinobacilhts actinomycetemcomitans. The disc‐plate procedure indicated that the growth of Actinomyces spp., E. sabwreum, C. ochracea, P. intermedia, P. loescheii, Porphyromonas gingivalis and T. dentieola were the most affected, more particularly by chymotrypsin and proteinase K. Interestingly, the growth of F. nucleatum was rather stimulated by proteolytic enzymes. The observations reported in this investigation indicate that specific and general proteolytic activities have the ability to lyse some oral bacterial species and to interfere with their growth. It is suggested that such effects could represent new mechanisms by which the bacterial ecology of subgingival sites may be affected.
Mitsuokella dentalis in human periodontitis
Authors: Flynn M. J. , Li G. , Slots J.
Published: December 2007

The occurrence of Mitsuokella dentalis in periodontitis was determined by culture and DNA probe detection. Subgingival paper‐point samples from 480 periodontitis patients were transported in VMGA III, plated onto brucella agar with 5% sheep blood and incubated anaerobically for 7 days. Presumptive identification was based on a colony morphology resembling a water drop and biochemical characteristics. DNA probe detection was performed on paper‐point samples using a digoxigenin‐labeled cellular M. dentalis DNA probe in a dot‐blot assay. Culture and DNA probe identified M. dentalis in 18.1% and in 80.7% of the study patients, respectively. M. dentalis isolates produced phosphatases, galactosidase, glucosidase and acetylglucosaminidase and showed high in vitro sensitivity to metronidazole. This study revealed that M. dentalis is a constituent of the pathogenic microbiota in human periodontitis. The periodontopathic potential of the organism is unknown.
Chemotaxis of human monocytes by synthetic peptides that mimic segments of Porphyromonas gingivalis fimbrial protein
Authors: Ogawa T. , Ogo H. , Hamada S.
Published: December 2007

Porphyromonas gingivalis strain 381 fimbriae and their synthetic peptide segments were assessed for migration‐stimulating activity on human peripheral blood monocytes by multiwell chemotaxis assay. P. gingivalis 381 fimbrial protein was found to markedly enhance migration of human monocytes. The observed increase in monocyte migration occurred mainly directed toward a positive stimulus (chemotaxis). Furthermore, lipopolysaccharides extracted from P. gingivalis 381 were shown to induce chemotaxis and chemokinesis. It was also revealed that the migration of monocytes was increased by specific synthetic peptide segments, FP381(61–80) and FP381(171–185), that correspond to GKTLAEVKALTTELTAENQE and DANYLTGSLTTFNGA, respectively, based on the ammo acid sequence of the fimbrial subunit protein proposed by Dickinson et al., and the migration stimulation was ascribed to chemotaxis. Furthermore, within the amino acid sequences, the LTXXLTXXN sequence may play an important role in binding the organisms to monocytes and macrophages and in the induction of migration‐stimulating activity.
Neutrophil function in juvenile periodontitis: induction of adherence
Authors: Agarwal S. , Suzuki J. B. , Piesco N. P. , Aichelmann‐Reidy M. B.
Published: December 2007

Polymorphonuclear leukocytes (neutrophils) in patients with localized juvenile periodontitis with decreased chemotaxis exhibit increased adherence. The molecular basis of increased adherence of neutrophils in localized juvenile periodontitis is not clear. We show that the neutrophils from localized juvenile periodontitis patients with decreased neutrophil chemotaxis and increased adherence exhibit increased expression of the CD 11/CD 18 family of adherence molecules, Mac‐1, leukocyte function‐associated antigen (LFA‐1) and p150,95, as compared with neutrophils obtained from systemically and periodontally healthy controls. Treatment of neutrophils obtained from healthy subjects with sera from localized juvenile periodontitis patients resulted in an increased expression of Mac‐1, leukocyte function‐associated antigen and p150,95 molecules. Sera obtained from healthy subjects, patients with adult periodontitis or localized juvenile periodontitis patients with normal chemotaxis did not increase the expression of CD11/CD18 molecules on the neutrophils obtained from healthy subjects. The ability of localized juvenile periodontitis sera to induce expression of adherence molecules was at least partially inhibited by pretreatment of localized juvenile periodontitis sera with anti‐tumor necrosis factor and anti‐interleukin‐1 antibodies. Furthermore, increasing concentrations of rh‐tumor necrosis factor‐α and interleukin‐1β also induced increased expression of the Mac‐1 molecule on neutrophils from localized juvenile periodontitis patients and healthy subjects. The increased expression of adherence molecules on neutrophils from localized juvenile periodontitis patients was sustained and could not be reversed by treatment of localized juvenile periodontitis neutrophils with sera obtained from healthy subjects. The upregulation of adherence molecules on neutrophils by localized juvenile periodontitis sera in these experiments emphasizes the physiologic importance of quantitatively small but biologically significant levels of cytokines in the modulation of neutrophil functions in localized juvenile periodontitis.
Transmission of oral Prevotella melaninogenica between a mother and her young child
Authors: Könönen E. , Saarela M. , Karjalainen J. , Jousimies‐Somer H. , Alaluusua S. , Asikainen S.
Published: December 2007

Most likely, young children acquire their oral microflora by frequent transfer of bacteria between family members. The possible transmission of obligately anaerobic Prevotella melaninogenica recovered from 11 mother‐child pairs was examined by ribotyping. One to 18 isolates (mean 13) per child from different oral sampling sites and 4 to 17 (mean 10) isolates per mother from stimulated salivary samples, collected on 2 occasions, were analyzed. On sampling, the mean ages of the children were 4 months and 32 months, respectively. Restriction endonucleases KpnI and ClaI were chosen for the digestion of chromosomal DNA. DNA fragments were electrophoretically separated, blotted onto a nylon membrane and hybridized with rRNA operon of Escherichia coli. DNA‐DNA hybrids were detected immunologically. Extensive genetic heterogeneity, 101 distinct ribotypes, was observed among 248 P. melaninogenica isolates studied. Both mothers and children harbored several (up to 7) ribotypes which, apart from 3 ribotypes, were distinguishable in unrelated subjects. Several P. melaninogenica ribotypes were detected on both sampling occasions over 2 years apart. Identical ribotypes were found in 6 of the 11 mother‐child pairs, 1 to 2 similar ribotypes per pair. This suggests the transmission of P. melaninogenica between the mother and her child, probably via maternal saliva. However, the unique ribotypes found in these children also indicate that other sources besides the mother influence the oral colonization of young children.
Kinetics of lactose‐reversible coadhesion of Actinomyces naeslundii WVU 398A and Streptococcus oralis 34 on the surface of hexadecane droplets
Authors: Ellen R. P. , Veisman H. , Buivids I. A. , Rosenberg M.
Published: December 2007

Most investigations of mechanisms accounting for intergeneric coaggregation have emphasized stereospecific rather than nonspecific interactions. The purpose of this investigation was to determine the relative importance of lectin‐carbohydrate and nonspecific hydrophobic and ionic interactions, using a model based on strains with one of the most well understood specific coaggregation mechanisms, the lactose‐reversible coaggregation of Actinomyces naeslundii and Streptococcus oralis. The kinetics of coadhesion and desorption of coadherent bacteria were studied using S. oralis 34 bound to hexadecane droplets as an affinity support for the adhesion of A. naeslundii WVU 398A. Light, confocal microscopy and transmission electron microscopy confirmed that A. naeslundii cells adhered only to the S. oralis cells, not to exposed hexadecane between the streptococci. Coadhesion was inhibited by lactose concentrations as low as 2.0 mM. The rate of coadhesion was halved at 60 mM lactose. The hydrophobicity inhibitors bovine serum albumin and defatted bovine serum albumin and the salts LiCl and KC1 failed to inhibit coadhesion in the hexadecane assay, and bovine serum albumin also failed to inhibit coaggregation in a bacterial aggregation assay on glass slides. High concentrations of the salts achieved a 50% rate decrease in A. naeslundii adhesion to the S. oralis‐coated droplets only when they were combined with > 20 mM lactose. Sodium dodecyl sulfate (SDS) and Tween 20 inhibition was tested by the slide coaggregation assay because they tended to emulsify the droplets; SDS was inhibitory. Lactose selectively desorbed A. naeslundii from S. oralis‐coated droplets at low concentrations equivalent to those that inhibited coadhesion. Neither LiCl nor KC1 desorbed A. naeslundii from the droplets, even at 500 mM. At low concentrations, SDS but not Tween 20 eluted both A. naeslundii and S. oralis from the droplets. Although the SDS results might suggest a degree of cooperative charge interactions, the results support the hypothesis that stereospecific, β‐galactoside‐sensitive interactions have a much greater impact than nonspecific interactions on the coadhesion of A. naeslundii and S. oralis.
Occurrence of enteric rods, staphylococci and Candida in subgingival samples
Authors: Dahlén G. , Wikström M.
Published: December 2007

The frequency and percentage of enteric rods, staphylococci and Candida were determined in 973 subgingival samples collected from 535 patients subjected to different periodontal treatment procedures. The analysis was performed with culture technique using selective and nonselective media. One or more organisms were detected in 65.5% of the samples and in 76.7% of the patients. In most samples enteric rods, staphylococci and/or Candida constituted a small amount of the total microbial viable count. Enteric rods exceeded 10% of the total viable count in 30 samples. Staphylococci occurred in more than 10% in only 3 samples. In these 3 samples, enterics constituted more than 10% of the total viable count. Candida was not found to exceed 10% of the total viable count in any of the samples. No statistically significant correlation was found between the presence of any of the target microorganisms and kind of periodontal treatment procedure received, antibiotic administration or sample transport time.
Inhibition of streptococcal growth, F‐ATPase and pyrophosphatase by diphosphonates
Authors: Hsu M.‐T. , Sturr M. G. , Curran T. M. , Marquis R. E.
Published: December 2007

1‐Hydroxyethane‐1, 1‐diphosphonate (EHDP) and a variety of other diphosphonates, and also pyrophosphate, at millimolar levels were found to inhibit the growth of Streptococcus mutans GS‐5. Inhibition appeared to be due mainly to chelation of Mg2+ and could be readily reversed through addition of Mg2+, or less effectively, by other divalent cations. The trianionic forms of the diphosphonates or pyrophosphate were more effective inhibitors than the dianionic forms. Diphosphonates and pyrophosphate did not inhibit glycolysis by S. mutans, assayed in terms of glucose utilization, or arginolysis by Streptoccus rattus FA‐1, assayed in terms of ammonia production. However, they did act as buffers to moderate pH changes. Diphosphonates also were inhibitors of the F‐ATPase of S. mutans by complex mechanisms only partly reversible with divalent cations. They also were inhibitors of the pyrophosphatase of the organism. However, intact cells were impermeable to the compounds, and inhibition of cytoplasmic or membrane enzymes did not appear to be involved in growth inhibition.
Detection of tet(M) and tet(Q) using the polymerase chain reaction in bacteria isolated from patients with periodontal disease
Authors: Olsvik B. , Olsen I. , Tenover F. C.
Published: December 2007

Olsvik B, Olsen I, Tenover FC. Detection of tet(M) and tet(O) using the polymerase chain reaction in bacteria isolated from patients with periodontal disease.

The polymerase chain reaction was used to examine 114 tetracycline‐resistant anaerobic and facultative anaerobic bacterial isolates from patients with periodontal disease for the tet(M) and tet(O) genes. A 740‐base‐pair fragment of the tet(M) gene was amplified from 84 of 114 isolates, and a 519‐base‐pair fragment of the tet(O) gene was amplified from 13 streptococcal isolates. Six of 7 tetracycline‐resistant isolates of Veillonella spp. and tetracycline‐resistant isolates of En‐bacterium spp. (n=3), Eubacterium saburreum (n=1), Streptococcus intermedius (n=5) and Gemella morbillorum (n=2) all harbored the tet(M) gene. The tet(M) and tet(O) negative as well as selected positive isolates were tested for the tet(K) and tet(L) genes using DNA probes. All isolates of Staphylococcus spp. (n=11) hybridized with the tet(K) probe. None of the isolates tested hybridized with the probe for tet(L). This is the first report of the tet(M) gene in the facultative bacterium G. morbillorum and in E. saburreum.
Delmopinol interactions with cell walls of gram‐negative and gram‐positive oral bacteria
Authors: Rundegren J. , Sjödin T. , Petersson L. , Hansson E. , Jonsson I.
Published: December 2007

The main purpose of the present study was to investigate the influence of delmopinol hydrochloride on the cell surface morphology of gram‐negative and gram‐positive bacterial cells by using transmission electron microscopy. A second purpose was to evaluate the extraction of cell wall material caused by delmopinol and the binding of radiolabelled delmopinol to the various strains. Fresh isolates and type strains of gram‐negative rods associated with periodontal disease, Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans, and strains of the gram‐positive streptococci Streptococcus sanguis, Streptococcus mutans and Streptococcus salivarius, were exposed to 3.2 mM (0.1 %) or 6.4 mM (0.2%) delmopinol hydrochloride from 1 to 90 min. For electron microscopy the cells were fixed and negatively contrast‐stained. Treatment with 6.4 mM delmopinol for 1 min resulted in marked ultrastructural changes of cell wall components and the outer cell membrane of the 3 gram‐negative species compared with control cells, whereas the gram‐positive streptococci treated with delmopinol showed little or no morphologic alteration as compared with untreated cells. The result from the electron microscopy was confirmed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of proteins released from bacterial cells treated as for electron microscopy. More material was extracted from the gram‐negative rods than from the gram‐positive cocci. Significantly more delmopinol bound to the gram‐negative rods than to the streptococci. It appears that the am‐phiphilic properties of delmopinol make gram‐negative rods more vulnerable to delmopinol than gram‐positive streptococci.
The influence of intracellular polyglucose and prior growth rate on the survival of Fusobacterium nucleatum under starvation conditions
Authors: Rogers A. H. , Zilm P. S.
Published: December 2007

Grown in a chemically denned medium containing glucose at a dilution rate of D=0.065 h−1, Fusobacterium nucleatum D212B‐2 produced large amounts of intracellular polyglucose. Aliquots of this culture were starved by anaerobic incubation at 37°C and at various times, assayed for intracellular polyglucose content and viability. This protocol was repeated using cells grown under the same conditions in a chemically defined medium, a medium lacking carbohydrate and in which the organism produced no intracellular polyglucose. Both cultures had 50% survival time values of about 1.5 h and were not eliminated even after 32 h of starvation. It was, therefore concluded that starvation‐survival is not influenced by intracellular polyglucose. Starvation‐survival was also determined for cells grown in a chemically defined medium at D=0.048 h‐1 and D=0.12 h‐1. The faster‐grown cells had a 50% survival time of 3.8 h but were completely eliminated after 8‐16 h of starvation. In contrast, slower‐grown cells had a 50% survival time of 1.5 h but were not completely eliminated after 32 h of starvation. This illustrates the importance of cell history and technique standardization in comparing the starvation‐survival of different organisms.
Multiple restriction fragment length polymorphism genotypes of Porphyromonas gingivalis in single periodontal pockets
Authors: Zhang Y. J. , Yasui S. , Yoshimura F. , Ishikawa I.
Published: December 2007

A total of 188 Porphyromonas gingivalis strains isolated from 13 periodontal pockets of 8 periodontitis patients were investigated by means of restriction fragment length polymorphism (RFLP) analysis using the fim A gene as a pobe. A total of 5 RFLP genotypes were identified, and over half of the isolates belonged to type I. Four of the 8 patients harbored only 1 RFLP genotype of P. gingivalis, and 1 patient harbored only 2 RFLP genotypes in different sites. On the other hand, in 3 other patients, multiple RFLP genotypes were found in single periodontal pockets. Further studies will be required to clarify whether multiple genotypes of P. gingivalis colonized a single periodontal pocket simultaneously or whether a mutation occurred in the fimbrilin gene locus of P. gingivalis.
Molecular and immunological characterization of a 64‐kDa protein of Actinobacillus actinomycetemcomitans
Authors: Nakano Y. , Inai Y. , Yamashita Y. , Nagaoka S. , Kusuzaki‐Nagira T. , Nishihara T. , Okahashi N. , Koga T.
Published: December 2007

The 64‐kDa protein to which about half the sera from patients with localized juvenile periodontitis and rapidly progressive periodontitis reacted strongly was purified from Actinobacillus actinomycetemcomitans Y4. Determination of the TV‐terminal sequence of the protein revealed that it was a GroEL‐like protein. The DNA fragment containing the groEL gene of A. actinomycetemcomitans was amplified by polymerase chain reaction, and the groESL operon was cloned by using colony hybridization with the amplified fragment from A. actinomycetemcomitans chromosomal DNA. Sequence analysis revealed that structures of the operon and its products were typical in gram‐negative bacteria. Rabbit polyclonal antibodies to the 64‐kDa protein cross‐reacted with approximately 65‐kDa proteins of Haemophilus aphrophilus, Haemophilus influenzae, Haemophilus paraphrophilus, Escherichia coli and Eikenella corrodens but not with any cellular proteins of Porphyromonas gingivalis, Prevotella intermedia and Fusobacteriuni nucleatum. It is possible that antibodies reactive to the 64‐kDa protein in periodontitis patients are induced by the cross‐reactivity with the hsp60 proteins of other bacteria.
Effect of sonicated material from Fusobacterium nucleatum on the functional capacity of accessory cells derived from dental pulp
Authors: Yoshida H. , Jontell M. , Sundqvist G. , Bergenholtz G.
Published: December 2007

Fusobacterium nucleatum‐derived components were studied for their effect on the ability of accessory pulpal cells to induce T‐lymphocyte proliferation. In initial experiments, spleen cells were used to establish concentration ranges at which testing of the effect of the bacterial components would be suitable. At low concentrations, bacterial substances induced a stimulatory effect on the proliferation rate of spleen cells, but not when purified T‐lymphocytes were incubated in the presence of concanavalin A and pulpal accessory cells. At increasing concentrations, a dose‐dependent reduction was observed. Pretreatment with the bacterial products for 4 h gave a stimulatory effect in the absence of concanavalin A for both spleen cells and T‐lymphocytes. Removal of low‐molecular‐weight components (<3.5 kDa) did not influence the inhibitory effect, whereas preheating the bacterial substances to 100°C eliminated the observed inhibition. Findings show that heat‐sensitive, high‐molecular‐weight components of F. nucleatum can evoke a concentration‐dependent stimulatory or suppressive effect on the proliferation rate of T‐lymphocytes in the presence of accessory cells derived from the dental pulp.
Insertional inactivation of binding determinants of Streptococcus crista CC5b using Tn916
Authors: Correia F. F. , DiRienzo J. M. , Lamont R. J. , Anderman C. , McKay T. L. , Rosan B.
Published: December 2007

Intermicrobial binding plays an important role in the ecology of the oral cavity because it represents one mechanism by which specific bacteria colonize dental plaque. The formation of “corncobs”, a morphologically distinct microbial unit composed of Streptococcus crista and Fusobacterium nucleatum, is a highly specific binding interaction that depends on the presence of polar tufts of fimbriae on the streptococci. We have used a genetic approach to examine the role of streptococcal cell surface components involved in the binding of S. crista to F. nudeatum. Such binding may be an important component of corncob formation. A method for the genetic transformation of S. crista was used to transfer the broad host range transposon, Tn916, into the bacteria. Cells were grown to early log phase in brain heart infusion broth containing 10% fetal calf serum. The competent cells were mixed with purified DNA from pDL916, a plasmid construct consisting of Tn916 and the streptococcal/Escherichia coli shuttle vector pDL278. Over 300 transformants were screened for a reduction in binding to F. nucleatum. Five of the transformants showed a change in binding ranging from 59% to 29% of the positive control values. Southern blots revealed that the binding‐deficient transformants contained the Tn916 element integrated into one of 4 different sites in the chromosome. The transposon, integrated into 4 different sites, appeared to be stable in the absence of selective pressure. Based on these findings, it appears that some strains of S. crista are naturally competent and that insertional inactivation methods can be used to facilitate the study of binding receptors in this group of oral streptococci.
The action of thymol on oral bacteria
Authors: Shapiro S. , Guggenheim B.
Published: December 2007

Several effects of thymol, a plant‐derived antimicrobial agent, on Porphyromonas gingivalis, Selenomonas artemidis and Streptococcus sobrinus were examined. The extremely rapid efflux of intracellular constituents evoked by thymol is consistent with its postulated membranotropic effects. Correlations between leakage‐inducing concentrations of thymol and minimal inhibitory concentrations and minimal bactericidal concentrations suggest that membrane perforation is a principal mode of action of this substance. The thymol‐induced decline in intracellular ATP in S. sobrinus appears to be entirely attributable to leakage, whereas in P. gingivalis thymol may also inhibit ATP‐generating pathways. Relative changes in the transmembrane potential of resting cells of S. sobrinus pulsed with glucose are as sensitive to thymol as is leakage from this organism. The effects of thymol on transmembrane potential are probably secondary to those arising from leakage of intracellular substances.
Molecular characterization of a 40‐kDa outer membrane protein, FomA, of Fusobacterium periodonticum and comparison with Fusobacterium nucleatum
Authors: Bolstad A. I. , Høgh B. T. , Jensen H. B.
Published: December 2007

The 40 kDa‐outer membrane protein FomA of Fusobacterium periodonticum ATCC 33693 was found to exhibit heat modifiable properties, typical for a porin, and N‐terminal sequencing indicated a close relationship to the porin FomA of Fusobacterium nucleatum. A polymerase chain reaction approach was therefore applied for sequencing the fomA gene of F. periodonticum, and nucleotide and deduced amino acid sequences were aligned and compared with the corresponding sequences of different strains of F. nucleatum. In all strains we found a common protein upstream of the fomA gene. The noncoding area upstream of the putative ‐35 region of the F. periodonticum fomA gene exhibited little sequence similarity with the F. nucleatum gene. The transcriptional unit of FomA, on the other hand, was very similar, with the similarities concentrated in domains that were interspersed with hypervariable regions. A topology model was made and compared with those made for F. nucleatum. This indicated that the great similarities reside in the membrane‐spanning segments of the protein, while most cell surface exposed loops were hypervariable. The results strongly support the proposed model for FomA and also indicate that these taxa are related but on a lower level than the subspecies level. The codon usage of F. periodonticum is comparable to that of F. nucleatum, and the triplet AGA is the only codon used for arginine.
Characterization of the immunodominant antigens of Porphyromonas gingivalis 381 in high‐responder patients
Authors: Boutsi E. A. , Koseki T. , Nishihara T. , Ishikawa I.
Published: December 2007

The immunodominant antigens of Porphyromonas gingivalis 381 whole cells that reacted with sera from high‐responder patients were examined in this study. Whole cells, phenol‐water extracted lipopolysaccharide, and fimbriae from P. gingivalis 381 were analyzed using sera from 14 patients with adult periodontitis, rapidly progressive periodontitis or juvenile periodontitis as well as from two healthy subjects. Western blot analysis and enzyme‐linked immunosorbent assay (ELISA) were performed. On Western blots, among many prominent protein bands, a smear was observed which was removed after adsorption of the sera with P. gingivalis phenol‐water extracted lipopolysaccharide. Two major protein bands of 43 kDa and 41 kDa were found to be prominent even at very high dilutions of sera, the latter of which showed the same molecular weight as the fimbrilin band. These two bands were resistant to treatment by papain and trypsin. ELISA titers remained high after adsorption of the sera with P. gingivalis phenol‐water extracted lipopolysaccharide. The results of this study suggest that the 43‐kDa and the fimbrilin (41 kDa) proteins may play an important role as immunodominant antigens of P. gingivalis 381.
Polymerase chain reaction detection of 8 putative periodontal pathogens in subgingival plaque of gingivitis and advanced periodontitis lesions
Authors: Ashimoto A. , Chen C. , Bakker I. , Slots J.
Published: December 2007

A 16S rRNA‐based polymerase chain reaction (PCR) detection method was used to determine the prevalence of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Campylobacter rectus, Eikenella corrodens, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and Treponema denticola in subgingival specimens of 50 advanced periodontitis, 50 adult gingivitis and 50 pediatric gingivitis subjects. The optimal PCR conditions were determined for each study species. Agarose gel electrophoresis of PCR products from each study species revealed a single band of the predicted size. Restriction enzyme digestion of amplicons confirmed the specificity of the amplification. PCR detection limits were in the range of 25–100 cells. No cross‐reactivity with other oral microorganisms or nonspecific amplification was observed. The prevalence by PCR in advanced periodontitis, adult gingivitis and pediatric gingivitis subjects was 30%, 14% and 14% for A. actinomycetemcomitans, 86%, 18% and 8% for B. forsythus, 74%, 52% and 78% for C. rectus, 80%, 70% and 66% for E. corrodens, 70%, 10% and 14% for P. gingivalis, 58%, 12% and 18% for P. intermedia, 52%, 20% and 22% for P. nigrescens, and 54%, 16% and 16% for T. denticola, respectively. The prevalence was higher in the advanced periodontitis group than in both adult gingivitis and pediatric gingivitis for A. actinomycetemcomitans, B. forsythus, P. gingivalis, P. intermedia, P. nigrescens and T. denticola at P<0.01, and for E. corrodens at P<0.05. The prevalence of C. rectus was significantly higher in the advanced periodontitis group than in the adult gingivitis group at P<0.01. Matching results between PCR and culture occurred in 28% (B. forsythus) to 71% (A. actinomycetemcomitans) of the samples; the major discrepancy occurred in the PCR‐positive/culture‐negative category. Matching results between PCR and DNA probe methods were found in 84% of the subjects (B. forsythus) and 70% (P. gingivalis). Odds ratio analysis revealed statistically significant positive associations between 17 of the 28 possible combinations (P<0.01). This study demonstrated the utility of a 16S rRNA‐based PCR detection method for identifying important subgingival microorganisms. The results indicated a strong association between the study species and periodontitis. Several previously unreported symbiotic relationships were found between the 8 species tested.
Detection and prevalence of the tetracycline resistance determinant Tet Q in the microbiota associated with adult periodontitis
Authors: Lacroix J.‐M. , Walker C. B.
Published: December 2007

Subgingival plaque samples were collected from 68 patients with a history of moderate to severe adult periodontitis and enumerated on Trypticase‐soy blood agar plates, with and without tetracycline at 4 μg/ml. Each different colony morphotype was enumerated, and a representative colony was subcultured for identification and examined for the tetracycline resistance gene tet(Q) by polymerase chain reaction (PCR) amplification and DNA hybridization, using a fragment of tetA(Q)2 from Bacteroides fragilis 1126. PCR primers (5′‐GGCTTCTACGACATCTATTA‐3’and 5′‐CATCAACATTTATCTCTCTG‐3′) were chosen to amplify a 755 bp region of tet(Q). The subgingival plaque samples were also tested by PCR. Approximately 12% of the total cultivable flora was resistant to tetracycline, and the percentage of the tetracycline‐resistant cultivable flora with the tet(Q) gene varied greatly from one patient to another with a range from 0.0 to 67%. Half of the 68 subgingival plaque samples were positive or weakly positive for tet(Q) by PCR. Approximately 15% of the 210 isolates subcultured with resistance to tetracycline, (4 μg/ml) contained tet(Q), and 0% contained tet(M). All of the tet(Q)‐resistant isolates were gram‐negative anaerobic bacilli and included all of the Prevotella and Bacteroides isolates.
Laminin binding to a heat‐modifiable outer membrane protein of Actinobacillus actinomycetemcomitans
Authors: Alugupalli K. R. , Kalfas S. , Forsgren A.
Published: December 2007

The interaction of Actinobacillus actinomycetemcomitans with the basement membrane protein, laminin, was examined in a 125I‐labeled protein‐binding assay. The binding of laminin increased by lowering the pH. The ability to bind laminin was decreased in cells at the stationary phase of growth and by the presence of blood in the culture medium. Laminin binding to this bacterium was saturable, and the affinity constant was 4.6 nM. Sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and Western blot (ligand blot) analysis of cell‐envelope and outer membrane of A. actinomycetemcomitans displayed a 125I‐laminin‐reactive protein band with a molecular weight of 29 k. The laminin‐binding protein was the previously described outer membrane protein A of A. actinomycetemcomitans. It was identified by its heat‐modifiable property, detergent‐solubility profile and reactivity with outer membrane protein A‐specific polyclonal antiserum. At acidic pH, 125I laminin bound to several cell‐envelope components of A. actinomycetemcomitans, but at neutral pH, laminin bound only to the heat‐modifiable protein. Despite the existence of the laminin‐binding protein, cells grown in blood‐containing media did not bind laminin. Several mammalian proteins interfered with laminin‐bacterial interaction, including lactoferrin, which binds to the same bacterial protein that inhibited and displaced the laminin‐bacterial interaction.
Lymphocyte adhesive interactions with cultured parotid salivary gland epithelial cells from rats
Authors: O'Sullivan N. L. , Montgomery P. C.
Published: December 2007

Cellular interactions control lymphocyte localization within salivary gland tissues and contribute to the immune defense of oral surfaces. We examined lymphocyte adherence to cultured parotid cells using an in vitro assay and found good correlation with previously reported binding to parotid gland frozen sections. Thoracic duct lymphocytes (TDL) bound to parotid cells in greater numbers than thymocytes (74 vs 11 cells/mm2). B cells showed preferential adherence compared to T cells (75% vs 28%). TDL binding was inhibited by sodium azide or cytochalasin B (60% and 80%, respectively). EDTA inhibition (63%) was restored by replacing calcium (9%) but not magnesium (65%). Binding was inhibited by fucoidin or phosphomannan (∼70%). Fibronectin peptides had no effect. Culture supernatants were inhibitory for TDL adherence (60%), suggesting that molecules involved in lymphocyte localization may be shed and that parotid cell cultures will be useful for ligand isolation and characterization.
Identification of human antigenic epitopes in an alanine‐rich repeating region using sera from hu‐PBL‐SCID mice immunized with a surface protein antigen of Streptococcus mutans
Authors: Senpuku H. , Iizima T. , Koga T. , Nisizawa T.
Published: December 2007

A 190‐kDa surface protein antigen (PAc) of Streptococcus mutans is considered to play an important role in dental caries. To identify antigenic epitopes of the PAc in humans, we immunized severe combined immunodeficient (SCID) mice with re‐combinant PAc that was transplanted with human peripheral blood mononuclear cells (PBMC). The reactivities of the sera from the immunized hu‐PBL‐SCID mice to the recombinant PAc and 24 19‐mer synthetic peptides covering the alanine‐rich repeating region (A‐region) presented in the PAc molecule were then examined. The results showed that the immunized mice produced a significant recombinant PAc‐specific human antibody, and among 24 19‐mer peptides, 6 19‐mer peptides showed a strong reaction with the antibodies. In addition, 4 19‐mer peptides containing human antigenic epitope in a donor were identified with enzyme‐linked immunosorbent assay (ELISA) inhibition assays using the recombinant PAc protein. In this study, the SCID mouse was useful in identifying human antigenic epitopes.
Similarity of salivary and subgingival Prevotella intermedia and Prevotella nigrescens isolates by arbitrarily primed polymerase chain reaction
Authors: Mättö J. , Saarela M. , von Troil‐Lindén B. , Alaluusua S. , Jousimies‐Somer H. , Asikainen S.
Published: December 2007

The distribution and the genetic similarity of Prevotella intermedia and Prevotella nigrescens in saliva and in subgingival samples recovered from the same subject were studied in 16 subjects with different periodontal status. The isolates (4 salivary and 4 subgingival P. intermedia/nigrescens group isolates per subject) were identified to species level by hybridization with species‐specific oligonucleotide probes, and the clonal analysis was performed using arbitrarily primed polymerase chain reaction (AP‐PCR) (all isolates) and ribotyping (isolates from 5 subjects). In addition, the applicability of AP‐PCR in differentiating between P. intermedia and P. nigrescens species was tested using 18 P. intermedia and 20 P. nigrescens isolates from 34 subjects. P. intermedia was detected in 7 and P. nigrescens in 14 of the 16 subjects. In all subjects the same species was found both in saliva and in subgingival plaque. In 15 of the 16 subjects, similar AP‐PCR types of P. intermedia and/or P. nigrescens between salivary and subgingival samples were found. The salivary and subgingival isolates that were similar by AP‐PCR were indistinguishable also by ribotyping. The AP‐PCR analysis revealed a P. intermedia or P. nigrescens species‐specific AP‐PCR product in most isolates. This study indicates that both P. intermedia and P. nigrescens were found both in salivary and in subgingival samples, and both sampling sites within the same individual were usually colonized with identical AP‐PCR types of the species. Thus, in addition to a subgingival sample a salivary sample seems to be suitable for detection and clonal analysis of these species. The AP‐PCR method proved to be a simple method applicable for differentiation and clonal analysis of P. intermedia and P. nigrescens.