Strains Of Bacteroides Gingivalis Research Articles

Toxicity of sonicated extracts of Bacteroides gingivalis on human pulpal cells and L929 cells in vitro

Human pulpal fibroblasts and L929 cells were treated with sonicated extracts of two strains of Bacteroides gingivalis (W83 and ATCC 33277). The cell reaction was evaluated by monitoring cell growth and DNA synthesis. Light and scanning electron microscopic analysis were used to evaluate morphological changes of the cells. Extracts from both bacterial strains exerted a growth inhibitory effect on the cells. The pulpal cells were more sensitive than L929 cells. The ATCC 33277 strain of B. gingivalis was more cytotoxic than the W83 strain. Pulpal cells appeared to be markedly affected on the microscopic level. The diffusion of these toxic bacterial by-products, through dentin to the pulp, may account for pulpal cell damage that contributes to the initiation of pulpal pathosis.

Comparative studies of the outer membranes of Bacteroides gingivalis, strains ATCC 33277, W50, W83,381

Outer membrane fractions from Bacteroides gingivalis strains ATCC 33277, 381, W50, and W83 were isolated by French pressure cell disruption and the distribution of major and minor proteins was determined by SDS-PAGE electrophoresis after treatment with 2% Sarkosyl and 2% Triton X-100. Heat-modifiability of the outer membrane proteins (OMPs) from these B. gingivalis strains was also determined after treatment at 100 degrees C and analysis by both 1- and 2-dimensional SDS-PAGE. The distribution of the OMPs on the surface of these B. gingivalis strains was determined by 125I labelling. For the most part of the OMPs of B. gingivalis presented a complex distribution, with OMPs observed between 123 kD and 13 kD. While the distribution of the OMPs was strain specific, OMPs common to all of the strains were observed. Two percent Sarkosyl treatment of the OMs at room temperature resulted in the solubilization of approximately 60% of the OMP. The Sarkosyl-insoluble MOMPs had relative molecular weights between 110 kD and 20 kD. Many of the OMPs which were separated at room temperature were heat-modified at either 65 degrees C or 100 degrees C. Heating of the OMs at 100 degrees C resulted in the heat modification of the majority of those OMPs observed at room temperature. Sarkosyl-100 degrees C OMs displayed MOMPs at apparent molecular weights between 90 kD and 15 kD. Radioiodination of the B. gingivalis strains ATCC 33277, 381, W83 and W50 revealed between 7 and 14 MOMPs at the cell surface depending upon the strain. The complexity of the OM of these B. gingivalis strains indicated the possibility of identifying and separating those OMPs involved in a variety of biological functions, including virulence, transport, and cell interaction.

Chemical, immunobiological and antigenic characterizations of lipopolysaccharides from Bacteroides gingivalis strains

Lipopolysaccharides (LPS) were extracted from whole cells of seven strains of Bacteroides gingivalis--381, ATCC 33277, BH18/10, OMZ314, OMZ406, 6/26 and HW24D-1--by the phenol/water procedure, and purified by treatment with nuclease and by repeated ultracentrifugation. These LPS were composed of hexoses, hexosamines, fatty acids, phosphorus and phosphorylated 2-keto-3-deoxyoctonate (KDO). The major components of the lipid portion of these LPS were hexadecanoic, 3-hydroxyhexadecanoic, branched 3-hydroxypentadecanoic and branched 3-hydroxyheptadecanoic acids. All the LPS preparations induced marked mitogenic and in vitro polyclonal B cell activation responses in spleen cells from both C3H/HeN and C3H/HeJ mice, exhibited no definitive preparatory activity in the local Shwartzman reaction in rabbits, but were active in the chromogenic Limulus amoebocyte lysate test. A monoclonal antibody (mAb) raised against the LPS from B. gingivalis strain 6/26 reacted with LPS from all other B. gingivalis strains tested. Other mAbs raised against LPS from B. gingivalis strains 381 and 6/26 reacted with the LPS from strains 381, ATCC 33277, BH18/10 and 6/26 (these strains were termed LPS serogroup I), as revealed by ELISA and immunodiffusion. The LPS from these strains except for 6/26 showed almost identical patterns in SDS-PAGE stained with ammoniacal silver. A mAb raised against the LPS from B. gingivalis HW24D-1 reacted with the LPS from strains OMZ314, HW24D-1 and OMZ409 (LPS serogroup II). These LPS, except OMZ409, exhibited very similar profiles in SDS-PAGE. These results indicate that there are at least two different antigenic groups present among LPS from B. gingivalis strains, as well as a common, species-specific antigen.

Inhibition of protease activities of periodontopathic bacteria by extracts of plants used in Kenya as chewing sticks (mswaki)

Extracts from five plants used as chewing sticks, and tannic acid, gallic acid and methyl ester of gallic acid, were tested for their ability to inhibit proteolytic activities of three strains of Bacteroides gingivalis, three strains of Bacteroides intermedius and two strains of Treponema denticola. Aqueous extract from the plants Rhus natalensis and Euclea divinorum were the most inhibitory of those tested, inhibiting by 50% the proteolytic activity of the test organisms, at concentrations of up to 200 μg/ml. Tannic and gallic acids had similar effects at concentrations of < 10 μg/ml, while the methyl ester of gallic acid was less inhibitory. These findings suggest that extracts from plants used as chewing sticks may possess enough inhibitory components to interfere with the virulence and growth of periodontopathic bacteria in vivo, provided they are able to gain access to the subgingival sites such bacteria preferentially inhabit.

Cytotoxicity against various cell lines of lipopolysaccharides purified from bacteroides, fusobacterium, and veillonella isolated from infected root canals

The cytotoxicity against two mesenchymal cell lines, L-929 and WI-38, and two epithelial cell lines, KB and HeLa S-3, of lipopolysaccharide (LPS) purified from strains of Bacteroides gingivalis, Fusobacterium nucleatum, and Veillonella parvula isolated from infected root canals was investigated. The inhibition of cell growth by these LPS's was considerable for mesenchymal cell lines, but mild for epithelial cell lines. The cytotoxic effect of F. nucleatum LPS was the greatest and that of B. gingivalis LPS was the least.

Characterization of a polysaccharide antigen from Bacteroides gingivalis.

A polysaccharide Ag (PS) was isolated from the phenol-water extract of Bacteroides gingivalis strain A7A1-28 and separated from LPS by Sephacryl S-400 HR chromatography. The PS was composed of glucose, glucosamine, galactosamine, and galactosaminuronic acid, while the LPS contained rhamnose, mannose, galactose, glucose, glucosamine, galactosamine, phosphate, and lipid, but not galactosaminuronic acid. The PS and LPS were immunochemically distinct by immunoelectrophoresis in agarose with homologous rabbit antiserum. The phenol-water extract from strain A7A1-28 was immunoreactive by immunoelectrophoresis against antisera to three additional strains of B. gingivalis, however, the PS was only reactive with homologous serum. Immunochemical characterization of decarboxylated and deacetylated PS derivatives suggest that the acetylation of the amino sugars, but not the presence of the carboxylate residue on galactosaminuronic acid contributes to major immunodeterminant expression.

Chemical characterization and biologic properties of lipopolysaccharide from Bacteroides gingivalis strains W50, W83, and ATCC 33277.

The chemistry and selected biological activity of lipopolysaccharide (LPS) from Bacteroides gingivalis strains W50, W83, and ATCC 33277 were compared, as well as the role of this molecule as a mediator of selected inflammatory responses. Chemically, the LPSs consisted of 47-58% Lipid A, 5-10% carbohydrate, 0.05% 3-deoxy 2-octulosonic acid, 0.3% heptose, 3.8-5.2% hexosamine, and 2% phosphate. Rhamnose represented the dominant sugar (26-36%), with lesser amounts of glucose (18-34%), galactose (18-25%), mannose (9-12%), glucosamine (7-11%), and galactosamine (2-5%). The major fatty acids were: 13-methyl-tetradecanoate (42-45%), 3-OH-heptadecanoate (21-23%), hexadecanoate (16-19%), and 12-methyl-tetradecanoate (6-8%). SDS-PAGE and sodium deoxycholate-PAGE revealed the LPS to be a smooth chemotype. Differences in migration patterns between the virulent and avirulent strain LPSs also occurred. C3H/HeN macrophages (Mø) exposed to 1 microgram/ml of LPS released 3.2-4.2 ng of prostaglandin E (PGE)/ml of supernatant, representing 236-278% of control. Interleukin-1 (IL-1) activity in C3H/HeN and C3H/HeJ Mø exposed to 50 micrograms of LPS/ml was 382-724% and 270-300% of control, respectively; similar Mø exposed to 10 micrograms of LPS/ml released 1.6-2.0 ng and 0.3-0.5 ng of tumor necrosis factor (TNF)/ml of supernatant, respectively. Maximum TNF release in C3H/HeN Mø occurred in response to 50 micrograms of LPS/ml, and was sustained for up to 96 hours. These results suggest that LPS from the B. gingivalis strains stimulate cytokine production from Mø which, in turn, may play a role in orchestrating the inflammatory response for the development of periodontal diseases.

Production of cell-bound and vesicle-associated trypsin-like protease, alkaline phosphatase and N-acetyl-beta-glucosaminidase by Bacteroides gingivalis strain W50.

Bacteroides gingivalis strain W50 was grown in batch and continuous culture on complex medium with haemin. In batch culture, cell-bound levels of trypsin-like protease (EC 3.4.21.4), alkaline phosphatase (EC 3.1.3.1) and N-acetyl-beta-glucosaminidase (EC 3.2.1.30) increased during the exponential phase of growth. These enzyme activities were also detected in extracellular vesicles and in extracellular soluble forms in the supernatant fluid, but in lower amounts per unit biomass compared to cell-bound levels. In continuous culture, at high relative growth rates (0.7-0.9 murel), the highest proportions of enzyme activities were cell-bound. In contrast, at low relative growth rates (0.1-0.2 murel), highest enzyme levels were detected in the extracellular vesicle fraction. Levels of extracellular soluble enzymes were always low compared to cell-bound or extracellular vesicle levels, but were highest at low relative growth rates. All three enzymes appeared to be relatively stable in their soluble forms. Vesicle production appeared to be associated with actively growing cells but was influenced by growth rate. The results are consistent with the hypothesis that cell-bound 'periplasmic' enzymes are encapsulated into vesicles which are subsequently released by the cells. Therefore, levels of total extracellular enzyme (extracellular vesicle plus extracellular soluble) may depend on the rate of vesicle formation superimposed on the rates of production of 'periplasmic' enzymes in the cell.

Failure of Bacteroides gingivalis W83 to accumulate bound C3 following opsonization with serum

Our previous studies have demonstrated that strains of Bacteroides gingivalis are capable of proteolytic degradation and inactivation of complement proteins including the third component of complement C3. Since a crucial step in the ability of complement to control bacterial infections is the binding of C3 fragments to the bacterial surface with subsequent enhancement of phagocytosis, further examination of the importance of the proteolytic capacity of Bacteroides in interactions with complement proteins was carried out by quantitating the amount of C3 bound to two proteolytic Bacteroides gingivalis strains. Pooled normal human serum (NHS) containing 125I-C3 was incubated with strains of B. gingivalis (W83 and ATCC 33277) and the non-proteolytic pathogen A. actinomycetemcomitans strain Y4, and samples of the reaction mixtures were removed at various time intervals for determination of bound C3. B. gingivalis 33277 bound only half the number of C3 molecules as did A. actinomycetemcomitans, while B. gingivalis W83 bound very little C3. A large increase in the number of C3 molecules bound to B. gingivalis W83 was noted in assays carried out in the presence of the protease inhibitor TLCK, indicating that bacterial proteases may be responsible for the lack of binding of C3 to strain W83. TLCK treatment modestly increased the accumulation of C3 on strain 33277, but had no effect on A. actinomycetemcomitans. Analysis of 125I-C3 in supernatants from reaction mixtures of strain 33277, W83, or a proteolytic strain of B. intermedius demonstrated no qualitative differences in the C3 fragments amongst the tested strains or in the presence or absence of TLCK.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of periodontal proteolytic Bacteroides species on proteins of the human complement system.

Proteolytic strains of Bacteroides gingivalis, Bacteroides intermedius, Bacteroides loeschii and Bacteroides denticola were assessed for their ability to inactivate select complement proteins in pooled human serum and for their effect on purified complement components C3, C4, and factor B. Under conditions of bacterial concentration resulting in limited C3 consumption in pooled serum, it was found that EDTA‐inhibitable consumption of C3, C4, and factor B occurred with nearly all strains. However, identical concentrations of B. gingivalis were also capable of inactivating purified C3, C4, and B. This property was not entirely inhibitable by EDTA. Further examination of the effect of B. gingivalis on the structure of C3 demonstrated that one strain (33277) converted purified fluid‐phase C3 to a C3b‐like molecule, while the other strain (W83) completely degraded the a‐chain of C3. In contrast, only minor cleavage of the α‐chain of C3 was apparent following incubation with B. intermedius, and no destruction of hemolytic complement activity was induced by the non‐proteolytic periodontal pathogen Actinobacillus actinomycetemcomitans. It was thus concluded that B. gingivalis is capable of proteolysis of purified complement proteins, but that the consumption of complement proteins in serum, which is completely inhibited by EDTA, is not appreciably affected by the proteolytic capacity of B. gingivalis.

Effect of immunization on experimental Bacteroides gingivalis infection in a murine model.

BALB/c mice were immunized with an invasive (A7A1-28) or noninvasive (381) Bacteroides gingivalis strain, Bacteroides intermedius, or Ringer solution. All immunized mice were subsequently challenged with the invasive B. gingivalis strain and examined for septicemia or secondary spread of the infection or both. Mice immunized with the invasive B. gingivalis strain localized the infection to the challenge site. Mice immunized with the noninvasive B. gingivalis strain, B. intermedius, or Ringer solution developed spreading infections. These data suggest that immunization with an invasive B. gingivalis strain can alter the course of subsequent infections.

Chemical composition and immunobiological activities of sodium dodecyl sulphate extracts from the cell envelopes of Actinobacillus actinomycetemcomitans, Bacteroides gingivalis and Fusobacterium nucleatum.

The chemical composition and immunobiological activities in vivo and in vitro of sodium dodecyl sulphate extracts (SDS-SE) derived from periodontopathic bacteria (three strains of Actinobacillus actinomycetemcomitans, two strains of Bacteroides gingivalis, and one strain of Fusobacterium nucleatum) were investigated. The main components of SDS-SE were protein and lipid, with negligible amounts of peptidoglycan and lipopolysaccharide. Immunopotentiating activity was detected in both delayed-type hypersensitivity and antibody formation against the elicitation of a protein antigen with the SDS-SE preparations of A. actinomycetemcomitans ATCC 29524 and B. gingivalis 381 and 1021. On the other hand the SDS-SE of A. actinomycetemcomitans ATCC 29522 enhanced only the induction of a delayed-type hypersensitivity response. All the SDS-SE preparations had mitogenic activity to splenocytes from BALB/c nu/nu, C3H/HeN and C3H/HeJ mice. Migration-stimulating activity for human peripheral blood monocytes was detected especially in the SDS-SE preparations of A. actinomycetemcomitans ATCC 29524 and Y4. All of the SDS-SE samples inhibited [3H]thymidine uptake in human gingival fibroblasts and caused degradation of the cells. The results suggest that the cell membrane components extractable with sodium dodecyl sulphate from periodontopathic bacteria are involved in the pathogenesis of periodontal disease.

Interactions of Bacteroides gingivalis with fibrinogen.

Results of previous studies from our laboratory have shown that a strain of Bacteroides intermedius isolated originally from a patient with acute necrotizing ulcerative gingivitis binds and degrades human fibrinogen (M.S. Lantz, L.M. Switalski, K.S. Kornman, and M. Hook, J. Bacteriol. 163:623-628, 1985). We report that strains of Bacteroides gingivalis, an organism implicated in the etiology of several forms of periodontitis, also bind and degrade fibrinogen. The binding is rapid, reversible, saturable, and specific. The number of fibrinogen-binding sites per cell varies from 500 to 1,500 in different batches of bacteria, and the dissociation constant for the complex is on the order of 10(-8) M. B. gingivalis possesses cell-associated fibrinogenolytic activity that is activated by dithiothreitol and blocked by thiol protease inhibitors. Interaction with fibrinogen may mediate colonization and establishment of these organisms in the periodontal microbiota.

Susceptibility of Bacteroides gingivalis to bactericidal activity of human serum.

Susceptibility of Bacteroides gingivalis strains to the complement-mediated bactericidal activity of human serum was examined under anaerobic conditions. Serum containing high concentrations of IgG antibody to B. gingivalis enhanced the bactericidal activity. No enhancement was found in serum which did not contain the specific antibody or in serum absorbed with intact cells. The sensitivity to the killing by pooled serum differed among B. gingivalis strains. Cells of B. gingivalis activated the pooled human serum complement not only through the classic pathway but also through an alternative pathway. It was found that the susceptibility of B. gingivalis to the bactericidal activity was classic-pathway-dependent.

Human and animal serotypes of Bacteroides gingivalis defined by crossed immunoelectrophoresis.

The antigenic complexity of three strains of Bacteroides gingivalis and four strains resembling B. gingivalis isolated from animals was analyzed and compared by crossed immunoelectrophoresis. Thirteen antigens of the human biotype were present in all human strains and six antigens of the animal biotype were present in all animal strains, indicating a marked serological homogeneity within each biotype. Four antigens cross-reacting between the human B. gingivalis and the animal strains were identified. This antigenic relatedness defined the serological homogeneity of the two biotypes within the species and allowed recognition of four species-specific antigens. Two antigens specific to the human strains and two antigens specific to the animal strains were identified, indicating that serotype-specific antigens can distinguish each biotype. It is thus proposed that the oral, black-pigmented asaccharolytic Bacteroides strains of animal origin be classified as catalase-positive variants of B. gingivalis. It is also proposed that two serotypes be recognized within the species B. gingivalis. Serotype 1 includes the catalase-negative human biotype, and serotype 2 includes the catalase-positive animal biotype.

Ability of oral bacteria to degrade fibronectin.

The fibronectin-degrading ability of 116, mainly oral, strains was assayed by using plasma-derived fibronectin adsorbed to a polystyrene surface. Ability to degrade fibronectin was revealed in strains of Bacteroides gingivalis, Bacteroides intermedius, Bacteroides loeschii, Staphylococcus aureus, Staphylococcus epidermidis, Peptococcus prevotii, Clostridium sporogenes, and Propionibacterium acnes. The fibronectinolytic activity of subgingival bacteriological samples was found to be related to the presence of B. gingivalis and B. intermedius. In addition, strains of the nonoral Bacteroides species B. asaccharolyticus and B. fragilis showed fibronectin-degrading ability. No such ability was detected in the oral strains tested of Streptococcus, Veillonella, Actinomyces, Lactobacillus, Actinobacillus, Capnocytophaga, Fusobacterium, or Haemophilus species.

The characterization of trypsin-positive black-pigmented Bacteroides isolated from sheep periodontal disease

Twenty trypsin‐positive black‐pigmented Bacteroides strains independently isolated from periodontal lesions in sheep were characterized by Gram stain, biochemical tests, haemagglutination, gas liquid chromatography, API ZYM system profiles and catalase activity. All strains reacted uniformly in these tests, the reactions closely resembling those reported for human strains of Bacteroides gingivalis.

An electron microscope survey of the surface structures and hydrophobicity of oral and non-oral species of the bacterial genus Bacteroides

Seventeen strains of Bacteroides representing 10 species were examined by negative staining; the majority were from the mouth but a few non-oral strains were included. Seven species had peritrichously-arranged, non-flagellar appendages which could be divided by morphology and ultra-structure into two subgroups, fibrils and fimbriae. Bacteroides asaccharolyticus strains B536 and B537 and Bacteroides gingivalis strains W50, W83, WPH15 and WPH35 had fimbriae with mean width of 4.4 nm and 0.5 to 6.0 μm long depending on the strain. The fimbrial length within each strain also varied. Fibrils were present on two fresh oral isolates of Bacteroides melaninogenicus, Bacteroides intermedius strains T588 and W09, Bacteroides corporis ATCC 33547, Bacteroides oralis ATCC 33269 and Bacteroides buccae ATCC 33574. Fibrils consistently clumped into bundles of variable thickness and formed a fringe around the cell periphery, ranging from 0.27 to 1.2 μm long depending on the strain. Fibril lengths of each strain were uniform. Fibrils had no measurable width and the clumps tapered towards the free ends. Bacteroides loeschii VPI 9085, Bacteroides pentosaceus strains NP333 and Jl and Bacteroides capillosus 29799 had no detectable surface appendages. Fimbriate strains had a layer outside the outer membrane, with a mean thickness of between 17.8 and 28.6 nm. Both fibrillar and fimbriate strains produced many small membranous vesicles budding from the outer membrane. There were two morphological forms of vesicles, ones with either fimbriae or fibrils (species-dependent) and ones with no attached appendages. Of eleven strains tested for cell-surface hydrophobicity by partitioning between hexadecane and buffer, all but one was non-hydrophobic.

Inactivation of guinea-pig serum proteinase inhibitors by Bacteroides gingivalis.

The activity of proteinase inhibitors of guinea-pig serum was evaluated by using trypsin as test enzyme. Strains of black-pigmented Bacteroides species were incubated with serum under anaerobic conditions. Strains of Bacteroides gingivalis inactivated the trypsin inhibitors of guinea-pig serum within 1 h, while the other strains reduced the activity of the trypsin inhibitors by less than 50% within 48 h. The inactivation of proteinase inhibitors of guinea-pig serum by B. gingivalis may be an important pathogenic mechanism in the spreading necrotic infections induced in guinea pigs by pure cultures of this species.

Isolation and characterization of the peptidoglycans from selected gram-positive and gram-negative periodontal pathogens.

The peptidoglycans from several Gram-negative and Gram-positive periodontal pathogens were isolated, purified, and characterized both morphologically and chemically. In addition, the effects of the mureolytic enzymes, lysozyme, M-1 N-acetyl-muramidase, and the AM-3 endopeptidase, on the peptidoglycans were examined. These enzymes were found to be highly effective in the degradation of the purified peptidoglycans; however, a Bacteroides capillus peptidoglycan-protein complex exhibited a greater resistance to these enzymes. Morphologically, the peptidoglycans consisted of large saccular sheets which, when viewed by scanning electron microscopy, contained numerous holes and tears. Chemically, the peptidoglycans consisted of muramic acid, glucosamine, alanine, glutamic acid, and meso-diaminopimelic acid (DAP). One Bacteroides species, Bacteroides gingivalis strain W, contained glycine and LL-DAP, suggestive of an indirectly cross-linked A3 gamma peptidoglycan.