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PubMed2006

Multiplex polymerase chain reaction detection of black-pigmented bacteria in infections of endodontic origin.

The purpose of this study was to detect the presence of Porphyromonas endodontalis, P. gingivalis, Prevotella intermedia, P. nigrescens, and P. tannerae from clinical samples using multiplex polymerase chain reactions (PCR). Two different multiplex PCR protocols were used (one for the two Porphyromonas species and the other for the three Prevotella species), each one using a primer pair specific for each target species. The results were compared to those of the conventional culture procedures. Microbial samples were taken aseptically from 40 infected root canals and abscesses from patients. Samples were cultured in an anaerobic condition for conventional identification using a Rapid ID 32 A kit. Multiplex PCR was processed using the DNA extracted from each sample. At least one of the five species of black-pigmented bacteria (BPB) were detected in 65% (26 of 40) of the samples using multiplex PCR, and in 15% (6 of 40) using the conventional culture procedures. Multiplex PCR was more rapid, sensitive, specific, and effective in detecting BPB than the conventional culture procedures.

Bacteroidaceae Infections↗
PubMed2006

Microbiology and antibiotic sensitivities of head and neck space infections of odontogenic origin.

PURPOSE: The purpose of this study is to assess the anatomical spaces and causative micro-organisms responsible for deep fascial space head and neck infections and evaluate the resistance of antibiotics used in the treatment of these infections. PATIENTS AND METHODS: A 6-year retrospective study evaluated hospital records of 103 patients. All patients in this study underwent surgical incision and drainage, received IV antibiotics, and had culture and sensitivity performed. Patient demographics reviewed were gender, age, involved fascial space(s), micro-organisms identified and antibiotic resistance from culture and sensitivity testing. RESULTS: There were 56 male (54%) and 47 (46%) female patients. The submandibular space was the most frequent location for a single space abscess (30%), followed by the buccal space (27.5%) and the lateral pharyngeal space (12.5%). Sixty-three patients presented with multiple space involvement, totaling 142 spaces involved. A total of 269 bacterial strains were isolated from 103 patients. The bacteria were found to be 63.5% gram-positive. Gram-positive cocci were isolated 57.7% of specimens and gram-negative rods were isolated in 33% of cultures. There were 178 aerobes (65.7%) and 91 anaerobes (34.3%) isolated. The most common bacteria isolated were Viridans streptococci, Provetella, Staphylococci, and Peptostreptococcus. Culture and sensitivities were reviewed on 101 patients. CONCLUSION: Patients who underwent surgical incision and drainage in the operating room had a tendency for involvement of multiple space abscesses with the submandibular space, submental, and lateral pharyngeal spaces effected most frequently. Cultures and sensitivities commonly showed greater growth in aerobes (65.7%) than in anaerobes. Gram positive cocci and gram negative rods had the greatest growth percentage in cultures.

Abscess↗
PubMed2003

Mixed infections with Porphyromonas gingivalis and Treponema denticola cause excessive inflammatory responses in a mouse pneumonia model compared with monoinfections.

Periodontopathic anaerobes such as Porphyromonas gingivalis are frequently found in aspiration pneumonia and lung abscesses. However, defense mechanisms and responses to these bacterial infections in the lung in vivo remain poorly understood. The coexistence of P. gingivalis with Treponema denticola has been found at higher levels and proportions in periodontally diseased sites. We hypothesized that mixed infections with P. gingivalis and T. denticola can cause severe respiratory disease. In the present study, inflammatory responses to mono- and mixed inoculations with P. gingivalis and T. denticola in the bronchoalveolar lavage (BAL) fluid were investigated. Acute pneumonia and lung abscesses in mice with the mixed infection resulted in a 40% mortality rate within 72 h, compared with only 10% mortality for the respective monoinfections. Pulmonary clearance of P. gingivalis was delayed in the mice with mixed infections with P. gingivalis and T. denticola. Tumor necrosis factor alpha (TNFalpha) interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) levels from BAL fluid of mice with mixed infections at 24 h after inoculation were significantly higher than those after P. gingivalis monoinfection (TNFalpha: P < 0.05, Il-1beta: P < 0.001, IL-6: P < 0.05). The chemokine KC level from BAL fluid of mice at 48 h (P < 0.05) and 72 h after mixed infection was also significantly increased when compared with that after P. gingivalis monoinfection (P < 0.001). The present study demonstrates that a mixed infection of P. gingivalis with T. denticola in mouse causes a marked bronchopneumonia and lung abscess in the mouse model.

Abscess↗
PubMed2005

Inflammatory responses of a macrophage/epithelial cell co-culture model to mono and mixed infections with Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia.

Accumulated evidence points to Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia as three major etiologic agents of chronic periodontitis. Epithelial cells and macrophages play a major role in the host response to periodontopathogens, and the secretion of inflammatory mediators and matrix metalloproteinases (MMPs) by these host cells is believed to contribute to periodontal tissue destruction. The aim of this study was to investigate the inflammatory response of a macrophage/epithelial cell co-culture model following mono or mixed infections with the above three periodontopathogens. An in vitro co-culture model composed of epithelial-like transformed cells (HeLa cell line) and macrophage-like cells (phorbol myristic acid-differentiated U937 monocytic cell line) was challenged with whole cells or lipopolysaccharides (LPS) of P. gingivalis, T. denticola, and T. forsythia, individually and in combination. Following stimulation, the production of interleukin-1 beta (IL-1beta), IL-6, IL-8, tumor necrosis factor alpha (TNF-alpha), regulated on activation normal T cell expressed and secreted (RANTES), prostaglandin E2 (PGE2), and MMP-9 were quantified by enzyme-linked immunoassays. We observed that mono or mixed infections of the co-culture model induced the secretion of IL-1beta, IL-6, IL-8, PGE2, and MMP-9. P. gingivalis and T. forsythia induced an increase in RANTES secretion, whereas T. denticola alone or in combination resulted in a significant decrease in RANTES levels. All LPS challenges induced an increase in chemokine, MMP-9, and PGE2 production. No synergistic effect on the production of cytokines, chemokines, PGE2, and MMP-9 was observed for any of the bacterial or LPS mixtures tested. This study supports the view that P. gingivalis, T. denticola, and T. forsythia may induce high levels of pro-inflammatory mediators and MMP-9 in periodontal lesions, thus contributing to the progression of periodontitis.

Bacteroidaceae↗
PubMed2006

Proinflammatory gene expression in mouse ST2 cell line in response to infection by Porphyromonas gingivalis.

Porphyromonas gingivalis is a predominant periodontal pathogen, whose infection causes inflammatory responses in periodontal tissue and alveolar bone resorption. Various virulence factors of this pathogen modulate host innate immune responses. It has been reported that gingipains degrade a wide variety of host cell proteins, and fimbriae are involved in bacterial adhesion to and invasion of host cells. In the present study, we profiled ST2 stromal cell gene expression following infection with the viable P. gingivalis strain ATCC33277 as well as with its gingipain- and fimbriae-deficient mutants, using microarray technology and quantitative real-time polymerase chain reaction. Using a mouse array of about 20,000 genes, we found that infection with the wild strain elicited a significant upregulation (greater than 2-fold) of expression of about 360 genes in ST2 cells, which included the chemokines CCL2, CCL5, and CXCL10, and other proinflammatory proteins such as interleukin-6 (IL-6) and matrix metalloproteinase-13 (MMP-13). Further, infection with the gingipain-deficient mutant elicited a reduced expression of the CXCL10, IL-6 and MMP-13 genes, suggesting that gingipains play an important role in inducing the expression of those genes following P. gingivalis infection. On the other hand, the pattern of global gene expression induced by the fimbriae-deficient mutant was similar to that by the wild strain. These results suggest that P. gingivalis infection induces gene expression of a wide variety of proinflammatory proteins in stromal cells/osteoblasts, and gingipains may be involved in inducing several of the proinflammatory factors.

Adhesins, Bacterial↗
PubMed2005

Genetic variation of a fimbrial protein from Porphyromonas gingivalis and its distribution in patients with periodontal diseases.

Pg-II fim from various strains of Porphyromonas gingivalis was classified on the basis of each nucleotide sequence, while the distribution of Pg-II fim types in 141 subgingival plaque samples was analyzed using PCR assays. Pg-II fim was divided into two types as follows: strains OMZ409, HG405, 381, ATCC 33277 and BH18/10 (type 1) and strains OMZ314 and HW24D-1 (type 2). The presence of P. gingivalis was demonstrated in 2.8% of healthy subjects and 56.1% of patients with periodontal diseases, and Pg-II fim was detected in 91.8% of the P. gingivalis-positive subjects. We also analyzed the distribution of the Pg-II fim types among Pg-II fim-positive patients, with the following results: type 1 (38.2%), type 2 (56.4%) and types 1 and 2 (5.4%). These findings strongly suggest that P. gingivalis organisms possessing Pg-II fim type 2 was principally detected in patients with periodontal diseases.

Adolescent↗
PubMed2006

[Pathogenic potential of Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia, the red bacterial complex associated with periodontitis].

Periodontitis are mixed bacterial infections leading to destruction of tooth-supporting tissues, including periodontal ligament and alveolar bone. Among over 500 bacterial species living in the oral cavity, a bacterial complex named "red complex" and made of Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia has been strongly related to advanced periodontal lesions. While periodontopathogenic bacteria are the primary etiologic factor of periodontitis, tissue destruction essentially results from the host immune response to the bacterial challenge. Members of the red complex are Gram negative anaerobic bacteria expressing numerous virulence factors allowing bacteria to colonize the subgingival sites, to disturb the host defense system, to invade and destroy periodontal tissue as well as to promote the immunodestructive host response. This article reviews current knowledge of the pathogenic mechanisms of bacteria of the red complex leading to tissue and alveolar bone destruction observed during periodontitis.

Animals↗
PubMed2005

Synthesis and assembly of an adjuvanted Porphyromonas gingivalis fimbrial antigen fusion protein in plants.

The gram-negative anaerobic oral bacterium Porphyromonas gingivalis initiates periodontal disease by binding to saliva-coated oral surfaces. To assess whether edible plants can synthesize biologically active P. gingivalis fimbrial antigen, for application as an oral vaccine, a cDNA fragment encoding the C-terminal binding portion of P. gingivalis fimbrial protein (FimA), was cloned into a plant expression vector immediately downstream of a cDNA fragment encoding the cholera toxin B subunit (CTB). The chimeric plasmid was transferred into potato (Solanum tuberosum) cells and the ctb-fimA cDNA fragment detected in transformed leaf genomic DNA by PCR amplification methods. A novel protein band of 21 kDa was detected in transformed potato tuber extracts by immunoblot analysis. Oligomeric CTB-FimA (266-337) fusion protein was identified in the extracts through the binding of anti-CTX and anti-native fimbriae antibodies. The pentameric structure of CTB-FimA fusion protein was confirmed by ELISA measurements of GM1 ganglioside receptor binding. Quantification of the CTB-FimA fusion protein by ELISA indicated that the chimeric protein made up about 0.33% of total soluble tuber protein. The biosynthesis of immunologically detectable CTB-FimA fusion proteins and the assembly of fusion protein monomers into biologically active pentamers in transformed potato tuber tissues demonstrate the feasibility of synthesizing adjuvanted fimbrial protein in edible plants for development of adjuvanted mucosal vaccines against P. gingivalis generated periodontal disease.

Adjuvants, Immunologic↗
PubMed2003

Antagonistic interactions among Fusobacterium nucleatum and Prevotella intermedia with oral lactobacilli.

Microbiota residing in oral ecosystems show great complexity because of the mutual interdependence of certain microorganisms that might influence their presence or absence at a given site. The aim of this work was to evaluate possible antagonistic interactions among Fusobacterium nucleatum and Prevotella intermedia, isolated from periodontal pockets, and Lactobacillus casei, L. salivarius, L. rhamnosus and L. plantarum isolated from saliva. Different tests were carried out using each strain as an effector as well as a sensitive strain, alternatively: (1) agar diffusion with paper disks embedded with supernatants and pellets in different culture media; (b) the well diffusion assay; (c) the double layer method; and (d) OD(560) measurements of the kinetic growth of Lactobacillus strains in LAPTg broth with different volumes of F. nucleatum supernatant. P. intermedia supernatant did not have an inhibitory effect on Lactobacillus growth, except for L. casei, when the double plaque method was used. F. nucleatum supernatant inhibited growth of all Lactobacillus strains when the well diffusion method was used. F. nucleatum inhibited L. casei growth whatever the method used. The substance responsible for inhibition was a thermoresistant extracellular protein which was hydrolyzed by trypsin-like proteases. None of the species of lactobacilli studied inhibited the growth of P. intermedia or F. nucleatum.

Antibiosis↗
PubMed2005

Prevotella massiliensis sp. nov. isolated from human blood.

We report a bacterial isolate (Marseille isolate) recovered from the blood of a patient hospitalized in an intensive care unit, presenting with severe trauma, fever and mechanical ventilation. Colonies appeared at 37 degrees C on blood agar after 72 h incubation. This isolate was a strictly anaerobic, Gram-negative rod phenotypically related to other Prevotella species described to date: non-motile, catalase-negative, oxidase-positive, non-glucose fermenting, resistant to vancomycin and susceptible to kanamycin. Cells exhibited a trilamellar membrane under electron microscopy. The fatty acid methyl ester profile was marginally related to that of Clostridium botulinum group A (distance: 26.27%) and Bifidobacterium bifidum GC subgroup B (distance: 26.38%). 16S rRNA gene sequence similarity was 90.0% with that of Prevotella oris and 89.1% with that of Prevotella melaninogenica. Partial rpoB gene sequence similarity was 84.5 and 86.4% with P. oris and P. melaninogenica, respectively. According to current standards, phenotypic traits, 16S rRNA and rpoB gene sequence analyses indicated that the Marseille isolate belonged to a previously unrecognized species of the genus Prevotella, and we propose classifying it in the new taxon "Prevotella massiliensis" sp. nov.

Adult↗
PubMed2004

Targeting of Porphyromonas gingivalis with a bispecific antibody directed to FcalphaRI (CD89) improves in vitro clearance by gingival crevicular neutrophils.

Phagocytosis and killing of pathogens by polymorphonuclear neutrophils (PMN) from gingival crevicular fluid (GCF) is diminished in chronic periodontitis patients. As an approach to improve targeting of PMN toward a periodontopathogen, Porphyromonas gingivalis, the efficacy of a bispecific antibody (BsAb) directed against both recombinant 130 kDa hemagglutinin domain (r130k-HMGD) of P. gingivalis, and PMN Fc receptor (FcR) was evaluated. GCF PMN exhibited higher IgA FcR (FcalphaRI) levels, and lower IgG FcR (FcgammaRIIa and FcgammaRIIIb) levels than PB PMN. Functional studies revealed that GCF PMN exhibited a higher capacity to phagocytose and kill P. gingivalis opsonized with a BsAb targeting P. gingivalis r130k-HMGD to FcalphaRI as compared to an anti-r130k-HMGD antibody. However, phagocytosis and killing activity of PB PMN that were incubated with the two antibodies proved comparable. These data support targeting of pathogens toward FcalphaRI as an option to improve antibacterial immunity in chronic periodontitis patients.

Adhesins, Bacterial↗
PubMed2005

Transcutaneous immunization with a 40-kDa outer membrane protein of Porphyromonas gingivalis induces specific antibodies which inhibit coaggregation by P. gingivalis.

This study seeks to assess the potential of a 40-kDa outer membrane protein of Porphyromonas gingivalis (40k-OMP) as a transcutaneous vaccine against chronic periodontitis. Transcutaneous immunization (TCI) of mice with 40k-OMP alone elicited 40k-OMP-specific IgG antibody (Ab) responses in both serum and saliva. When administered with cholera toxin (CT) as adjuvant, TCI with 40k-OMP not only elevated IgG Abs as noted above, but also induced IgA responses in serum but not in saliva. Salivary IgG from mice given 40k-OMP alone or 40k-OMP plus CT showed higher binding levels to the 40k-OMP than did that of non-immunized mice. Ab-forming cell (AFC) analysis revealed high numbers of 40k-OMP-specific IgG AFCs in the spleen but low numbers in the salivary glands of mice given 40k-OMP alone or 40k-OMP plus CT. Since 40k-OMP-specific IgG inhibited the coaggregation of P. gingivalis vesicles and S. gordonii, TCI with 40k-OMP may be a useful tool in the quest to prevent P. gingivalis infection.

Adjuvants, Immunologic↗
PubMed2006

Vaccination with recombinant adhesins from the RgpA-Kgp proteinase-adhesin complex protects against Porphyromonas gingivalis infection.

Porphyromonas gingivalis is a pathogen associated with chronic periodontitis, an inflammatory disease of the supporting tissues of the teeth. A major virulence factor for P. gingivalis is the RgpA-Kgp proteinase-adhesin complex. We have prepared the following recombinant proteins corresponding to domains/regions of the RgpA-Kgp complex; rRgpA(cat), rRgpA(A1), rRgpA(A2), rRgpA(A4), rRgpA(A1)(784-1009), rRgpA(A1)(911-1009), rKgp(A1) and rKgp(A1)(759-989). The ability of each recombinant protein to attenuate P. gingivalis infection, when used as a vaccine, in the murine lesion model was determined. All of the recombinant adhesin domains were found to significantly attenuate P. gingivalis infection with the most effective being rKgp(A1) and rKgp(A1)(759-989) followed by rRgpA(A1), rRgpA(A1)(784-1009) and rRgpA(A1)(911-1009). The predominant antibody isotype was IgG1. The A1 adhesins, which gave the best protection, contain specific motifs implicated in binding to host tissue. Immunisation with rRgpA(cat) had no effect on P. gingivalis infection. As well as detecting the Kgp(A1) adhesin in a Western blot, the rKgp(A1) and rKgp(A1)(759-989) antisera were also immunoreactive with the A1 and A3 adhesins of RgpA and HagA. Flow cytometric analysis indicated that rKgp(A1) and rRgpA(A1) antisera recognised native antigen on P. gingivalis whole cells. Furthermore, the rKgp(A1) and rKgp(A1)(759-989) antisera exhibited a similar immunoreactive profile with outer membrane preparations of all P. gingivalis serotypes and clinical isolates tested. The recombinant A1 adhesin therefore has potential in the development of a P. gingivalis vaccine.

Adhesins, Bacterial↗
PubMed2005

Pigmented-anaerobic bacteria associated with canine periodontitis.

The etiology of human periodontal disease has been the focus of considerable research, yet relatively little is known about the causative agents of companion animal periodontitis. In humans, Porphyromonas gingivalis, a black-pigmented anaerobic bacteria (BPAB), has been implicated as the primary periopathogen. It has been demonstrated that BPAB are also found in companion animal periodontal pockets. While some animal BPAB have been individually identified, a study to identify the most frequently isolated subgingival BPAB has not been completed using genetic tools. The objective of this work was to identify the types and relative frequencies of pigmented anaerobic bacteria found in the periodontal pockets of dogs. Porphyromonas salivosa, Porphyromonas denticanis (a novel species) and Porphyromonas gulae were found to be the most frequently isolated BPAB associated with canine periodontitis.

Animals↗
PubMed2004

Systemic up-regulation of sTNFR2 and IL-6 in Porphyromonas gingivalis pneumonia in mice.

Aspiration pneumonia is a common cause of death in older people, and the pathophysiology is a chronic respiratory failure with a mild airway inflammation. In this study, we established a mild inflammatory pneumonia model using Porphyromonas gingivalis (Pg) pathogen-infected mice. It elucidated the effects of Pg-infected pneumonia on proinflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin-6 (IL-6), and IL-1beta production in both lung tissue and serum. We also elucidated production of soluble (s) TNF receptor (R) s, because TNF-alpha is considered to be a dominant inflammatory mediator. Lung TNF-alpha levels significantly increased at 2 h after infection, and rapidly returned to basal level at 24 h. Consistent with increase of TNF-alpha, remarkable increase of sTNFR2 but not sTNFR1 was detected in lung tissue from 2 to 72 h. Interestingly, sTNFR2/sTNFR1 ratio was significantly enhanced at 2 h in serum. In addition, lung IL-1beta and IL-6 levels also significantly increased from 2 to 24 h. Importantly, we found that IL-6 levels in serum reflected its local level. These results may suggest that systemically produced sTNFR2 and IL-6 could be a key role to modulate proinflammatory activities of TNF-alpha in Pg-induced lung inflammation simulated aspiration pneumonia.

Animals↗
PubMed1999

T-cell antigen specificity in humans following stimulation with Porphyromonas gingivalis.

The effects of Porphyromonas gingivalis stimulation on T-cell clonality and cytokine mRNA expression in peripheral blood mononuclear cells from individuals with gingivitis and periodontitis were investigated. Clonality of T cells was investigated by reverse transcription-polymerase chain reaction (RT-PCR) and single-strand conformation polymorphism analysis. Cytokine mRNA expression was investigated by RT-PCR. Whereas unstimulated mononuclear cells did not demonstrate obvious clonality, clonal expansion was found in most Vbeta families after stimulation. However, there was no relation between clonal change and disease category or the presence of P. gingivalis infection. Messenger RNA for interferon-gamma and interleukin-13 was upregulated whereas interleukin-4 and -10 were downregulated following P. gingivalis stimulation. Interleukin-12p35 demonstrated no consistent pattern. This study supports the concept that P. gingivalis stimulates T cells in an antigen-specific fashion. It further suggests that peripheral blood T cells may preferentially produce interferon-gamma and interleukin-13 in response to P. gingivalis stimulation irrespective of disease or P. gingivalis status.

Bacteroidaceae Infections↗
PubMed1997

Structural studies of the extracellular polysaccharide from Butyrivibrio fibrisolvens strain CF3.

The structure of the Butyrivibrio fibrisolvens strain CF3 capsular polysaccharide has been investigated mainly by sugar and methylation analyses, Smith degradation, NMR spectroscopy, and mass spectrometry. The results indicate that the polysaccharide is composed of pentasaccharide repeating units having the following structure: -->4)-beta-L-Altp-(1-->4)-beta-D-Glcp-(1-->3)-4-O-[(R)-1-carboxyet hyl]-beta- D-Glcp-(1-->4)-6-O-[(R)-1-carboxyethyl]-alpha-D-Galp-(1--> 2 increases 1 beta-D-Glcp.

Bacteroidaceae↗