Adult periodontitis: an ecological perspective of mixed infections.
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Adult↗
Biomedical subjects
D Grenier
Publications and source records attributed to D Grenier.
Demonstration of adherence properties of Porphyromonas gingivalis outer membrane vesicles using a new microassay.
Vesicles made by Porphyromonas gingivalis possess several biological activities, including the ability to adhere to oral surfaces and to bacteria. In this study, a new and simple method was developed to measure the adherence capability of outer membrane vesicles from P. gingivalis. Vesicles were conjugated to fluorescent microspheres (0.7 micron) and added to wells of a Teflon-coated microscope slide previously covered with a variety of soluble ligands. After incubation and washes, the number of fluorescent microspheres per microscopic field were counted. Vesicle-coated microspheres attached best to gelatin (> 200 per field), whereas other compounds (such as fibronectin, fibrinogen, collagen and laminin) provided moderate attachment, and no attachment was observed to bovine serum albumin. Adherence to any of the tested ligands was not observed when fluorescent microspheres were conjugated to bovine serum albumin or lipopolysaccharides from P. gingivalis. The adherence of vesicle-coated microspheres to ligands was not significantly affected when the pH of the reaction mixture was between 4 and 10. None of the tested carbohydrates lowered the attachment capability of vesicle-coated microspheres to substrates. When vesicle-coated microspheres were treated with trypsin and chymotrypsin or heated, this resulted in a significant loss of attachment, suggesting a possible involvement of proteinaceous molecules in the process. The present study confirms that vesicles of P. gingivalis are capable of attachment to various molecules and indicate their potential role in colonization.
Animals↗
Characterization of immunoglobulin G-degrading proteases of Prevotella intermedia and Prevotella nigrescens.
Degradation of immunoglobulins is thought to be an important factor in the causation of periodontal diseases by hindering local host defenses and by providing nutrients to the periodontal microflora. In this study, we characterized the proteolytic activity against human immunoglobulin G (IgG) of 20 strains of Prevotella intermedia and Prevotella nigrescens isolated from periodontal pockets and oral abscesses. IgG degradation was studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. All strains degraded IgG within 48 h after growth in trypticase-yeast extract medium (TY) supplemented with 0.3% IgG. Incorporating IgG in TY broth enhanced bacterial growth. Protease profiles (zymography), which revealed the presence of 1-4 IgG-degrading proteolytic bands in bacterial cell extracts, became more complex after growth in the presence of IgG. A 38-kDa protease capable of degrading IgG nonspecifically was present in almost all strains. The proteolytic activity was mainly located on the surface of the cell envelope. Two strains of P. intermedia and P. nigrescens ATCC 33563 were selected for further studies. Bacterial cell suspensions in phosphate-buffered saline completely degraded human IgG, IgA and IgM within 24 h. This activity depended on reducing conditions and was inhibited at temperatures above 50 degrees C. The pH optimum of immunoglobulin degradation was at pH 7. Strains cultured at 42 degrees C showed a markedly reduced capacity to degrade IgG. Inhibition studies revealed that breakdown of IgG was caused by a cysteine protease(s). The capacity of P. intermedia and P. nigrescens to degrade immunoglobulins may explain their association with polymicrobial oral diseases.
Animals↗
Porphyromonas gingivalis outer membrane vesicles promote bacterial resistance to chlorhexidine.
Porphyromonas gingivalis has been frequently associated with some types of periodontal diseases and possesses various mechanisms favoring the pathogenic process. It has been recently observed that vesicles elaborated by P. gingivalis are able to protect bacteria from the bactericidal activity of human serum. The aim of the present investigation was to evaluate the ability of vesicles from P. gingivalis in protecting oral bacteria against chlorhexidine. Data indicate that vesicles released by P. gingivalis may bind chlorhexidine, thus allowing protection for itself and for other oral bacterial species. It has also been demonstrated that lipopolysaccharides are the major component involved in the binding of chlorhexidine by vesicles. The mechanism of resistance reported in this study indicates that bacterial interactions in the oral cavity may influence the sensitivity of microbes.
Anti-Infective Agents, Local↗
Quantitative analysis of bacterial aerosols in two different dental clinic environments.
Microbial aerosols are generated during dental treatments and may represent an important source of infection. This study was designed to quantify bacterial air contamination during dental treatments in both a closed dental operatory and a multichair dental clinic. Air was sampled by using a slit type of biological air sampler. Following air sampling, blood-supplemented Trypticase soy agar plates were incubated at 37 degrees C under anaerobic conditions for 7 days. The maximum levels of air contamination in the closed dental operatory were observed while dental treatments were being performed (four trials; 216 +/- 75 CFU/m3 for ultrasonic scaling treatments and 75 +/- 22 CFU/m3 for operative treatments). At 2 h after completion of the treatments, the bacterial counts were about the same as the pretreatment levels (12 to 14 CFU/m3). In the second part of the study, a multichair dental clinic was divided into four areas, and air contamination was monitored at each site. Three sites were located in active dental treatment areas, whereas no dental treatments were performed within an 11-m radius of the fourth site. At 3 h after the beginning of dental treatments, the highest bacterial counts were obtained in the three active dental treatment areas (76 to 114 CFU/m3). However, there was noticeable contamination in the inactive dental treatment area (42 CFU/m3). Thus, bacterial aerosols were able to spread into areas where there was no dental activity. My data show that dental treatments significantly increased the levels of bacterial air contamination in both a closed dental operatory and a multichair dental clinic.(ABSTRACT TRUNCATED AT 250 WORDS)
Aerosols↗
Characterization of the human immunoglobulin G Fc-binding activity in Prevotella intermedia.
Many pathogenic bacteria possess cell surface receptors which can bind immunoglobulins via the Fc portion. The aim of this study was to characterize the human immunoglobulin G (IgG) Fc-binding activity of Prevotella intermedia, a suspected etiologic agent of adult chronic periodontitis. The Fc-binding activity of P. intermedia on whole cells and on extracellular vesicles was demonstrated. Incubation of P. intermedia cells in the presence of Zwittergent 3-14 allowed complete solubilization of the Fc receptor from the cell surface. This cell envelope extract was thus used to characterize the Fc-binding activity. A microtiter plate assay using alkaline phosphatase-labeled Fc fragments showed that preincubation of the cell envelope extract with human IgG, human IgG Fc fragments, or human serum completely inhibited the Fc-binding activity. Partial inhibition was obtained with human IgG F(ab')2 fragments, whereas no inhibition occurred following preincubation with human IgA, carbohydrates, and selected proteins. Preincubation of the cell envelope extract with IgG from a variety of animals demonstrated that rabbit, mouse, rat, goat, and sheep IgG did not inhibit Fc-binding activity, whereas cow, pig, and dog IgG partially inhibited Fc-binding activity. A strong inhibition comparable to that obtained with human IgG was noted with monkey IgG. The Fc receptor of P. intermedia is thus different from the six types previously reported in other nonoral bacteria. Polyacrylamide gel electrophoresis and Western blotting (immunoblotting) analysis of the cell envelope extract revealed a major band with a molecular mass of approximately 65 kDa which reacted with peroxidase-labeled human IgG Fe fragments. Transmission electron microscopy showed a uniform distribution of the Fc receptor on the bacterial surface, as revealed by gold labeling. The Fc-binding activity demonstrated in this study may act as an additional virulence factor for P. intermedia by reducing IgG reactions with the bacterial cell.
Bacterial Proteins↗
[Patients with heart valve prostheses: dental care, dental procedures and prevention].
Patients with birth or rheumatic heart defects, or wearing prosthetic cardiac valves, are at high risk of developing bacterial endocarditis following dental treatment. Two hundred patients with cardiac valves were surveyed with respect to both their oral hygiene habits and their last visit to the dentist. More than half of the respondents (112) were fully edentulous, and most of them had not seen a dentist for many years. Of the 88 respondents who had natural teeth, 23 had not been to a dentist since surgery. For the purpose of this study, focus was placed primarily on the answers given by the 65 patients who reported having been to a dentist after surgery. Only 41 of them (63 per cent) reported having received a prophylactic antibiotic therapy while being treated with a risk of bacteremia. Of the 24 others who did not receive prophylactic antibiotic therapy, 20 were given dental treatments with a risk of bacteremia. In light of this study, there should be recommendations to improve the health care given to patients predisposed to bacterial endocarditis.
Antibiotic Prophylaxis↗
Selective growth inhibition of Porphyromonas gingivalis by bestatin.
Recent work in our laboratory indicates that selected protease/peptidase inhibitors interfere with the growth of Porphyromonas gingivalis. The aim of the present study was to further investigate the inhibitory effect of bestatin on the growth of P. gingivalis. Complete growth inhibition of P. gingivalis (11 strains) was observed when bestatin was incorporated at 2.5 micrograms ml-1 in a complex broth medium. Fifty percent inhibition was still obtained with bestatin at a final concentration of 0.5 microgram ml-1. The inhibitory effect of bestatin was highly specific as the growth of 20 different oral bacterial species, including Gram-positive and Gram-negative as well as saccharolytic and asaccharoltic bacteria, was not affected even at bestatin concentrations up to 50 micrograms ml-1. Bestatin did not significantly affect the viability of P. gingivalis indicating that it has a bacteriostatic rather than a bactericidal effect. Growth assays using other specific inhibitors suggested that the effect of bestatin on the growth of P. gingivalis was unlikely to be related to its aminopeptidase inhibitor activity. Cultivation of P. gingivalis with a subinhibitory concentration of bestatin did not modify the cell envelope protein profile, as determined by SDS-PAGE analysis, but significantly decreased the number of extracellular vesicles produced. The present study indicated that bestatin is a highly effective inhibitor of cell growth of P. gingivalis. Additional studies will indicate whether bestatin should be considered as a potential drug in the control of P. gingivalis, a suspected pathogen in adult chronic periodontitis.
Anti-Bacterial Agents↗
Detection of stress proteins in Porphyromonas gingivalis and other oral bacteria by western immunoblotting analysis.
Detection of stress proteins in Porphyromonas gingivalis was investigated by SDS-PAGE and Western immunoblotting procedure using a polyclonal antibody (anti hsp60) and a monoclonal antibody (anti-Dnak). Results indicate that P. gingivalis can elicit a hsp60-like stress protein when submitted to different environmental stresses such as a heat shift from 35 degrees C to 43 degrees C, a pH drop from 7.2 to 6.0 or an increase in oxygen concentration. Virulent and non-virulent strains of P. gingivalis responded the same way. The other bacterial species tested also showed an increased synthesis of a GroEL-like protein after heat shock, as detected by the anti hsp60 antibody. However, the monoclonal anti-Dnak recognized an hsp70-like protein only in two of the tested species.
Adult↗
Characteristics of a protease inhibitor produced by Prevotella intermedia.
The characterization of a protease inhibitor produced by a strain of Prevotella intermedia is reported in the present study. The protease inhibitor was secreted into the culture medium during the exponential growth phase and was also present in significant amounts in the periplasmic space. The inhibitor was active against a wide variety of proteolytic enzymes, including the trypsin-like activity of Porphyromonas gingivalis. The inhibitory compound, a heat labile protein, was isolated by non-denaturing preparative polyacrylamide gel electrophoresis, using concentrated culture supernatant as starting material. The exact functions and the ecological roles of the protease inhibitor produced by P. intermedia remain to be determined.
Bacteroides↗
Hydrolytic enzymes and lectin-binding activity of black-pigmented anaerobic rods.
Recent taxonomic studies on black-pigmented anaerobic rods, a group of bacteria found on mucosal surfaces of humans and animals, led to the subdivision of existing species and to the creation of new species. The aim of this study was to characterize all 11 currently recognized species of black-pigmented bacteria (55 strains) for their ability to hydrolyse a variety of natural and synthetic substrates and for their lectin reactivity. Although most of the strains demonstrated some activity against proteinaceous substrates, Porphyromonas gingivalis was the only species able to hydrolyse type I collagen. Most strains possessed glycylprolyl protease activity, elastase-like activity and phospholipase C activity, whereas trypsin-like activity was restricted to P. gingivalis, Porphyromonas salivosa and Bacteroides macacae. beta-Lactamase activity was demonstrated in five strains belonging to the saccharolytic group. The lectin reactivity of the bacteria was determined by a dot-blot procedure using horseradish-peroxidase-conjugated lectins. Three lectins, LOTUS A, RCA-I and ConA, failed to react with any of the bacteria tested. WGA reacted strongly with the cell surface of human biotypes of asaccharolytic black-pigmented bacteria (P. gingivalis, Porphyromonas asaccharolytica and Porphyromonas endodontalis) and Prevotella intermedia. The animal biotype strains of P. gingivalis showed a higher affinity for SBA and PNA than for WGA.
Amino Acid Sequence↗
Effect of proteolytic enzymes on the lysis and growth of oral bacteria.
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 proteinase 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, Prevotella intermedia, Capnocytophaga ochracea, Fusobacterium nucleatum, Prevotella loescheii, Treponema denticola and Actinobacillus actinomycetemcomitans. The disc-plate procedure indicated that the growth of Actinomyces spp., E. saburreum, C. ochracea, P. intermedia, P. loescheii, Porphyromonas gingivalis and T. denticola 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.
Actinomyces↗
Occurrence and identity of proteolytic bacteria in adult periodontitis.
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Adult↗
Demonstration of human immunoglobulin G Fc-binding activity in oral bacteria.
Nonimmune binding of immunoglobulins via the Fc fragment may reduce opsonization and phagocytosis of bacteria and is thus considered a virulence factor. The aim of this study was to investigate a wide range of oral bacterial strains for the presence of human immunoglobulin G (IgG) Fc-binding activity. A total of 132 strains representing 40 different gram-positive and gram negative bacterial species were tested for IgG Fc-binding activity by using a fast and simple dot blot procedure with horseradish peroxidase-conjugated Fc fragments from human IgG. Neither the human nor animal biotype of Porphyromonas gingivalis possessed IgG Fc-binding activity. The strongest positive reaction of gram-negative species with the IgG Fc fragments were obtained with strains of Prevotella intermedia and Fusobacterium nucleatum. Among the gram-positive bacteria tested, Peptostreptococcus micros, Lactobacillus spp., and several species of streptococci possessed IgG Fc-binding activity. In the present investigation, the ability of several oral bacterial species to bind IgG Fc fragments was demonstrated. This factor represents a potential virulence determinant as it may help pathogenic oral bacteria escape host defense mechanisms.
Binding Sites, Antibody↗
Cytotoxic effect of peptidoglycan from Treponema denticola.
Peptidoglycan isolated from pathogenic bacteria has been previously found to exhibit various biological activities. The aim of this investigation was to evaluate the toxicity of Treponema denticola peptidoglycan towards epithelial cells. The cytotoxicity of a lipopolysaccharide-like material was also determined. Epithelial cells were incubated with the test substances and cell viability was assayed by measuring lactate dehydrogenase activity and the conversion of tetrazolium salt into blue formazan. Morphological changes in the epithelial cells were monitored by scanning and transmission electron microscopy. While lipopolysaccharide-like material exerted negligible toxic effects on the epithelial cells, peptidoglycan was highly toxic. This cytotoxicity was both time- and concentration-dependent and was higher in the presence of serum. The epithelial cells appeared to be unable to recover following a short period of incubation with peptidoglycan. The study demonstrated that peptidoglycan from T. denticola is a potent toxic factor for epithelial cells. This represents a potential new virulence factor for this suspected periodontopathogen.
Animals↗
Characterization of the wheat germ agglutinin-binding property of Treponema denticola.
Lectins were used to characterize glycoconjugates on the cell surface of Treponema denticola, a suspected periodontopathogen. Bacteria were first screened by light microscopy using fluorescein isothiocyanate-coupled lectins. Wheat germ agglutinin (WGA) showed a high reactivity to T. denticola. While the WGA-binding activity was accentuated following heating or detergent treatments of bacterial cells, the reaction was inhibited by incorporation of competing carbohydrates. Scanning and transmission electron microscope studies were conducted in order to characterize the distribution of the WGA-binding sites on the cell surface of T. denticola. Data from these studies confirmed that heat treatment increases the percentage of labeled profiles and suggest that the WGA-binding sites are concentrated on specific regions on the spirochete surface. Initial biochemical analysis indicated that the high reactivity to WGA resides in a peptidoglycan fraction.
Bacterial Adhesion↗
Evidence for the absence of hyaluronidase activity in Porphyromonas gingivalis.
The aim of the present study was to evaluate the ability of Porphyromonas gingivalis to degrade hyaluronic acid. No hyaluronidase activity was detected using a turbidimetric method, whereas a standard plate assay showed a positive reaction for P. gingivalis. We postulated that the high proteolytic activity of P. gingivalis may account for this observation. A modified plate assay was designed to avoid false-positive reactions caused by proteolytic bacteria. The new assay, based on the formation of a water-insoluble salt between hyaluronic acid and the polyanion cetylpyridinium chloride, indicated that P. gingivalis does not have hyaluronidase activity. By this modified plate method, it was found that among 24 different oral bacterial species tested, Propionibacterium acnes and Prevotella oris were the only species that possess hyaluronidase activity.
Bacteriological Techniques↗
Reduction of proteolytic degradation by chlorhexidine.
The present investigation was designed to evaluate the effect of the antimicrobial agent chlorhexidine on proteolytic degradation. Chlorhexidine, at a final concentration of 0.01%, significantly (p = 0.05) affected the degradation of the general chromogenic substrate azocoll by both commercial proteases and cell-bound bacterial proteases. Reduction of proteolytic degradation was observed following pre-treatment of either azocoll or bacterial cells with chlorhexidine. Chlorhexidine was also found to prevent degradation of type I collagen by Porphyromonas gingivalis cells. The mechanism of inhibition of proteolytic degradation appears to be related to an electrostatic chlorhexidine-protein interaction.
Azo Compounds↗