Predominant bacteria recovered from a periodontitis site in a hamster model raised by silk-ligature with Porphyromonas gingivalis infection

We isolated oral bacteria that coexisted with Porphyromonas gingivalis in a hamster periodontitis model. As predominant bacteria in the periodontitis site, Collinsella-reltaed strains, Eubacterium-reltaed strains, Streptococcus suis-related strains, and Veillonella parvula-reltaed strains were detected. In addition, Actinomyces, Bacteroides, and P. gingivalis were also isolated predominantly. The results suggest that the bacterial composition of the periodontitis site in hamsters is complex, as in human periodontitis.     

Expression of the short fimbriae of Porphyromonas gingivalis is regulated in oral bacterial consortia

The Mfa1 protein of Porphyromonas gingivalis is the structural subunit of the short fimbriae and mediates coadhesion between P. gingivalis and Streptococcus gordonii. We utilized a promoter-lacZ reporter construct to examine the regulation of mfa1 expression in consortia with common oral plaque bacteria. Promoter activity of mfa1 was inhibited by S. gordonii, Streptococcus sanguinis and Streptococcus mitis. In contrast, Streptococcus mutans, Streptococcus cristatus, Actinomyces naeslundii, Actinobacillus actinomycetemcomitans and Fusobacterium nucleatum did not affect mfa1 expression. Expression of SspA/B, the streptococcal receptor for Mfa1, was not required for regulation of mfa1 promoter activity. Proteinaceous molecule(s) in oral streptococci may be responsible for regulation of Mfa1 expression. Porphyromonas gingivalis is capable of detecting heterologous organisms, and responds to selected organisms by specific gene regulation.     

Effect of saliva viscosity on the co-aggregation between oral streptococci and Actinomyces naeslundii

doi: 10.1111/j.1741‐2358.2011.00595.x Effect of saliva viscosity on the co‐aggregation between oral streptococci and Actinomyces naeslundii Background: The co‐aggregation of oral bacteria leads to their clearance from the oral cavity. Poor oral hygiene and high saliva viscosity are common amongst the elderly; thus, they frequently suffer from pneumonia caused by the aspiration of oral microorganisms. Objectives: To examine the direct effect of saliva viscosity on the co‐aggregation of oral streptococci with actinomyces. Materials and methods: Fifteen oral streptococcal and a single actinomyces strain were used. Co‐aggregation was assessed by a visual assay in phosphate buffer and a spectrophotometric assay in the same buffer containing 0–60% glycerol or whole saliva. Results: Nine oral streptococci co‐aggregated with Actinomyces naeslundii ATCC12104 in the visual assay and were subsequently used for the spectrophotometric analysis. All tested strains displayed a decrease in co‐aggregation with increasing amounts of glycerol in the buffer. The co‐aggregation of Streptococcus oralis with A. naeslundii recovered to baseline level following the removal of glycerol. The per cent co‐aggregation of S. oralis with A. naeslundii was significantly correlated with the viscosity in unstimulated and stimulated whole saliva samples (correlation coefficients: ?0.52 and ?0.48, respectively). Conclusion: This study suggests that saliva viscosity affects the co‐aggregation of oral streptococci with actinomyces and that bacterial co‐aggregation decreases with increasing saliva viscosity.     

Porphyromonas gingivalis-induced platelet aggregation in plasma depends on Hgp44 adhesin but not Rgp proteinase

Evidence from recent epidemiological studies suggests a link between periodontal infections and increased risk of atherosclerosis and related cardiovascular and cerebrovascular events in human subjects. One of the major pathogens of periodontitis, Porphyromonas gingivalis, has the ability to aggregate human platelets in platelet-rich plasma (PRP). Mechanism of P. gingivalis-induced platelet aggregation in PRP was investigated. Proteinase inhibitors toward Arg-gingipain (Rgp) and Lys-gingipain (Kgp) did not suppress P. gingivalis-induced platelet aggregation in PRP, whereas the Rgp inhibitor markedly inhibited P. gingivalis-induced platelet aggregation using washed platelets. Mutant analysis revealed that P. gingivalis-induced platelet aggregation in PRP depended on Rgp-, Kgp- and haemagglutinin A (HagA)-encoding genes that intragenically coded for adhesins such as Hgp44. Hgp44 adhesin on the bacterial cell surface, which was processed by Rgp and Kgp proteinases, was essential for P. gingivalis-induced platelet aggregation in PRP. P. gingivalis cell-reactive IgG in plasma, and FcgammaRIIa receptor and to a lesser extent GPIbalpha receptor on platelets were found to be a prerequisite for P. gingivalis-induced platelet aggregation in PRP. These results reveal a novel mechanism of platelet aggregation by P. gingivalis.     

Coaggregation of Porphyromonas gingivalis and Prevotella intermedia.

Porphyromonas gingivalis cells coaggregated with Prevotella intermedia cells. The coaggregation was inhibited with L-arginine, L-lysine, Nalpha-p-tosyl-L-lysine chloromethyl ketone, trypsin inhibitor, and leupeptin. Heat- and proteinase K-treated P. gingivalis cells showed no coaggregation with P. intermedia cells, whereas heat and proteinase K treatments of P. intermedia cells did not affect the coaggregation. The vesicles from P. gingivalis culture supernatant aggregated with P. intermedia cells, and this aggregation was also inhibited by addition of L-arginine or L-lysine and by heat treatment of the vesicles. The rgpA rgpB, rgpA kgp, rgpA rgpB kgp, and rgpA kgp hagA mutants of P. gingivalis did not coaggregate with P. intermedia. On the other hand, the fimA mutant lacking the FimA fimbriae showed coaggregation with P. intermedia as well as the wild type parent. These results strongly imply that a heat-labile and proteinous factor on the cell surface of P gingivalis, most likely the gingipain-adhesin complex, is involved in coaggregation of P. gingivalis and P. intermedia.     

Porphyromonas gingivalis prevalence related to other micro-organisms in adult refractory periodontitis.

Forty-six adult periodontal patients, selected on the basis of clinical examination, and 46 adult healthy subjects were examined. The subgingival plaque samples from one inflammatory and one non-inflammatory site of each periodontal patient were studied to determine Porphyromonas gingivalis prevalence related to other periodontal micro-organisms and to periodontal tissue destruction. The results showed Porphyromonas gingivalis as the main pathogenic micro-organism isolated in the inflammatory sites together with Bacteroides forsythus. Peptostreptococcus sp., Actinomyces sp. and Prevotella sp. were found as a normal oral flora in the healthy subjects. Fusobacterium nucleatum, Prevotella intermedia, Campylobacter rectus and Eikenella corrodens were detected both in inflammatory and in non-inflammatory sites of periodontal patients as well as in the healthy subjects.     

Adhesion of Actinomyces viscosus to Porphyromonas (Bacteroides) gingivalis-coated hexadecane droplets.

Interbacterial adhesion (coadhesion) is considered a major determinant of dental plaque ecology. In this report, we studied several aspects of the adhesion of Porphyromonas (Bacteroides) gingivalis to hexadecane in order to use the liquid hydrocarbon as a convenient substratum for coadhesion assays. Washed suspensions of hydrophobic P. gingivalis 2561 cells were vortexed with hexadecane to yield highly stable cell-coated droplets. Kinetics of coadhesion between Actinomyces viscosus cells and P. gingivalis-coated hexadecane droplets (PCHD) was subsequently studied. Aliquots of PCHD were added to A. viscosus suspensions, and the mixtures were gently rotated. Avid adhesion of A. viscosus cells to the immobilized P. gingivalis layer could be readily measured by the decrease in turbidity in the aqueous phase, following phase separation. Despite the ability of A. viscosus cells to adsorb to hexadecane following vigorous mixing, gentle mixing did not appreciably promote adhesion to bare hexadecane. Moreover, extensive microscopic examinations revealed that A. viscosus cells adhered exclusively to the bound P. gingivalis cells rather than to exposed areas of hexadecane. Coadhesion of A. viscosus to the PCHD appeared to follow first-order kinetics, attaining 80% levels within 30 min. Electron micrographs revealed A. viscosus cells adhering to the P. gingivalis cell layer adsorbed at the hexadecane-water interface. Interestingly, P. gingivalis cells did not appear to penetrate the hexadecane. A viscosus mutants lacking type 1 or type 2 fimbriae or both were still able to bind to the PCHD. No obvious correlation was observed between relative hydrophobicity of A. viscosus strains and their binding to PCHD. However, defatted bovine serum albumin, an inhibitor of hydrophobic interactions, was the most potent inhibitor among those tested. The data suggest that this approach provides a simple, quantitative technique for studying kinetics of bacterial coadhesion which is amenable to both light and electron microscopic observation.     

Red light kills bacteria via photodynamic action.

With the increase in the number of antibiotic resistant strains of microorganism, the search for alternative treatments of microbial infections becomes all the more important. We report a novel method for bacterial inactivation based on the optical excitation of the naturally occurring (endogenous) photosensitzing porphyrins by red light. In particular, the pathogenic Gram-positive porphyrin producing ATCC strains Propionibacterium acnes, Actinomyces odontolyticus and Porphyromonas gingivalis were investigated. Sensitive autofluorescence spectroscopy revealed that these bacteria naturally synthezise the fluorescent photosensitizer protoporphyrin IX. In addition, bacterial plaque samples of periodontitis patients were studied. Non-labeled fluorescent bacterial colonies were exposed to red light at 632.8 nm, 100 mW/cm2 light intensity and 360 J/cm2 energy density using a helium-neon laser. The survival rate after a single phototreatment with red light was found to be 0.58 +/- 0.09 in the case of Propionibacterium acnes, 0.30 +/- 0.04 in Actinomyces odontolyticus and 0.59 +/- 0.10 in Porphyrormonas gingivalis compared to non-exposed bacteria suspensions. No photoeffect was found for the bacterium Streptococcus mutans which exhibited no detectable porphyrin autofluorescence. Red-light exposed plaque samples of patients showed significant reduction of colony forming units by 50% as well as a pronounced photoeffect on the pigmented species Prevotella intermedia. Taken together, these results suggest the treatment with red light can be potentially employed as an therapeutic method to inactivate certain pathogenic strains of porphyrin producing bacteria without the use of external photosensitizers.     

Evidence that Porphyromonas (Bacteroides) gingivalis fimbriae function in adhesion to Actinomyces viscosus.

Porphyromonas (Bacteroides) gingivalis adheres to gram-positive bacteria, such as Actinomyces viscosus, when colonizing the tooth surface. However, little is known of the adhesins responsible for this interaction. A series of experiments were performed to determine whether P. gingivalis fimbriae function in its coadhesion with A. viscosus. Fimbriae typical of P. gingivalis were isolated from strain 2561 (ATCC 33277) by the method of Yoshimura et al. (F. Yoshimura, K. Takahashi, Y. Nodasaka, and T. Suzuki, J. Bacteriol. 160:949-957, 1984) in fractions enriched with a 40-kDa subunit, the fimbrillin monomer, P. gingivalis-A. viscosus coaggregation was inhibited by purified rabbit antifimbrial immunoglobulin G (IgG) at dilutions eightfold higher than those of preimmune IgG, providing indirect evidence implicating P. gingivalis fimbriae in coadhesion. Three types of direct binding assays further supported this observation. (i) Mixtures of isolated P. gingivalis fimbriae and A. viscosus WVU627 cells were incubated for 1 h, washed vigorously with phosphate-buffered saline (pH 7.2), and subjected to electrophoresis. Transblots onto nitrocellulose were probed with antifimbrial antiserum. Fimbrillin labeled positively on these blots. No reaction occurred with the control protein, porcine serum albumin, when blots were exposed to anti-porcine serum albumin, (ii) A. viscosus cells incubated with P. gingivalis fimbriae were agglutinated only after the addition of antifimbrial antibodies. (iii) Binding curves generated from an enzyme immunoassay demonstrated concentration-dependent binding of P. gingivalis fimbriae to A. viscosus cells. From these lines of evidence, P. gingivalis fimbriae appear to be capable of binding to A. viscosus and mediating the coadhesion of these species.     

Characterization of three bioenergetically active respiratory terminal oxidases in the cyanobacterium Synechocystis sp. strain PCC 6803

The adhesion properties of the recombinant fimbriae (r-fimbriae) recovered from a YH522 transformant of Porphyromonas gingivalis which harbors a chimeric plasmid, pYHF2, containing the fimA gene of strain 381 were compared with those of the endogenous fimA fimbriae of strain 33277. The adhesion level of the r-fimbriae to Actinomyces viscosus was clearly lower than that of the endogenous fimbriae. In addition, the r-fimbriae were shown to lack some minor components detectable in the endogenous fimbriae. The plasmid pYHF2 prepared from the YH522 transformant was then transformed into six different P. gingivalis strains and the resultant pYHF2-containing strains were examined for their fimbrial expression. In spite of the presence of a considerable diversity in the expression level of the r-fimbriae among these transformants, it was evident that the strains expressing higher levels of the r-fimbriae exhibited a greater decrease in adhesion activity to other bacteria and to oral epithelial cells, as well as in self-aggregation.     

不同浓度臭氧水对牙周致病菌的体外抑菌作用

摘要：目的 探讨不同浓度臭氧水对4种牙周致病菌的体外抑菌效果。方法 采用定量悬液法,分别用1.02 ppm、2.03 ppm和3.88 ppm三种浓度臭氧水,0 ppm无臭氧水溶液（阴性对照组）及3%双氧水（阳性对照组）,对常见的4种牙周致病菌牙龈卟啉单胞菌（Porphyromonas gingivalis）、具核梭杆菌（Fusobacterium nucleatum）、中间普氏菌（Prevotella intermedia）和黏性放线菌（Actinomyces viscosus）分别作用30 s、60 s后,观察不同作用时间下3种不同浓度臭氧水的抑菌效果。结果 对P. gingivalis、F. nucleatum、P. intermedia和A. viscosus作用30 s和60 s,与阴性对照组比较,1.02 ppm、2.03 ppm和3.88 ppm三种浓度臭氧水均具有明显的抑菌效果,3.88 ppm臭氧水与3%双氧水对4种细菌作用60 s的抑菌效果相同。 结论 实验所用的3种不同浓度臭氧水对牙周致病菌P. gingivalis、F. nucleatum、P. intermedia和A. viscosus均有抑菌作用,浓度越高,抑菌效果越好,臭氧水应用于牙龈牙周疾病临床防治有一定的价值和可行性。     

羧甲基壳聚糖对口腔重要厌氧菌的抑菌性能评价

目的 :评价羧甲基壳聚糖对口腔重要厌氧菌的抑菌性能。方法 :选择与口腔疾病密切相关的厌氧菌 11株 ,采用梯度稀释法测定羧甲基壳聚糖的最低抑菌浓度 (MIC)。结果 :羧甲基壳聚糖对牙龈卟啉菌、放线共生放线菌、中间普氏菌、牙龈嗜二氧化碳纤维菌、黄褐嗜二氧化碳纤维菌、产黑色素普氏菌、白色念珠菌、牙髓卟啉菌、小齿普氏菌、变形链球菌、远缘链球菌、粘性放线菌的 MIC分别为 2 0 ,10 ,5,80 ,2 0 ,>80 ,2 0 ,5,2 0 ,10 ,60 ,40 mg/ml。结论 :羧甲基壳聚糖对多数与口腔疾病密切相关的厌氧菌有一定抑制作用 ,而对产黑色素普氏菌的抑菌性不明显     

Roles of Arg- and Lys-gingipains in coaggregation of Porphyromonas gingivalis: identification of its responsible molecules in translation products of rgpA, kgp, and hagA genes

Arg- (Rgp) and Lys-gingipains (Kgp) are two individual cysteine proteinases produced by Porphyromonas gingivalis , an oral anaerobic bacterium, and are implicated as major virulence factors in a wide range of pathologies of adult periodontitis. Coaggregation of this bacterium with other oral bacteria is an initial and critical step in infectious processes, yet the factors and mechanisms responsible for this process remain elusive. Here we show that the initial translation products of the rgpA , kgp and hemagglutinin hagA genes are responsible for coaggregation of P. gingivalis and that the proteolytic activity of Rgp and Kgp is indispensable in this process. The rgpA rgpB kgp- and rgpA kgp hagA -deficient triple mutants exhibited no coaggregation activity with Actinomyces viscosus , whereas the kgp -null and rgpA rgpB -deficient double mutants significantly retained this activity. Consistently, the combined action of Rgp- and Kgp-specific inhibitors strongly inhibited the coaggregation activity of the bacterium, although single use of Rgp- or Kgp-specific inhibitor significantly retained this activity. We also demonstrate that the 47- and 43-kDa proteins produced from the translation products of the rgpA , kgp , and hagA genes by proteolytic activity of both Rgp and Kgp are responsible for the coaggregation of P. gingivalis.     

Influence of sample preparation technique on two-dimensional gel electrophoresis of proteins from Porphyromonas gingivalis

Sample preparation methods were compared for two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of cellular proteins from the proteolytic bacterium Porphyromonas gingivalis. Standard solubilization buffer yielded poorly resolved protein spots, but pre-treatment of cells with trichloroacetic acid or inclusion of the protease inhibitor TLCK during solubilization improved definition and separation. The latter approach allowed reliable detection of a 55 kDa immunodominant surface antigen by Western immunoblotting. Further improvements in resolution occurred when SDS was included in the sample preparation. Thus, controlling proteolysis and optimizing protein solubilization were essential for reproducible separations and maximal protein recovery during 2D-PAGE of P. gingivalis.     

Characterization of a bacteriocin produced by Prevotella nigrescens ATCC 25261

AIMS: To characterize the antimicrobial activity produced by Prevotella nigrescens ATCC 25261, and to evaluate its safety on cultured gingival fibroblasts. METHODS AND RESULTS: An antimicrobial activity was obtained from purifying the culture supernatant of Pr. nigrescens ATCC 25261. Purification of the active compound was achieved with ammonium sulphate precipitation followed by anion-exchange and gel filtration chromatography. As revealed by SDS-PAGE, the active fraction was relatively homogeneous, showing a protein with an approximate molecular weight of 41 kDa. The antimicrobial compound, named nigrescin, exhibited a bactericidal mode of action against Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis, and Actinomyces spp. Nigrescin was stable in a pH range between 6.5 and 9.5, at 100 degrees C for 10 min, and resistant to lyophilization. But its activity was lost after proteinase K treatment. Despite at very high concentrations beyond the minimum inhibitory concentration (MIC), nigrescin was not toxic to the gingival fibroblasts. CONCLUSION: Nigrescin is a novel bacteriocin produced by Pr. nigrescens ATCC 25261. It exhibits antimicrobial activity against species that are implicated in periodontal diseases. The absence of toxicity on the gingival fibroblasts suggests the possibility in using of nigrescin for an application in periodontal treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: Novel evidence on nigrescin would make Pr. nigrescens ATCC 25261 attractive in biotechnological applications as an antimicrobial agent in clinical dentistry.     

In vitro evaluation of the effect of nicotine, cotinine, and caffeine on oral microorganisms

The aim of this in vitro study was to evaluate the effects of nicotine, cotinine, and caffeine on the viability of some oral bacterial species. It also evaluated the ability of these bacteria to metabolize those substances. Single-species biofilms of Streptococcus gordonii, Porphyromonas gingivalis, or Fusobacterium nucleatum and dual-species biofilms of S. gordonii -- F. nucleatum and F. nucleatum -- P. gingivalis were grown on hydroxyapatite discs. Seven species were studied as planktonic cells, including Streptococcus oralis, Streptococcus mitis, Propionibacterium acnes, Actinomyces naeslundii, and the species mentioned above. The viability of planktonic cells and biofilms was analyzed by susceptibility tests and time-kill assays, respectively, against different concentrations of nicotine, cotinine, and caffeine. High-performance liquid chromatography was performed to quantify nicotine, cotinine, and caffeine concentrations in the culture media after the assays. Susceptibility tests and viability assays showed that nicotine, cotinine, and caffeine cannot reduce or stimulate bacterial growth. High-performance liquid chromatography results showed that nicotine, cotinine, and caffeine concentrations were not altered after bacteria exposure. These findings indicate that nicotine, cotinine, and caffeine, in the concentrations used, cannot affect significantly the growth of these oral bacterial strains. Moreover, these species do not seem to metabolize these substances.     

Aggregation of poliovirus and reovirus by dilution in water.

Poliovirus and reovirus were found to aggregate into clumps of up to several hundred particles when diluted 10-fold into distilled water from a stock preparation of minimal aggregation in 0.05 M phosphate buffer, pH 7.2, plus 22 to 30% sucrose. Reovirus was also found to aggregate when diluted into phosphate-buffered saline. The aggregation was concentration dependent and did not occur when either virus was diluted into water 100-fold or greater. The aggregation of poliovirus was reversible by further addition of saline and produced a dispersed preparation of virus. Reovirus aggregation was not reversible. Both viruses aggregated when diluted into buffers at pH 5 and 3, and poliovirus aggregated at pH 6, and this aggregation of both viruses was reversible when returned to pH 7. Aggregation did not occur at alkaline pH values. Aggregation at low pH could be caused aggregation of either virus at pH 7. Calcium ions, however, were found to aggregate both viruses at a concentration of 0.01 M.     

Aggregation of poliovirus and reovirus by dilution in water.

Poliovirus and reovirus were found to aggregate into clumps of up to several hundred particles when diluted 10-fold into distilled water from a stock preparation of minimal aggregation in 0.05 M phosphate buffer, pH 7.2, plus 22 to 30% sucrose. Reovirus was also found to aggregate when diluted into phosphate-buffered saline. The aggregation was concentration dependent and did not occur when either virus was diluted into water 100-fold or greater. The aggregation of poliovirus was reversible by further addition of saline and produced a dispersed preparation of virus. Reovirus aggregation was not reversible. Both viruses aggregated when diluted into buffers at pH 5 and 3, and poliovirus aggregated at pH 6, and this aggregation of both viruses was reversible when returned to pH 7. Aggregation did not occur at alkaline pH values. Aggregation at low pH could be caused aggregation of either virus at pH 7. Calcium ions, however, were found to aggregate both viruses at a concentration of 0.01 M.     

Transformation of Actinomyces spp. by a gram-negative broad-host-range plasmid.

The gram-negative broad-host-range vector pJRD215 was transferred by electroporation into strains of Actinomyces viscosus or Actinomyces naeslundii at efficiencies which ranged from 10(2) to 10(7) transformants per microgram of plasmid DNA. The Actinomyces transformants expressed pJRD215-encoded resistance to kanamycin and streptomycin. Moreover, the transforming plasmid DNA had not undergone any deletions or rearrangements, nor had it integrated into the genomes of these strains.     

Lactoferrampin: a novel antimicrobial peptide in the N1-domain of bovine lactoferrin

The antimicrobial activity of bovine lactoferrin is attributed to lactoferricin, situated in the N1-domain. Based on common features of antimicrobial peptides, a second putative antimicrobial domain was identified in the N1-domain of lactoferrin, designated lactoferrampin. This novel peptide exhibited candidacidal activity, which was substantially higher than the activity of lactoferrin. Furthermore, lactoferrampin was active against Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa, but not against the fermenting bacteria Actinomyces naeslundii, Porphyromonas gingivalis, Streptococcus mutans and Streptococcus sanguis. Notably, lactoferrampin is located in the N1-domain in close proximity to lactoferricin, which plays a crucial role in membrane-mediated activities of lactoferrin.