Identification and localization of extraradicular biofilm-forming bacteria associated with refractory endodontic pathogens

Bacterial biofilms have been found to develop on root surfaces outside the apical foramen and be associated with refractory periapical periodontitis. However, it is unknown which bacterial species form extraradicular biofilms. The present study aimed to investigate the identity and localization of bacteria in human extraradicular biofilms. Twenty extraradicular biofilms, used to identify bacteria using a PCR-based 16S rRNA gene assay, and seven root-tips, used to observe immunohistochemical localization of three selected bacterial species, were taken from 27 patients with refractory periapical periodontitis. Bacterial DNA was detected from 14 of the 20 samples, and 113 bacterial species were isolated. Fusobacterium nucleatum (14 of 14), Porphyromonas gingivalis (12 of 14), and Tannellera forsythensis (8 of 14) were frequently detected. Unidentified and uncultured bacterial DNA was also detected in 11 of the 14 samples in which DNA was detected. In the biofilms, P. gingivalis was immunohistochemically detected in all parts of the extraradicular biofilms. Positive reactions to anti-F. nucleatum and anti-T. forsythensis sera were found at specific portions of the biofilm. These findings suggested that P. gingivalis, T. forsythensis, and F. nucleatum were associated with extraradicular biofilm formation and refractory periapical periodontitis.     

Localization of the Fusobacterium nucleatum T18 adhesin activity mediating coaggregation with Porphyromonas gingivalis T22.

Adherence of pathogenic bacteria is often an essential first step in the infectious process. The ability of bacteria to adhere to one another, or to coaggregate, may be an important factor in their ability to colonize and function as pathogens in the periodontal pocket. Previously, a strong and specific coaggregation was demonstrated between two putative periodontal pathogens, Fusobacterium nucleatum and Porphyromonas gingivalis. The interaction appeared to be mediated by a protein adhesin on the F. nucleatum cells and a carbohydrate receptor on the P. gingivalis cells. In this investigation, we have localized the adhesin activity of F. nucleatum T18 to the outer membrane on the basis of the ability of F. nucleatum T18 vesicles to coaggregate with whole cells of P. gingivalis T22 and the ability of the outer membrane fraction of F. nucleatum T18 to inhibit coaggregation between whole cells of F. nucleatum T18 and P. gingivalis T22. Proteolytic pretreatment of the F. nucleatum T18 outer membrane fraction resulted in a loss of coaggregation inhibition, confirming the proteinaceous nature of the adhesin. The F. nucleatum T18 outer membrane fraction was found to be enriched for several proteins, including a 42-kDa major outer membrane protein which appeared to be exposed on the bacterial cell surface. Fab fragments prepared from antiserum raised to the 42-kDa outer membrane protein were found to partially but specifically block coaggregation. These data support the conclusion that the 42-kDa major outer membrane protein of F. nucleatum T18 plays a role in mediating coaggregation with P. gingivalis T22.     

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.     

Fluorescence based measurements of Fusobacterium nucleatum coaggregation and of fusobacterial attachment to mammalian cells

Fusobacterium nucleatum is a common oral anaerobe associated with gingivitis, periodontal disease and preterm deliveries. Coaggregation among oral bacteria is considered to be a significant factor in dental plaque development. Adhesion to host cells was suggested to be important for the F. nucleatum virulence associated with oral inflammation and with preterm births. An uncharacterized fusobacterial galactose inhibitible adhesin mediates coaggregation of F. nucleatum 12230 and F. nucleatum PK1594 with the periodontal pathogen Porphyromonas gingivalis. This adhesin is also involved with the attachment of both fusobacterial strains to host cells. However, it has been suggested that additional unidentified fusobacterial adhesins are involved in F. nucleatum virulence associated with preterm births. In this study, a fluorescence-based high throughput sensitive and reproducible method was developed for measuring bacterial coaggregation and bacterial attachment to mammalian cells. Using this method we found that coaggregation of F. nucleatum 4H with P. gingivalis and its attachment to murine macrophages is less inhibitible by galactose than that of F. nucleatum PK1594. These findings suggest that F. nucleatum 4H can serve as a model organism for identifying nongalactose inhibitible F. nucleatum adhesins considered to be involved in fusobacterial attachment to mammalian cells.     

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。     

Coaggregation of Porphyromonas gingivalis and Fusobacterium nucleatum PK 1594 is mediated by capsular polysaccharide and lipopolysaccharide

Previous reports have shown that coaggregation between Porphyromonas gingivalis and Fusobacterium nucleatum, two important periodontopathogens, is mediated by a galactoside on the surface of P. gingivalis and a lectin on F. nucleatum. In the present study, purified capsular polysaccharide (CPS) and lipopolysaccharide (LPS) of P. gingivalis PK 1924 (serotype K5) were found to be able to bind to F. nucleatum cells and to inhibit binding of F. nucleatum to P. gingivalis serotype K5. Sugar binding studies showed that the requirements for binding of P. gingivalis serotype K5 CPS and LPS to the F. nucleatum lectin are: the presence of a metal divalent ion, an axial free hydroxyl group at position 4 and free equatorial hydroxyl groups at position 3 and 6 of d-galactose. These data suggest that P. gingivalis serotype K5- CPS and LPS act as receptors mediating coaggregation between P. gingivalis and fusobacteria.     

甘草提取物对四种牙周常见致病菌的抑菌作用

目的体外评价甘草提取物对牙龈卟啉单胞菌、中间普氏菌、具核梭杆菌和伴放线放线杆菌四种牙周常见致病菌的抑制效果。方法以牙龈卟啉单胞菌、中间普氏菌、具核梭杆菌和伴放线放线杆菌四种牙周常见致病菌作为供试菌,采用液体稀释法,考察甘草提取物对这四种细菌的最小抑菌浓度(MIC)和最小杀菌浓度(MBC);并采用不同浓度的甘草提取物溶液,绘制甘草提取物对四种牙周致病菌的时间-杀菌曲线。结果甘草提取物对牙龈卟啉单胞菌、中间普氏菌、具核梭杆菌和伴放线放线杆菌的MIC值分别为1.50、1.50、0.75和1.50mg/mL,MBC值分别为6、3、3和3mg/mL。当甘草提取物达到对四种细菌的MBC值时,对于牙龈卟啉单胞菌、中间普氏菌、伴放线放线杆菌可在2h后可达到杀菌效果,对于具核梭杆菌可在4h后达到杀菌效果。结论甘草提取物对以上四种牙周常见致病菌具有良好的抑菌及杀菌作用。     

Physiological alterations of a Fusobacterium nucleatum strain exposed to oxidative stress

AIM: The purpose of this study was to investigate the effect of oxidative stress on physiological and genetic characteristics of Fusobacterium nucleatum and its interference on this microbial identification methods. METHODS AND RESULTS: Fus. nucleatum ssp. nucleatum ATCC 25586 (wt-strain) and an oxidative-stress-adapted strain derived from the wt-strain (aero-strain) were employed in the study. Cell-free crude protein extracts were obtained from both strains and differentially expressed proteins were identified by two-dimensional electrophoresis. Bacterium identification was performed by conventional biochemical tests, automated Rapid ID 32A system and specific PCR analysis. Genetic diversity between wt- and aero-strain was assessed by arbitrarily-primed (AP)-PCR. There were significant changes in the protein profile of aero-strain. The identification of the wt-strain was confirmed by all methods employed. Similar results were obtained for aero-strain when conventional biochemical tests and PCR were used. However, aero-strain was identified as Fusobacterium varium when submitted to Rapid ID 32A system. According to AP-PCR analysis, no significant genetic alteration was detected in aero-strain. CONCLUSIONS: The adaptive response of Fus. nucleatum to oxidative stress is associated with changes on its biology, which may lead to misidentification of the organism, according to the conventional identification methods. SIGNIFICANCE AND IMPACT OF THE STUDY: Oxidative stress may act as a cause of adaptive response in Fus. nucleatum with consequences to its biology, such as alterations on biochemical and physiological profile.     

Conversion of DL-threonine, D-threonine and 2-oxobutyrate into propionate and 2-hydroxybutyrate by Fusobacterium species

The present investigation examined DL -threonine, D- threonine and 2-oxobutyrate conversion into propionate and 2-hydroxybutyrate by various type strains and clinical isolates of Fusobacterium. Except for Fus. naviforme , the type strains were able to produce varying degrees of propionate and/or 2-hydroxybutyrate from DL -threonine. Additionally, D -threonine was converted into an equimolar amount of propionate by Fus. necrophorum subsp. necrophorum , Fus. nucleatum subsp. nucleatum and Fus. varium , and to a lower but significant amount by Fus. mortiferum and Fus. perfoetens. However, the level of propionate remained unchanged for Fus. nucleatum subsp. fusiforme , Fus. nucleatum subsp. vincentii , Fus. naviforme , Fus. necrophorum subsp. funduliforme , Fus. gonidiaforme and Fus. russii. 2-Oxobutyrate was fermented to propionate by all type strains, although Fus. russii reduced it mainly to 2-hydroxybutyrate. Thus, an attempt was made to make use of these features in order to identify clinical isolates.     

The Fusobacterium nucleatum outer membrane protein RadD is an arginine-inhibitable adhesin required for inter-species adherence and the structured architecture of multispecies biofilm

A defining characteristic of the suspected periodontal pathogen Fusobacterium nucleatum is its ability to adhere to a plethora of oral bacteria. This distinguishing feature is suggested to play an important role in oral biofilm formation and pathogenesis, with fusobacteria proposed to serve as central 'bridging organisms' in the architecture of the oral biofilm bringing together species which would not interact otherwise. Previous studies indicate that these bacterial interactions are mediated by galactose- or arginine-inhibitable adhesins although genetic evidence for the role and nature of these proposed adhesins remains elusive. To characterize these adhesins at the molecular level, the genetically transformable F. nucleatum strain ATCC 23726 was screened for adherence properties, and arginine-inhibitable adhesion was evident, while galactose-inhibitable adhesion was not detected. Six potential arginine-binding proteins were isolated from the membrane fraction of F. nucleatum ATCC 23726 and identified via mass spectroscopy as members of the outer membrane family of proteins in F. nucleatum . Inactivation of the genes encoding these six candidates for arginine-inhibitable adhesion and two additional homologues revealed that only a mutant derivative carrying an insertion in Fn1526 (now designated as radD ) demonstrated significantly decreased co-aggregation with representatives of the Gram-positive 'early oral colonizers'. Lack of the 350 kDa outer membrane protein encoded by radD resulted in the failure to form the extensive structured biofilm observed with the parent strain when grown in the presence of Streptococcus sanguinis ATCC 10556. These findings indicate that radD is responsible for arginine-inhibitable adherence of F. nucleatum and provides definitive molecular evidence that F. nucleatum adhesins play a vital role in inter-species adherence and multispecies biofilm formation.     

Interactions between salivary Bifidobacterium adolescentis and other oral bacteria: in vitro coaggregation and coadhesion assays

Coaggregation assays were performed to investigate interactions between oral Bifidobacterium adolescentis and other oral bacterial species. Bifidobacterium adolescentis OLB6410 isolated from the saliva of healthy humans did not coaggregate with Actinomyces naeslundii JCM8350, Streptococcus mitis OLS3293, Streptococcus sanguinis JCM5708, Veillonella parvula ATCC17745 or Porphyromonas gingivalis OB7124, but it did coaggregate with Fusobacterium nucleatum JCM8532. Subsequent examination of biofilm formation on saliva-coated hydroxyapatite discs using FISH revealed that B. adolescentis OLB6410 could not directly adhere to the coated discs. It did, however, adhere to biofilms of A. naeslundii, V. parvula, and F. nucleatum, although it did not coaggregate with A. naeslundii nor with V. parvula. These results suggest that the adhesion of B. adolescentis to tooth surfaces is mediated by other oral bacteria. Heat- or proteinase K-treated F. nucleatum could not coaggregate with B. adolescentis. Similarly, the coaggregation and coadhesion of proteinase K-treated B. adolescentis were strongly inhibited. It is therefore probable that proteinaceous factors on the cellular surface of B. adolescentis and F. nucleatum are involved in their interaction. The data presented in this study add to our understanding of bifidobacterial colonization in the human oral cavity.     

Interactions of Anaerobic Bacteria with Dental Stem Cells: An In Vitro Study

Background

In patients with periodontitis, it is highly likely that local (progenitor) cells encounter pathogenic bacteria. The purpose of this in vitro study was to elucidate how human dental follicle stem cells (hDFSC) react towards a direct challenge with anaerobic periodontal pathogens under their natural oxygen-free atmosphere. HDFSC were compared to human bone marrow mesenchymal stem cells (hBMSC) and differentiated primary human gingival fibroblasts (hGiF), as well as permanent gingival carcinoma cells (Ca9-22).

Methodology/Principal Findings

The different cell types were investigated in a co-culture system with Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum). The viability of the cells and pathogens under anaerobic conditions, as well as interactions in terms of adherence and internalization, were examined. Additionally, the release of pro-inflammatory interleukin-8 (IL-8) and anti-inflammatory interleukin-10 (IL-10) was quantified via enzyme-linked immunosorbent assay. The bacteria adhered less efficiently to hDFSC compared to Ca9-22 (P. gingivalis: 0.18% adherence to hDFSC; 3.1% adherence to Ca9-22). Similar results were observed for host cell internalization (F. nucleatum: 0.002% internalization into hDFSC; 0.09% internalization into Ca9-22). Statistically significantly less IL-8 was secreted from hDFSC after stimulation with F. nucleatum and P. gingivalis in comparison with hGiF (F. nucleatum: 2080.0 pg/ml – hGiF; 19.7 pg/ml – hDFSC). The IL-10 response of the differentiated cells was found to be low in relation to their pro-inflammatory IL-8 response.

Conclusions/Significance

The results indicate that dental stem cells are less prone to interactions with pathogenic bacteria than differentiated cells in an anaerobic environment. Moreover, during bacterial challenge, the stem cell immune response seems to be more towards an anti-inflammatory reaction. For a potential future therapeutic use of hDFSC, these findings support the idea of a save application.     

Gingipains inactivate a cell surface ligand on Porphyromonas gingivalis that induces TLR2-and TLR4-independent signaling

Arginine-specific gingipain and lysine-specific gingipain are two major cysteine proteinases produced by Porphyromonas gingivalis. To clarify the role of gingipains in the interaction between P. gingivalis and the innate immune system, CHO reporter cells expressing TLR2 or TLR4 were stimulated with wildtype or gingipain-deficient P. gingivalis cells and activation of nuclear factor-kappaB in these cells was examined. While CHO/CD14 cells and 7.19 cells, an MD-2-defective mutant derived from CHO/CD14 cells, failed to respond to wild-type P. gingivalis, they responded to gingipain-deficient P. gingivalis. On the other hand, CHO/CD14/TLR2 cells responded to both wild-type and gingipain-deficient P. gingivalis. These results suggested that gingipains have no effects on TLR2-dependent signaling from P. gingivalis but have inhibitory effects on TLR2-and TLR4-independent signaling in CHO cells. Indeed, the activity of gingipain-deficient P. gingivalis to induce the activation of 7.19 cells was diminished after treatment of the bacterial cells with gingipains. We next partially purified bacterial cell components activating 7.19 cells from gingipain-deficient P. gingivalis. The activity of the partially purified components was diminished by treatment with heat or gingipains. It is also noteworthy that anti-CD14 mAb inhibited the activation of 7.19 cells induced by the partially purified components. These results indicated that the components of P. gingivalis that were able to induce TLR2-and TLR4-independent signaling were inactivated by gingipains before being recognized by CD14. The inactivation of the components would be helpful for P. gingivalis to escape from the innate immune system.     

抗菌肽对种植体周围炎主要致病菌生长抑制作用的研究

目的研究抗菌肽KSL及其衍生物KSL—W对种植体周围炎主要致病菌的体外抑菌效果。方法应用二倍稀释法检测KSL和KSL—W对血链球菌、具梭核杆菌和牙龈卟啉单胞菌的最小抑菌浓度（MIC）和最小杀菌浓度（MBC）;MTT法检测KSL和KSL—W对成骨样细胞MG-63的细胞毒性。结果KSL和KSL—W对具梭核杆菌的MIC和MBC分别为0．0156mg／mL和0．0313mg／mL,对牙龈卟啉单胞菌的MIC和MBC分别为0．125mg／mL和0．5mg／mL,在0．5mg／mL的浓度范围内对血链球菌没有抑制作用;KSL和KSL-W在0．5mg／mL的浓度范围内没有细胞毒性。结论KSL和KSL—W没有细胞毒性,对具梭核杆菌和牙龈卟啉单胞菌具有抑制作用。     

Further evidence for a possible role of trypsin-like activity in the adherence of Porphyromonas gingivalis.

The present study was undertaken to evaluate a possible correlation between the level of trypsin-like activity and the adherence properties of Porphyromonas gingivalis. It was demonstrated that strains with high cell-associated trypsin-like activity attach in higher numbers to human epithelial cells than strains with low levels of trypsin-like activity. To a lesser extent, the same tendency was also noted for the agglutination of human erythrocytes. The ability of P. gingivalis to attach to erythrocytes and epithelial cells was found to be affected by the presence of arginine and thiol protease inhibitors (leupeptin, p-chloromercuriphenylsulfonic acid). The inhibition profile was partially dependent on the age of the bacterial cells used in the adherence assay. It is suggested that adherence of mid-log P. gingivalis cells involves primarily trypsin-like proteases, whereas 2-day-old cells possess additional specific attachment mechanism(s).     

Serum antibodies of periodontitis patients compared to the lipopolysaccharides of Porphyromonas gingivalis and Fusobacterium nucleatum

Serum antibody titers against the lipopolysaccharides (LPSs) of Porphyromonas gingivalis and Fusobacterium nucleatum were compared between 9 periodontitis patients and 24 healthy persons. The IgG titers against the LPSs of P. gingivalis ATCC 33277(T) and W50 were clearly higher in the patients than in the healthy persons. However, IgM titers against the LPSs of P. gingivalis strains were relatively low, and no significant difference was observed between the patients and healthy persons. On the other hand, IgG and IgM titers against the LPS of Fusobacterium nucleatum JCM 8532(T) in some patients were significantly higher than those in the healthy persons, although the difference in IgG titers was not large compared to that of the LPS of P. gingivalis. These results suggest that the antibody measurement of patients' sera against the LPS of periodontal bacteria can be applied for the diagnosis of periodontitis.     

Comparison of culture methods and multiplex PCR for the detection of periodontopathogenic bacteria in biofilm associated with severe forms of periodontitis

Conventional culture methods and Multiplex PCR, both of which we have been used for a long time in our clinical microbiology laboratory, were compared for their ability to detect a selected panel of periodontopathic bacteria: Aggregatibacter actinomycetemcomitans, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia. Tests were performed in a single subgingival sample taken from a periodontal diseased site with a probing depth equal to or greater than 6mm. The results were compared site-by-site, taking into account the quality and the presence or absence of pathogens. 529 samples of subgingival plaque were analysed and the prevalence of the six species monitored varied in relation to the species itself and the method of detection. The most represented species is F. nucleatum, with a percentage of positive variability between 44.9% PCR and 46.5% culture test. Generally, the lowest prevalence was determined by culture test, with the exception of E. corrodens and F. nucleatum, which, unlike other bacteria, have been seen in higher percentages in culture with respect to PCR. For both methods, there was a good degree of accuracy in the determination of A. actinomycetemcomitans, C. rectus, E. corrodens, and P. gingivalis. It becomes weak for F. nucleatum and P. intermedia. Both culture and PCR techniques introduced many methodological problems when applied in oral microbiology, but the ideal technique for accurate detection of pathogens in subgingival plaque samples has yet to be developed.     

Expression of saliva-binding epitopes of the Porphyromonas gingivalis FimA protein on the surface of Streptococcus gordonii

Porphyromonas gingivalis, a gram-negative oral anaerobic bacterium, has been implicated in the onset and development of periodontitis. The P. gingivalis fimbriae which mediate bacterial adherence to host oral sites and induce host inflammatory responses have been suggested as a potential antigen candidate. for vaccine development. This study was undertaken to generate Streptococcus gordonii vectors expressing the major subunit protein (FimA) of P. gingivalis fimbriae for testing as a potential live vaccine against periodontitis. We report here the expression of the C-terminal saliva-binding epitopes of P. gingivalis FimA on the surface of S. gordonii and demonstrate that domains containing free cysteine residues are poorly expressed on the surface of S. gordonii.     

Effects of Porphyromonas gingivalis culture products on human polymorphonuclear leukocyte function

Abstract Porphyromonas gingivalis culture supernate was found to induce hemotypic agglutination of human polymorphonuclear leukocytes (PMN). Pretreatment of PMN with P. gingivalis supernate inhibited both the rate and the degree of aglutination induced by the secretagogues PMA and FMLP. Lipopolysaccaharide from P. gingivalis upregulated the CR3 (Mac-1, CD11b) receptors of PMN. Treatment of glass-adherent PMN with P. gingivalis supernate did not alter their phagocytic capacity fot P. gingivalis cells but when PMN were treated in suspension the cells adhered less well to glass and phagocytosis of those PMN that did adhere was reduced. P. gingivalis supernate treatment of PMN induced lysozyme release but the amount released during phagocytosis when supernate was present did not change. Neither P. gingivalis supernate nor LPS were cytotoxic for PMN. The data suggest that P. gingivalis factors could interfere with PMN elimination of this organism at the site of infection by inappropriately stimulating PMN, depressing phagocytosis and causing enhanced CR3 expression. The consequent agglutinatin or enhanced adherence could also lead to decreased phagocytic capacity of the adherant or agglutinated cells.     

Effects of Porphyromonas gingivalis culture products on human polymorphonuclear leukocyte function

Porphyromonas gingivalis culture supernate was found to induce homotypic agglutination of human polymorphonuclear leukocytes (PMN). Pretreatment of PMN with P. gingivalis supernate inhibited both the rate and the degree of agglutination induced by the secretagogues PMA and FMLP. Lipopolysaccharide from P. gingivalis upregulated the CR3 (Mac-1, CD11b) receptors on PMN. Treatment of glass-adherent PMN with P. gingivalis supernate did not alter their phagocytic capacity for P. gingivalis cells but when PMN were pretreated in suspension the cells adhered less well to glass and phagocytosis of those PMN that did adhere was reduced. P. gingivalis supernate treatment of PMN induced lysozyme release but the amount released during phagocytosis when supernate was present did not change. Neither P. gingivalis supernate nor LPS were cytotoxic for PMN. The data suggest that P. gingivalis factors could interfere with PMN elimination of this organism at the site of infection by inappropriately stimulating PMN, depressing phagocytosis and causing enhanced CR3 expression. The consequent agglutination or enhanced adherence could also lead to decreased phagocytic capacity of the adherent or agglutinated cells.