[THE CHARACTERISTICS OF MORPHOLOGY OF BIOFILM OF PERIODONTIUM UNDER INFLAMMATORY DISEASES OF GUMS (CHRONIC CATARRHAL GINGIVITIS, CHRONIC PERIODONTITIS, CANDIDA-ASSOCIATED PERIODONTITIS) ACCORDING RESULTS OF ELECTRONIC MICROSCOPY].

The study was carried out to analyze morphology of biofilm of periodontium and to develop electronic microscopic criteria of differentiated diagnostic of inflammatory diseases of gums. The scanning electronic microscopy was applied to analyze samples of bioflm of periodont from 70 patients. Including ten patients with every nosologic form of groups with chronic catarrhal periodontitis. of light, mean and severe degree, chronic catarrhal gingivitis, Candida-associated paroperiodontitis and 20 healthy persons with intact periodontium. The analysis was implemented using dual-beam scanning electronic microscope Quanta 200 3D (FEI company, USA) and walk-through electronic micJEM 100B (JEOL, Japan). To detect marker DNA of periodont pathogenic bacteria in analyzed samples the kit of reagentsfor polymerase chain reaction "MultiDent-5" ("GenLab", Russia). The scanning electronic microscopy in combination with transmission electronic microscopy and polymerase chain reaction permits analyzing structure, composition and degree of development of biofilm of periodontium and to apply differentiated diagnostic of different nosologic forms of inflammatory diseases of periodontium, including light form of chronic periodontitis and gingivitis. The electronic microscopical indications of diseases ofperiodontium of inflammatory character are established: catarrhal gingivitis, (coccal morphological alternate), chronic periodontitis (bacillary morphological alternate), Candida-associated periodontitis (Candida morphological alternate of biofilm ofperiodontium).

Phenotypic changes in nonfimbriated smooth strains of Aggregatibacter actinomycetemcomitans grown in low-humidity solid medium.

Aggregatibacter actinomycetemcomitans is the primary etiologic agent of localized aggressive periodontitis. In vitro, it can undergo fimbriated rough to nonfimbriated smooth phenotypic transition, accompanied by an increase in invasive ability and a decrease in adhesive ability. No opposite direction phenotypic transition was reported. To better understand its pathogenicity, the authors studied the morphological changes of nonfimbriated smooth strains induced by growth environmental humidity. Transmission electron microscopy was used to identify fimbriae expression change. It was found that the lower medium humidity, the more fimbriae reexpressed. In conclusion, the smooth strain of A. actinomycetemcomitans can reexpress the fimbriae in lower humidity environment.

Ultrastructural changes in bacterial membranes induced by nano-assemblies ß-cyclodextrin chlorhexidine: SEM, AFM, and TEM evaluation.

Chemical hosts bind their guests by the same physical mechanisms as biomolecules and often display similarly subtle structure activity relationships. The cyclodextrins have found increasing application as inert, nontoxic carriers of active compounds in drug formulations. The present study was conducted to prepare inclusion complexes of chlorhexidine:ß-cyclodextrin (Cx:ß-cd), and evaluate their interactions with bacterial membrane through: scanning electron microscopy (SEM) and transmission electron microscopy (TEM); and measuring morphology alterations, roughness values, and cell weights by atomic force microscopy (AFM). It was found that the antimicrobial activity was significantly enhanced by cyclodextrin encapsulation. SEM analysis images demonstrated recognizable cell membrane structural changes and ultrastructural membrane swelling. By TEM, cellular alterations such as vacuolization, cellular leakage, and membrane defects were observed; these effects were enhanced at 1:3 and 1:4 Cx:ß-cd. In addition, AFM analysis at these ratios showed substantially more membrane disruption and large aggregates mixing with microorganism remains. In conclusion, nanoaggregates formed by cyclodextrin inclusion compounds create cluster-like structures with the cell membrane, possibly due to a hydrogen rich bonding interaction system with increasing surface roughness and possibly increasing the electrostatic interaction between cationic chlorhexidine with the lipopolysaccharides of Gram negative bacteria.

Interleukin-1ß is internalised by viable Aggregatibacter actinomycetemcomitans biofilm and locates to the outer edges of nucleoids.

The opportunistic pathogen Aggregatibacter actinomycetemcomitans causes periodontitis, which is a biofilm infection that destroys tooth-supportive tissues. Interleukin (IL)-1ß, a central proinflammatory cytokine of periodontitis, is an essential first line cytokine for local inflammation that modulates the cell proliferation and anti-pathogen response of human gingival keratinocytes. Previously, we demonstrated that A. actinomycetemcomitans biofilms bind IL-1ß; however, whether this binding is an active process is not known. In this study, we showed for the first time with immuno-electron microscopy that viable bacterial biofilm cells internalised IL-1ß when co-cultured with an organotypic mucosa. Decreased biofilm viability hindered the ability of biofilm to sequester IL-1ß and caused IL-1ß leakage into the culture medium. In some A. actinomycetemcomitans cells, intracellular IL-1ß localized to the outer edges of the nucleoids. We identified the DNA-binding protein HU as an IL-1ß interacting protein with mass spectroscopy and showed the interaction of recombinant HU and IL-1ßin vitro using enzyme-linked immunosorbent assay (ELISA). Close contact with a viable A. actinomycetemcomitans biofilm decreased the proliferation and apoptosis of human gingival keratinocytes as demonstrated using Ki-67 and the terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) staining, respectively. Our results suggest that viable A. actinomycetemcomitans biofilms may disturb the critical first steps of local inflammation in periodontitis by binding and internalising IL-1ß. The interaction of IL-1ß with conserved HU provides a potential mechanism for shaping bacterial gene expression.

In vitro efficacy of diallyl sulfides against the periodontopathogen Aggregatibacter actinomycetemcomitans.

The in vitro antibacterial effects of diallyl sulfide (DAS) against the Gram-negative periodontopathogen Aggregatibacter actinomycetemcomitans, the key etiologic agent of the severe form of localized aggressive periodontitis and other nonoral infections, were studied. A. actinomycetemcomitans was treated with garlic extract, allicin, or DAS, and the anti-A. actinomycetemcomitans effects of the treatment were evaluated. Garlic extract, allicin, and DAS significantly inhibited the growth of A. actinomycetemcomitans (greater than 3 log; P < 0.01) compared to control cells. Heat inactivation of the garlic extracts significantly reduced the protein concentration; however, the antimicrobial effect was retained. Purified proteins from garlic extract did not exhibit antimicrobial activity. Allicin lost all its antimicrobial effect when it was subjected to heat treatment, whereas DAS demonstrated an antimicrobial effect similar to that of the garlic extract, suggesting that the antimicrobial activity of garlic extract is mainly due to DAS. An A. actinomycetemcomitans biofilm-killing assay performed with DAS showed a significant reduction in biofilm cell numbers, as evidenced by both confocal microscopy and culture. Scanning electron microscopy (SEM) analysis of DAS-treated A. actinomycetemcomitans biofilms showed alterations of colony architecture indicating severe stress. Flow cytometry analysis of OBA9 cells did not demonstrate apoptosis or cell cycle arrest at therapeutic concentrations of DAS (0.01 and 0.1 µg/ml). DAS-treated A. actinomycetemcomitans cells demonstrated complete inhibition of glutathione (GSH) S-transferase (GST) activity. However, OBA9 cells, when exposed to DAS at similar concentrations, showed no significant differences in GST activity, suggesting that DAS-induced GST inhibition might be involved in A. actinomycetemcomitans cell death. These findings demonstrate that DAS exhibits significant antibacterial activity against A. actinomycetemcomitans and that this property might be utilized for exploring its therapeutic potential in treatment of A. actinomycetemcomitans-associated oral and nonoral infections.

[Invasion of four common periodontal pathogens into vascular endothelial cells in vitro].

OBJECTIVE: To investigate the adhesive and invasive ability of four common periodontal pathogens, Pg33277, Pi25611, Aa29522 and Fn10953 in human umbilical vein endothelial cells (HUVEC). METHODS: The model of infection of HUVEC by periodontal pathogens was established in vitro. The invasive ability of four periodontal pathogens in HUVEC was tested by scanning electron microscope (SEM) and antibiotic protection assays-colony-forming units (CFU). RESULTS: All of the four periodontal pathogens were found to adhere to HUVEC by SEM and invaded HUVEC at invasion numbers of (0.8 +/- 0.1) x 10(8), (4.1 +/- 0.5) x 10(6), (1.6 +/- 0.3) x 10(6) and (5.0 +/- 0.4) x 10(6) CFU/L respectively by antibiotic protection assays-CFU. The invasion efficiencies were (0.400 +/- 0.050)%, (0.021 +/- 0.003)%, (0.008 +/- 0.002)% and (0.025 +/- 0.002)%, respectively. The invasive ability of Pg33277 was significantly greater than those of the other three periodontal pathogens (P < 0.001). There was no difference in invasive abilities among Pi25611, Aa29522 and Fn10953 (P > 0.05). CONCLUSIONS: All of the four common periodontal pathogens, Pg33277, Pi25611, Aa29522 and Fn10953 could adhere to and invaded HUVEC, with Pg33277 being the strongest.

Antibacterial activity of polysaccharide gel extract from fruit rinds of Durio zibethinus Murr. against oral pathogenic bacteria.

AIM: The polysaccharide gel (PG) extract from durian fruit rinds (Durio zibethinus Murr. "Monthong") is a pectic polysaccharide with antibacterial activity. This study aimed to investigate the in vitro antibacterial activity of PG against oral pathogens, Streptococcus mutans (S.mutans) and Aggregatibacter actinomycetemcomitans (A.actinomycetemcomitans). METHODS: The inhibitory activity of PG at 50, 100, and 150 mg/mL against S.mutans (American Tissue Culture Collection 25175) and A. actinomycetemcomitans (American Tissue Culture Collection 43718) was determined after 1- and 5-min exposure by broth macrodilution susceptibility test and scanning electron microscopy. Normal saline or culture broth medium and 0.1% chlorhexidine were used as negative and positive controls, respectively. RESULTS: For 1-min exposure, 150 mg/mL PG or 0.1% chlorhexidine significantly possessed bactericidal activity against both tested bacteria (P=0.037), while PG at 100 mg/mL possessed significant bactericidal activity against S.mutans (P=0.037) and inhibitory activity against A.actinomycetemcomitans (P=0.05). Blebs, irregular-shaped cells, and disrupted cells were found in bacteria treated with either 0.1% chlorhexidine or 50-150mg/mL PG under scanning electron microscopy. CONCLUSION: The bactericidal activity of PG at 150 mg/mL against oral bacteria at 1-min exposure suggests its possibility to be used as a natural antibacterial ingredient in oral hygiene products.

Membrane morphology and leukotoxin secretion are associated with a novel membrane protein of Aggregatibacter actinomycetemcomitans.

Gram-negative bacteria display either a flat or an irregular outer membrane. The periodontal pathogen Aggregatibacter (Actinobacillus) actinomycetemcomitans has an irregular outer membrane. We have identified a gene that is associated with the biogenesis of this morphology. The gene is part of a three-gene operon and codes for a 141-kDa protein designated morphogenesis protein C (MorC), which is conserved in several gram-negative bacteria including Haemophilus influenzae and Pasteurella multocida. Insertional inactivation of this gene resulted in the conversion of an irregularly shaped membrane to a flat membrane. Associated with this morphological change were the autoaggregation of the bacteria during planktonic growth and a concomitant increase in the surface hydrophobicity of the bacterium. The absence of MorC also resulted in the loss of the secretion of leukotoxin but not the ltxA transcription. Our findings suggest that MorC is critical for membrane morphology and leukotoxin secretion in A. actinomycetemcomitans.

Vesicle-independent extracellular release of a proinflammatory outer membrane lipoprotein in free-soluble form.

BACKGROUND: Aggregatibacter actinomycetemcomitans is an oral bacterium associated with aggressively progressing periodontitis. Extracellular release of bacterial outer membrane proteins has been suggested to mainly occur via outer membrane vesicles. This study investigated the presence and conservation of peptidoglycan-associated lipoprotein (AaPAL) among A. actinomycetemcomitans strains, the immunostimulatory effect of AaPAL, and whether live cells release this structural outer membrane lipoprotein in free-soluble form independent of vesicles. RESULTS: The pal locus and its gene product were confirmed in clinical A. actinomycetemcomitans strains by PCR-restriction fragment length polymorphism and immunoblotting. Culturing under different growth conditions revealed no apparent requirement for the AaPAL expression. Inactivation of pal in a wild-type strain (D7S) and in its spontaneous laboratory variant (D7SS) resulted in pleiotropic cellular effects. In a cell culture insert model (filter pore size 0.02 mum), AaPAL was detected from filtrates when strains D7S and D7SS were incubated in serum or broth in the inserts. Electron microscopy showed that A. actinomycetemcomitans vesicles (0.05-0.2 mum) were larger than the filter pores and that there were no vesicles in the filtrates. The filtrates were immunoblot negative for a cytoplasmic marker, cyclic AMP (cAMP) receptor protein. An ex vivo model indicated cytokine production from human whole blood stimulated by AaPAL. CONCLUSION: Free-soluble AaPAL can be extracellularly released in a process independent of vesicles.

Actinobacillus actinomycetemcomitans adheres to human gingival fibroblasts and modifies cytoskeletal organization.

Adherence of Actinobacillus actinomycetemcomitans to human gingival fibroblast cells induces cytoskeletal reorganization. A. actinomycetemcomitans is considered a pathogenic bacteria involved in localized aggressive periodontitis. Studies with epithelial cells have shown an adherent capacity of bacteria that is increased under anaerobic conditions. For adherence to take place, there is a need for interaction between extracellular vesicles and bacterial fimbriae. However, molecular events associated with the adherence process are still unknown. The aim of this study was to investigate whether A. actinomycetemcomitans adherence to human gingival fibroblasts promotes cytoskeletal reorganization. Adherence was determined with light microscopy and scanning electron microscopy. For F-actin visualization, cells were treated with fluorescein-isothiocyanate-phalloidin and samples were examined with epifluorescence optics. Fluorescent was recorded on Kodak T-Max 400 film. We showed that A. actinomycetemcomitans adheres to human gingival fibroblast primary cultures, this property stimulating an increase in the intracellular calcium levels. In human gingival fibroblast primary cultures, we observed that maximal A. actinomycetemcomitans adherence took place 1.5h after culture infection occurred and remained for 6h. The adherence was associated with morphologic alterations and an increased in the intracellular calcium levels. These experiments suggest that A. actinomycetemcomitans adherence cause morphological alterations, induce actin stress fibers and recruitment of intracellular calcium levels.

Susceptibility of Actinobacillus actinomycetemcomitans to six antibiotics decreases as biofilm matures.

OBJECTIVES: Actinobacillus actinomycetemcomitans is a major causative agent of chronic and aggressive periodontitis. Freshly isolated strains of A. actinomycetemcomitans display rough-type colonies and initiate biofilm formation on glass surfaces. The purpose of this study was to determine the antibiotic susceptibility of A. actinomycetemcomitans biofilm during different phases of maturation. METHODS: Using 96-well microtitre plates, we determined the antibiotic susceptibility of rough-type strain 310a to concentrations from 0.1 to 10 mg/L each of erythromycin, ofloxacin, ampicillin, cefalexin, tetracycline and minocycline during biofilm formation. Antibiotics were added at the start of the culture (early phase) and after 24 h of cultivation (mature phase). RESULTS: Adding 10 mg/L of ampicillin, 10 mg/L of cefalexin, 0.1 or 1 mg/L of tetracycline, or 0.1 mg/L of minocycline significantly inhibited 310a biofilm formation in the early phase, but not in the mature phase. Although adding 10 mg/L of erythromycin, tetracycline or minocycline reduced biofilm development in the early phase, it enhanced 310a biofilm development in the mature phase. Ofloxacin exerted a strong inhibitory effect in both the early and mature phases of biofilm formation throughout all experiments. CONCLUSIONS: The present study demonstrated that the susceptibility of A. actinomycetemcomitans to many antibiotics decreased after biofilm maturation.

A simple viability-maintaining method produces homogenic cell suspensions of autoaggregating wild-type Actinobacillus actinomycetemcomitans.

Tenacious adherence and autoaggregation of wild-type Actinobacillus actinomycetemcomitans strains jeopardize reliability of determined cell concentrations, e.g. for studies on bacteria-host interactions. We first compared the efficacy of two methods, an indirect and a direct method, for homogenizing cell suspensions of a wild-type, autoaggregating (SA269) strain and of a non-autoaggregating laboratory variant (ATCC 43718) used as a reference. Since the direct method left visible clumps in SA269 suspension, only the indirect method was further tested. In serial dilutions of the homogenized cell suspensions of strains SA269 and ATCC 43718, the OD600 values (R2=0.99, R2=0.99, respectively) and protein concentrations (R2=0.93, R2=0.95, respectively) correlated significantly (all P<0.002) with the dilution factor. There were no differences (P>0.05) in the bacterial viable counts between the two strains or between suspending solutions, i.e., PBS and water, the cell concentrations demonstrating 1x10(9) cells/ml at OD600=1. Repeated microscopic cell counts did not differ (P>0.05) from each other. Large aggregates occurred as 1% of cell units counted. Dispersing bacterial mass indirectly to solution leads to homogeneous cell suspensions with repeatable cell concentrations. Viability of A. actinomycetemcomitans was also maintained when cells were suspended in water.

Novel surface structures are associated with the adhesion of Actinobacillus actinomycetemcomitans to collagen.

Actinobacillus actinomycetemcomitans is a gram-negative, facultative, anaerobic bacterium that colonizes the human oral cavity and the upper respiratory tract. This bacterium is strongly associated with localized aggressive periodontitis and adult periodontitis and is the causative agent for other serious systemic infections. Recently, we have identified a protein, EmaA (extracellular matrix protein adhesin A), that mediates the adhesion of A. actinomycetemcomitans to collagen. The conserved sequence and predicted secondary structure suggest that EmaA is an orthologue of the Yersinia enterocolitica adhesin YadA. Electron microscopy examinations of A. actinomycetemcomitans have identified antenna-like protrusions associated with the surface of the bacterium. These structures are absent on emaA mutant strains and can be restored by transformation of the mutant strain with emaA in trans. The loss of these structures is associated with a decrease in the binding of this bacterium to collagen. The antenna-like structures are composed of a long rod that terminates in an ellipsoidal head region. The analysis of these structures using image processing techniques has provided an initial estimate of the overall dimensions, which suggests that the appendages are oligomeric structures formed by either three or four subunits. Together, the data suggest that emaA is required for the expression of novel appendages on the surface of A. actinomycetemcomitans that mediate the adhesion of the bacterium to collagen.

Environmental influences on Actinobacillus actinomycetemcomitans biofilm formation.

Fresh clinical isolates of the periodontal pathogen Actinobacillus actinomycetemcomitans have an adherent, rough colony morphology that transforms into a minimally adherent, smooth colony phenotype during successive in vitro passage. The objectives of this study were: (1) to compare biofilm formation of the rough (RVs) and smooth variants (SVs) of several strains of A. actinomycetemcomitans grown under various environmental conditions and (2) to examine the dynamics of biofilm formation. A microtitre plate biofilm assay was used to evaluate biofilm formation of strains grown in broth with modified salt concentration and pH, and to evaluate the effect of pre-conditioning films. Scanning electron microscopy (SEM) was used to monitor microscopic changes in morphology. Dynamics of biofilm formation were measured in a flowcell monitored by confocal microscopy. The RVs generally produced greater biofilm than the SVs. However, medium-dependent differences in biofilm formation were evident for some rough/smooth pairs. The RVs were more tolerant to changes in salt and pH, and more resistant to chlorhexidine than the SVs. Horse serum virtually eliminated, and saliva significantly reduced, biofilm formation by the SVs in contrast to the RVs. SEM revealed no alteration in morphology with change of environment. In a flowcell, the RVs produced towers of microcolonies anchored by a small contact area, whereas the SVs produced an open architecture of reduced height. After 7 days in a flowcell, the rough to smooth phenotype transition could be demonstrated. In conclusion, strain, growth medium and conditioning film all affect biofilm formation. The RVs produce biofilms of unique architecture that may serve to protect the bacterium from environmental perturbations.

Genetic basis for conversion of rough-to-smooth colony morphology in Actinobacillus actinomycetemcomitans.

The basis of the rough-to-smooth conversion of Actinobacillus actinomycetemcomitans was examined. Smooth variants often contained mutations at the flp promoter region. Replacing the mutated flp promoter with the wild-type promoter restored the rough phenotype. The expression level of the flp promoter was approximately 100-fold lower in smooth than in rough strains. Mutations of the flp promoter are a cause of the rough-to-smooth conversion.

Mutation analysis of the flp operon in Actinobacillus actinomycetemcomitans.

Fresh clinical isolates of the periodontal pathogen Actinobacillus actinomycetemcomitans live as autoaggregates, in which cells are densely packed and embedded in an extracellular matrix composed of bundled fimbriae, exopolymers, and vesicles. The expression of fimbriae is known to be determined by the flp operon of 14 genes, flp-1-flp-2-tadV-rcpCAB-tadZABCDEFG. We generated mutations of each gene of this operon in A. actinomycetemcomitans strain D7S. All mutants expressed some changes in the production of extracellular matrix materials that include vesicles, exopolymers, and fimbriae. The expression of fimbriae required the function of flp-1, rcpA, rcpB, tadB, tadD, tadE, and tadF. Mutants of flp-2, tadZ, tadA, tadC, and tadG expressed reduced levels of fimbriae, or fimbriae that had different gross appearance. Importantly, the expression of the non-fimbrial matrix materials was affected by all mutations, suggesting that the flp operon was involved in production of these materials. The flp locus apparently plays a central role in autoaggregation of A. actinomycetemcomitans, which may be the primary survival strategy of this bacterium in vivo.

Apoptotic activity and sub-cellular localization of a T4SS-associated CagE-homologue in Actinobacillus actinomycetemcomitans.

A potent virulence factor, cagE homologue of Actinobacillus actinomycetemcomitans, was identified via an expression cloning strategy and periodontitis-associated CD4(+)T-cells of a humanized mouse model. Through the immuno-gold labeling with transmission electron microscopy, immunofluorescent staining, in vitro co-cultures and Western blot studies, the resulting data clearly demonstrate that: (i) in CagE-homologue treated human epithelia in vitro, there are ultra-structural features of plasma membrane blebbing, sub-cellular disorganization with condensed and marginalized chromatins along the nuclear membrane, consistent with the pro-apoptotic characteristics, (ii) the disturbed membrane integrity detected above is associated with localization of the CagE proteins on target cell surface, and (iii) CagE-homologue is located in the cytoplasm of A. actinomycetemcomitans and associated with a bacterial type-IV secretion system (T4SS), suggesting that its translocation is required for secretion. Thus, CagE-homologue may be critically involved in A. actinomycetemcomitans-induced tissue destruction, inflammation and subsequent adverse immunity in periodontal pathogenesis.

Detection of subgingival calculus and dentine caries by laser fluorescence.

OBJECTIVES: Detection of subgingival calculus and dentine caries in the bottom of deep periodontal pockets is often difficult without visual observation. We thus examined the possibility of its detection using autofluorescence induced by laser irritation. METHODS: Autofluorescence was measured at various excitation and emission wavelength settings in five specimens each of sound dentine and enamel, subgingival calculus, and root caries. Periodontopathic model teeth with bacterial cells and blood clots were also irritated by laser to obtain autofluorescent images. RESULTS: Subgingival calculus and dentine caries showed a characteristic 700 nm emission when excited at 635 nm or a 720 nm emission when excited at 655 nm; sound dentine or enamel, however, did not. The calculus differentiation power, however, was higher with excitation at 635 nm than at 655 nm. The autofluorescent images photographed at an excitation of 633 nm provided clear calculus identification in periodontopathic model teeth when a 700 nm band-pass filter or a 700 nm high-pass filter was used. However, fluorescence intensity was masked when the calculus surface was covered by bacterial cells or blood clots. For clinical use, it would be important to remove subgingival plaque and debris from root surfaces before attempting to detect subgingival calculus and root caries with this manner. CONCLUSION: The autofluorescence method employing excitation of 633-635 nm was found to be a powerful tool for detecting subgingival calculus and root caries.

Outer membrane protein 100, a versatile virulence factor of Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans (Aa) is one of the pathogenic bacteria involved in periodontal diseases. We have previously identified six major outer membrane proteins (Omps) of Aa Y4. Among them is an Omp with high molecular mass, designated Omp100, which has homology to a variety of virulence factors. Electron microscopic observation indicated that Omp100 is randomly localized on the cell surface of Aa. Aa Y4 has been shown to adhere and invade KB or normal human gingival keratinocytes. Anti-Omp100 antibody inhibited 50% of adhesion and 70% of invasion of Aa Y4 to KB cells. An Omp100 knock-out mutant had a decreased adhesion and invasion efficiency of 60%, compared with that of the wild type. Escherichia coli HB101 expressing Omp100 adhered twofold and invaded 10-fold more than the wild-type E. coli HB101. HB101 expressing Omp100 showed resistance to serum by trapping factor H, an inhibitor for C3b, with Omp100. Omp100 induced inflammatory cytokine responses of interleukin (IL)-8, IL-6 and tumour necrosis factor (TNF)alpha in epithelial cells, and induced IL-1beta and TNFalpha production in mouse macrophages. These results indicate that Omp100 is a versatile virulence factor that may demonstrate potential significance in the onset of periodontal diseases related to Aa.

Biofilm growth and detachment of Actinobacillus actinomycetemcomitans.

The gram-negative, oral bacterium Actinobacillus actinomycetemcomitans has been implicated as the causative agent of several forms of periodontal disease in humans. When cultured in broth, fresh clinical isolates of A. actinomycetemcomitans form tenacious biofilms on surfaces such as glass, plastic, and saliva-coated hydroxyapatite, a property that probably plays an important role in the ability of this bacterium to colonize the oral cavity and cause disease. We examined the morphology of A. actinomycetemcomitans biofilm colonies grown on glass slides and in polystyrene petri dishes by using light microscopy and scanning and transmission electron microscopy. We found that A. actinomycetemcomitans developed asymmetric, lobed biofilm colonies that displayed complex architectural features, including a layer of densely packed cells on the outside of the colony and nonaggregated cells and large, transparent cavities on the inside of the colony. Mature biofilm colonies released single cells or small clusters of cells into the medium. These released cells adhered to the surface of the culture vessel and formed new colonies, enabling the biofilm to spread. We isolated three transposon insertion mutants which produced biofilm colonies that lacked internal, nonaggregated cells and were unable to release cells into the medium. All three transposon insertions mapped to genes required for the synthesis of the O polysaccharide (O-PS) component of lipopolysaccharide. Plasmids carrying the complementary wild-type genes restored the ability of mutant strains to synthesize O-PS and release cells into the medium. Our findings suggest that A. actinomycetemcomitans biofilm growth and detachment are discrete processes and that biofilm cell detachment evidently involves the formation of nonaggregated cells inside the biofilm colony that are destined for release from the colony.