Evidence for invasion of a human oral cell line by Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans, an oral bacterial species associated with periodontal disease, was found to invade human cell lines. Invasion was demonstrated by recovery of viable organisms from gentamicin-treated KB cell monolayers and by light and electron microscopy. Internalization occurred through a cytochalasin D-sensitive process. Invasion efficiencies of some A. actinomycetemcomitans strains were comparable to those of invasive members of the family Enterobacteriaceae. Differences in invasiveness were correlated with bacterial colonial morphology. Smooth variants invaded more proficiently than rough variants. A. actinomycetemcomitans can undergo a smooth-to-rough colonial morphology shift which results in the loss of invasiveness. Coordinated regulation of genes involved in the rough-to-smooth phenotypic transitions may play a role in the episodic nature of periodontal disease.

A model for demonstrating the adhesion of Actinobacillus seminis to epithelial cells.

The objective of this study was to demonstrate that a field isolate of Actinobacillus seminis (As8C) will adhere to epithelial cells and that this adhesion can be inhibited by pretreating the bacteria with mouse serum containing polyclonal antibodies (PoAbs) prepared against this isolate. An indirect fluorescent antibody test, transmission electron microscopy, and phase-contrast microscopy confirmed the adhesion of As8C to an established culture of bovine kidney epithelial cells (BKECs). In a bacterial adhesion assay, 40 As8C were estimated to adhere to each BKEC after 60 min. Using a bacterial inhibition assay, PoAbs diluted 10(-2) or 10(-3) inhibited the adhesion of As8C to BKECs by approximately 90%. Bacterial inhibition decreased to about 50% when the PoAbs were diluted to 10(-4). There was less than 10% inhibition of adhesion of As8C to BKECs when higher dilutions of PoAbs were used. The inhibition of As8C adhesion to BKECs was less than 20% following pretreatment of BKECs with 10(-2) to 10(-5) dilutions of PoAbs. Moreover, pretreatment of As8C with a 10(-2) dilution of PoAbs did not appear to adversely affect bacterial growth on agar. It is likely that the PoAbs interrupted the adhesion of As8C to BKECs by sterically interfering with a bacterial adhesin-epithelial cell receptor interaction.

Adherence of Actinobacillus pleuropneumoniae to porcine tracheal epithelial cells and frozen lung sections.

The ability of 23 different Actinobacillus pleuropneumoniae isolates to adhere in vitro to porcine tracheal epithelial cells and to porcine frozen lung sections was examined. It was found that A. pleuropneumoniae adhered poorly to isolated tracheal epithelial cells. On the other hand, A. pleuropneumoniae adhered to frozen lung sections and marked variations were observed between and within serotypes. Adherence to lung sections did not seem related to the hemagglutinating activity of the isolate. Two noncapsulated variants adhered to lung sections in greater numbers than their capsulated parent strains. Adherence to lung sections was not inhibited by the extracellular matrix components tested namely, laminin, fibronectin, and collagen, but was inhibited by homologous serotype-specific antiserum. The data indicated that the A. pleuropneumoniae isolates tested possess the ability to adhere to porcine lung tissue, a property which did not seem to be related to the serotype and did not seem to involve the capsular material or the hemagglutinins of the isolates.

Identification and localization of surface sialylated glycoconjugates in Actinobacillus pleuropneumoniae by direct enzyme-colloidal gold cytochemistry.

Electron microscopic examination of ultrathin sections of 5 strains of Actinobacillus pleuropneumoniae stained for polysaccharides with ruthenium red revealed considerable variability in the amounts of preserved capsular material among the 5 serotypes studied. The amount of capsule was inversely related to the extent of outer membrane-associated sialylated glycoconjugate as evidenced by the degree of binding by colloidal gold-labelled neuraminidase at the cell surface. Serotypes 1, 3, and 5 possessed a well-developed and continuous capsular layer. In serotypes 2 and 7, the capsule consisted of a broken patchy layer that left much of the underlying outer membrane exposed. Morphometric analyses of the mean frequencies of neuraminidase-conjugated gold particles over the perimeters of the A. pleuropneumoniae cells showed that the lowest mean frequencies were observed in serotypes 1, 3, and 5, whereas the second highest and highest mean frequencies were observed in serotypes 7 and 2, respectively. Evidence suggested a serotypic difference in the amount of capsule present and this correlated inversely with the number of sialylated glycoconjugates, which appear to be localized in the outer membranes of the A. pleuropneumoniae cells.

Identification of Actinobacillus actinomycetemcomitans by gold immunolabeling and scanning electron microscopy.

Recent evidence suggests that dental plaque is not a homogeneous bacterial mass but, on the contrary, specific bacterial morphotypes and species may be preferentially located within certain microenvironments. The aim of the present study was to develop combined gold immunolabeling and scanning electron microscopic (SEM) techniques for the identification of periodontal pathogens in subgingival dental plaque and on the root surfaces of extracted teeth. Suspensions of pure A. actinomycetemcomitans cultures or suspensions of A. actinomycetemcomitans mixed together with other oral bacteria were prepared, labeled with goat anti-rabbit IgG conjugated with 5 nm or 40 nm colloidal gold particles, and observed by SEM using both secondary and back-scattered imaging. Scanning electron microscopy demonstrated that the A. actinomycetemcomitans bacterial cell surface was specifically labelled. There was no cross-reaction with any of the other bacterial morphotypes. Only the labeled A. actinomycetemcomitans were visible in mixtures examined by back-scattered imaging scanning electron microscopy. The combined techniques of gold immunolabeling and SEM may, therefore, be useful in identifying A. actinomycetemcomitans in subgingival plaque samples and on the root surfaces of extracted teeth as well as in studies of bacterial ecology in dental plaque, in general.

Characterization of an attenuated strain of Actinobacillus pleuropneumoniae, serotype 1.

Pleuropneumonia is an important disease of swine caused by Actinobacillus pleuropneumoniae. Putative virulence determinants include capsule, lipopolysaccharide, and cytotoxin. We studied the virulence and virulence determinants of 2 strains: CM5 and CM5A of serotype 1. Strain CM5 was isolated from a pig with pleuropneumonia and passaged once in vitro; strain CM5A was a substrain of CM5 passaged 70 times in vitro. Pigs challenge exposed to an aerosol of 1.3 x 10(7) colony-forming units of CM5/ml died within 30 hours; pigs challenge exposed to an aerosol of 1.6 x 10(8) colony-forming units of CM5A/ml survived. The average thickness of the capsular layer was 137 nm in strain CM5 and 53 nm in strain CM5A in bacteria treated with homologous antibody and examined by transmission electron microscopy. Similarly, capsular material binding polycationic ferritin was found in colonies of strain CM5, but not in strain CM5A. The ratio of hexosamine to protein in extracted capsule of CM5 was more than twice that of CM5A. The polyacrylamide gel electrophoretic profile of the lipopolysaccharide, outer membrane proteins, and whole cell proteins did not differ between the 2 strains. Also, the amount of cytotoxin or endotoxin produced by the 2 strains during the logarithmic growth phase was not different. The electrophoretic profile of restriction endonuclease digested DNA was similar, with the exception of bands in the 750- and 620-basepair regions. It was concluded that attenuation of strain CM5A during in vitro passage was a result of reduced capsule production and that encapsulation is an important virulence determinant of A pleuropneumoniae, serotype 1.

Colonial variation and fimbriation of Actinobacillus actinomycetemcomitans.

Three colonial variants of Actinobacillus actinomycetemcomitans, which formed transparent rough (TR)-, transparent smooth (TS)-, and opaque smooth (OS)-surfaced colonies, were described in relation to their fimbriation. TR- and TS-cells were adhesive to agar and glass surfaces but not the OS-cells. The examination by electron microscopy revealed that TR-cells were highly fimbriated but not TS- and OS-cells. Thus, TS-cells seemed to be an intermediate type. The fimbriae were isolated from TR-cells by suspending in 0.15 M ethanolamine-HCl buffer (pH 10.5) and purified by dissolving non-fimbrial components in 0.5% deoxycholate and 0.7% n-octyl-beta-D-glucopyranoside. The relative molecular mass of the fimbrial subunit protein was 54,000.

A new method of bacterial identification using gold immunolabelling and scanning electron microscopy.

This study sought to develop an immunolabelling technique to identify specific bacteria by scanning electron microscopy. Bacterial suspensions were prepared of Actinobacillus actinomycetemcomitans, Porphyromonas (Bacteroides) gingivalis and mixtures of these species with other genera of common oral microorganisms. This method was also used to examine subgingival plaques from 6 subjects with chronic adult periodontitis. Sample preparation consisted of prefixation of the bacterial suspensions with 0.2% glutaraldehyde, incubation with species-specific rabbit antisera and goat anti-rabbit IgG conjugated with colloidal gold particles, postfixation in 2% glutaraldehyde and dehydration in ethanol. Finally, the samples were dried, coated with evaporated carbon and examined by scanning electron microscopy. Pure cultures and artificial mixtures of A. actinomycetemcomitans and Bact. gingivalis were specifically labelled by gold probes as demonstrated by both secondary and back-scattered imaging. These species were also evident in samples of subgingival plaque. The findings indicate that this new technique can be used to identify specific microorganisms both in plaque samples and on the root surface of extracted teeth.

Surface hydrophobicity and surface free energy of Actinobacillus actinomycetemcomitans strains from human periodontitis.

The surface properties of five freshly isolated clinical strains of Actinobacillus actinomycetemcomitans and a non-sticky, non-cohesive colonial variant were characterized. On growth in serum-containing and serum-free media, the primary isolates had a high surface free energy, corresponding with the low hydrophobicity observed, using as assay adsorption to hexadecane. The dispersion component of the surface free energy was significantly lower upon growth in serum-free medium. In the electron microscope the cells appeared covered with vesicles. In sharp contrast, the colonial variant had a very low surface free energy but still showed a low hydrophobicity in the hexadecane assay. Expression of the surface free energy characteristics in serum-grown cultures appeared strongly dependent on cell treatment, suggesting a significant influence of an adsorbed proteinaceous layer. The cells of the variant strain generally carried fewer surface vesicles. No major difference was observed in the outer membrane protein composition between parent and variant strain.

Outer membranous vesicles and leukotoxic activity of Actinobacillus actinomycetemcomitans from subjects with different periodontal status.

Strains of A. actinomycetemcomitans (A.a) from juvenile periodontitis patients (JP), adult periodontitis patients (AP), and 14-yr-old healthy children were tested for the correlation between leukotoxin activity and the number of outer membranous vesicles measured in electron micrographs. To determine the potential for connective tissue destruction following the interaction of polymorphonuclear leukocytes (PMN) with the bacteria, the lysosomal release of neutrophil elastase was assessed. The highest potential to kill leukocytes and to release lysosomal elastase from them was observed in the strains isolated from JP patients. No correlation existed between leukotoxic activity and the number of outer membranous vesicles per bacterium when the data from A.a. strains from all sources were combined. Furthermore, no significant differences were found between the numbers of outer membranous vesicles in the three groups tested. The only significant correlation between the number of vesicles and leukotoxicity was found in the A.a. strains derived from the mouths of healthy children.

Hemagglutinating properties of Actinobacillus pleuropneumoniae.

A total of 26 isolates of Actinobacillus pleuropneumoniae were tested for their ability to agglutinate erythrocytes of different origins. Seven different hemagglutination patterns were found. Ten (38%) isolates did not agglutinate any of the erythrocytes tested. The remaining 16 (62%) isolates agglutinated human erythrocytes, and among these, 12 also agglutinated rat, cat, dog, guinea pig, or bovine erythrocytes. No correlation was found between the seven different hemagglutination patterns observed and the serotypes. Hemagglutination activity was destroyed by heating at 100 degrees C as well as by formaldehyde treatment, but was not affected by heating at 60 degrees C, by treatment with trypsin or pronase, or by homogenization of bacterial cells. No fimbriae were observed on examination of bacterial cells negatively stained with phosphotungstate using electron microscopy. Hydrophobic surface properties of the isolates were evaluated. All the isolates appear to possess a hydrophilic cell surface. The present study provides evidence that certain isolates of A. pleuropneumoniae possess hemagglutinating properties which do not appear to be mediated by fimbriae or to involve hydrophobic interactions.

Electron microscopic examination of capsular material from various serotypes of Actinobacillus pleuropneumoniae.

The capsular material on PPLO broth-grown cells of Actinobacillus pleuropneumoniae representing serotypes 1 to 10 was visualized by transmission electron microscopy after polycationic ferritin labeling and also after stabilization with specific antibodies. All the isolates examined were covered with a layer of capsular material whose thickness varied between 80 to 90 nm and 210 to 230 nm when examined by immunostabilization. We were also able to visualize A. pleuropneumoniae in lungs of infected pigs and to estimate the amount of capsular material covering the cells. Our results indicate that differences in capsular structure exist among the different A. pleuropneumoniae serotypes, and this result may explain in part why the serotypes are not equally virulent.

Effect of anaerobiosis on the surface ultrastructure and surface proteins of Actinobacillus actinomycetemcomitans (Haemophilus actinomycetemcomitans).

The ultrastructures and surface protein profiles of aerobically cultured Actinobacillus actinomycetemcomitans (Haemophilus actinomycetemcomitans) differed from those of cells cultured anaerobically. Similar ultrastructural differences were also observed when aerobic and anaerobic cultures of a strain of Escherichia coli were compared. These results suggest that oxygen-related variations in the bacterial cell surface may play a role in the adaptation of oral bacteria to different host environments.

Tissue localization of Actinobacillus actinomycetemcomitans in human periodontitis. I. Light, immunofluorescence and electron microscopic studies.

Invasion of periodontal tissues by different bacterial morphotypes has been reported in human periodontitis; however, limited information is available as to prevalence, localization and the bacterial species involved. The present study determined prevalence and gingival localization of Actinobacillus (Haemophilus) actinomycetemcomitans in periodontal lesions of juvenile periodontitis patients. Thirty-five gingival biopsies were obtained from 12 juvenile periodontitis patients at the time of periodontal therapy. One additional control biopsy was obtained from each of two adult periodontally healthy subjects, one adult periodontitis patient and one periodontally healthy monkey (Macaca fosibolius). The biopsies were carefully processed to avoid mechanical introduction of bacteria into the tissues and were examined using light and electron microscopy. Rabbit antisera specific for the three A. actinomycetemcomitans serotypes were used for immunofluorescence microscopic localization of A. actinomycetemcomitans antigens in the gingival sections. Immunofluorescence microscopy showed A. actinomycetemcomitans specific antigens in the gingival tissues of 11 of the 12 juvenile patients examined. None of the control specimens showed evidence of A. actinomycetemcomitans antigens in the gingival connective tissue. One specimen from a periodontally healthy subject and the monkey biopsy, however, showed A. actinomycetemcomitans antigens in bacterial plaque on the surface of the crevicular epithelium. Transmission electron microscopic examination showed microcolonies of small gram-negative rods in the connective tissue, as well as single bacterial cells between collagen fibers and in areas of cell debris. In addition to these extracellular bacterial cells, evidence of bacterial cells was also found within gingival connective tissue phagocytic cells. The data from the present study suggest that the gingival tissue in juvenile periodontitis lesions harbors A. actinomycetemcomitans.

Cell surface hydrophobicity of Actinobacillus actinomycetemcomitans Y4.

Oral bacteria colonize the dento-gingival tissues in a selective manner. Hydrophobic reactions have been suggested as one of the major mechanisms of adhesion. Hydrophobicity of Actinobacillus actinomycetemcomitans Y4 (Aa) cells was studied in vitro using adherence to the liquid hydrocarbon, octane. Adherence of Aa cells to octane varied from 60-90%, depending on the medium in which they were grown, age of the culture and the buffer in which the assay was carried out. These data suggest that Aa is a hydrophobic bacterium, the hydrophobicity of which is expressed to a varying degree, and may have a rôle in its adherence to oral tissues.

Association between bacteriophage-infected Actinobacillus actinomycetemcomitans and rapid periodontal destruction.

Actinobacillus actinomycetemcomitans was isolated from periodontal pockets in a patient suffering from prepubertal periodontitis. Electron microscopy revealed 3 different groups of bacteriophages in filtrates of subgingival plaque from all the active periodontal lesions. Phage infected A. actinomycetemcomitans in this patient was restricted to periodontal pockets which, according to standardized roentgenograms, had shown bone destruction during the past 12 months. A follow-up study of 7 months revealed that a "burned out" site which harbored noninfected A. actinomycetemcomitans, turned into an active site at the same time as the A. actinomycetemcomitans of that site became infected with the phages. These findings indicate a relationship between rapid prepubertal periodontal destruction and phage-infected A. actinomycetemcomitans.