Biofilm Growth and Detachment of Actinobacillus actinomycetemcomitans

Abstract

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. Actinobacillus actinomycetemcomitans is a gram-negative, nonmotile coccobacillus that colonizes the human oral cavity (20). A. actinomycetemcomitans has been implicated as the causative agent of several forms of severe periodontal disease, including localized juvenile periodontitis, early-onset periodontitis, and rapidly progressive periodontitis (37). Infrequently, A. actinomycetemcomitans can enter the submucosa and cause nonoral infections, including bacteremias, infective endocarditis, and localized abscesses (17). When cultured in broth, fresh clinical isolates of A. actinomycetemcomitans form extremely tenacious biofilms on surfaces such as glass, plastic, and saliva-coated hydroxyapatite (7, 8, 11, 13-16, 20, 29, 30). Nearly all of the cells grow attached to the surface, while the broth remains clear and is often sterile (7). The dense biofilm that forms on the surface is resistant to removal by agents such as detergents, proteases, heat, sonication, and vortex agitation (7) and exhibits increased resistance to antimicrobial agents compared with that exhibited by cells grown in planktonic form (6). Tight adherence has been shown to play an important role in the ability of A. actinomycetemcomitans to colonize the mouths of rats (9) and probably plays an equally important role in its ability to colonize humans. Tenacious surface attachment is dependent on the presence of long, bundled adhesive pili (fimbriae) that form on the surface of the cell (29). Mutations in flp-1, which encodes the major fimbrial protein subunit, result in cells that fail to produce fimbriae or attach to surfaces (15).Kaplan and Fine have recently shown that A. actinomycetemcomitans biofilm colonies are capable of releasi...

Identification of critical amino acid residues in the plague biofilm Hms proteins

“…Second, PgaC was able to restore the CR-binding phenotype of an hmsR mutant when expressed from a highcopy-number plasmid (Jones et al, 1999). Third, DspB, a b-hexosaminidase and biofilm-dispersing enzyme of A. actinomycetemcomitans (Kaplan et al, 2003), shown to specifically hydrolyse the glycosidic linkages of poly-b-Nacetyl-D-glucosamine, caused complete inhibition of biofilm formation by Y. pestis KIM6+ (Itoh et al, 2005). Our results further support this conclusion.…”

Biofilm Formation by a Fimbriae‐Deficient Mutant of Actinobacillus actinomycetemcomitans

“…3a). Very recently, Kaplan et al also observed similar microcolony formation by an A. actinomycetemcomitans clinical isolate (13). Electron micrographs with higher magnification show that TR cells are more tightly packed within the biofilm than are DF cells (Fig.…”

Interactions between the Aggregatibacter actinomycetemcomitans secretin HofQ and host cytokines indicate a link between natural competence and interleukin-8 uptake

“…The role of HofQ in biofilm formation was studied with a crystal violet staining procedure [50] as described earlier [17]. Briefly, a 48-well microtiter plate was inoculated with diluted starter cultures of the A. actinomycetemcomitans D7S or D7S Δ hofQ strains so that each well contained 3.8 × 10 7 CFU.…”