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. 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.

FIG. 1.

Microscopic analyses of A. actinomycetemcomitans CU1000 biofilm colonies. (A to D) Scanning electron micrographs of day 0.5 (A), day 1 (B), day 2 (C), and day 4 (D) colonies grown on glass slides. (E to H) Thin sections of day 1 (E), day 2 (F), day 3 (G), and day 4 (H) colonies grown on polystyrene. Panels E and F show multiple biofilm colonies. (I and J) Transmission electron micrographs of the outer surface of day 3 (I) and day 4 (J) colonies grown on polystyrene. O, outside of the colony; L, the internal lacuna. Bars = 25 μm (E), 100 μm (F to H), and 10 μm (J).

FIG. 2.

Dispersal of A. actinomycetemcomitans CU1000 biofilm colonies in broth cultures. (A) Pictures, taken 1, 2, and 3 days after inoculation, of a single colony from a 35-mm-diameter petri dish inoculated with ≈25 CFU. Bar = 1 mm. (B) A 100-mm-diameter petri dish inoculated with 1 CFU of strain CU1000 and incubated for 3 days. Colonies appear as white spots on a dark background. (C) Scanning electron micrograph of the surface of a glass slide approximately 5 mm away from a mature biofilm colony after 2 days of growth. The glass slide contained five mature colonies.

FIG. 3.

Phenotypes of A. actinomycetemcomitans wild-type strain CU1000N (left side of panels) and biofilm dispersal mutant JK1017 (right side of panels). (A to C) Shown are colony morphology on agar (bar = 2 mm) (A), biofilm colony morphology in broth (bar = 1 mm) (B), and growth in 100-mm-diameter petri dishes (C), all after 3 days of growth. (D) Thin sections of 2-day-old biofilm colonies grown on polystyrene (bar = 100 μm).

FIG. 4.

Characterization of A. actinomycetemcomitans biofilm dispersal mutants. (A) Genetic map of an 8.7-kb region of the gene cluster responsible for the synthesis of serotype f-specific O-PS in strain CU1000 (19). Open arrows indicate open reading frames and direction of transcription. Gene names are shown above the arrows, and the filled arrowheads indicate the location and direction of transcription of IS903φkan insertions in three different mutant strains. (B) Adherence to polystyrene was measured using a 96-well microtiter plate binding assay (15). Adherence is proportional to the optical density (O.D.) values; mean values plus standard errors for triplicate samples are shown. Strain JK1009 contains a transposon insertion in flp-1 which results in complete loss of surface attachment (15). (C) Synthesis of O-PS in wild-type and mutant strains. O-PS was detected with an enzyme-linked immunosorbent assay using anti-CU1000 rabbit antiserum as previously described (19). The amount of O-PS is proportional to the O.D. values; mean values plus standard errors for triplicate samples are shown. Filled bars indicate the levels of O-PS from strains carrying vector plasmid pJAK16, and open bars indicate the levels from mutants JK1002, JK1017, and JK1022 carrying complementing plasmids pJK595, pJK597, and pJK596, respectively. (D) Detachment of cells from biofilm colonies grown on polystyrene rods as measured by a 96-well biofilm detachment assay. Cell detachment is proportional to the O.D. values; mean results plus standard errors for 8 to 15 wells for each strain are shown. Bars are as described for panel C.