Vol. 173, No. 8

JOURNAL OF BACTERIOLOGY, Apr. 1991, p. 2581-2589

0021-9193/91/082581-09$02.00/0 Copyright C) 1991, American Society for Microbiology

Adhesion of Actinomyces viscosus to Porphyromonas (Bacteroides) gingivalis-Coated Hexadecane Droplets MEL ROSENBERG,"2 ILZE A. BUIVIDS,l AND RICHARD P. ELLEN'* Faculty of Dentistry, University of Toronto, 124 Edward Street, Toronto, Ontario MSG JG6, Canada,' and The Maurice and Gabriela Goldschleger School of Dental Medicine, and Department of Human Microbiology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat-Aviv, Israel' Received 11 September 1990/Accepted 30 January 1991

Interbacterial adhesion (coadhesion) is considered a major determinant of dental plaque ecology. In this report, we studied several aspects of the adhesion of Porphyromonas (Bacteroides) gingivalis to hexadecane in order to use the liquid hydrocarbon as a convenient substratum for coadhesion assays. Washed suspensions of hydrophobic P. gingivalis 2561 cells were vortexed with hexadecane to yield highly stable cell-coated droplets. Kinetics of coadhesion between Actinomyces viscosus cells and P. gingivalis-coated hexadecane droplets (PCHD) was subsequently studied. Aliquots of PCHD were added to A. viscosus suspensions, and the mixtures were gently rotated. Avid adhesion of A. viscosus cells to the immobilized P. gingivalis layer could be readily measured by the decrease in turbidity in the aqueous phase, following phase separation. Despite the ability of A. viscosus cells to adsorb to hexadecane following vigorous mixing, gentle mixing did not appreciably promote adhesion to bare hexadecane. Moreover, extensive microscopic examinations revealed that A. viscosus cells adhered exclusively to the bound P. gingivalis cells rather than to exposed areas of hexadecane. Coadhesion of A. viscosus to the PCHD appeared to follow first-order kinetics, attaining 80% levels within 30 min. Electron micrographs revealed A. viscosus cells adhering to the P. gingivalis cell layer adsorbed at the hexadecane-water interface. Interestingly, P. gingivalis cells did not appear to penetrate the hexadecane. A. viscosus mutants lacking type 1 or type 2 fimbriae or both were still able to bind to the PCHD. No obvious correlation was observed between relative hydrophobicity of A. viscosus strains and their binding to PCHD. However, defatted bovine serum albumin, an inhibitor of hydrophobic interactions, was the most potent inhibitor among those tested. The data suggest that this approach provides a simple, quantitative technique for studying kinetics of bacterial coadhesion which is amenable to both light and electron microscopic observation.

Surfaces submerged in an aqueous environment are prone to rapid colonization by adhering microbial populations. Initial microbial adsorption may be followed by adhesion of additional microorganisms from the bulk aqueous phase to the developing microbial layer. The buildup of adherent macroscopic masses of attached microbiota requires intimate cell-cell interactions which protect the community from being dislodged. Such biofilms are common in the oral cavity, particularly on tooth surfaces (dental plaque). In recent years, the role of interbacterial adhesion (coadhesion) in dental plaque ecology has drawn increasing interest (15). Whereas adhesion to freshly cleaned tooth surfaces occurs rapidly, the majority of microorganisms in maturing dental plaque are bound to one another rather than to the salivary pellicle on the tooth surface (5, 7, 15). Certain bacteria may adhere preferentially to preformed plaque rather than directly to the tooth surface (5). Finally, coadhesion between interdependent species in dental plaque may promote synergistic physiological relations (7, 17). Interbacterial adhesion of oral species was first demonstrated by Gibbons and Nygaard (12). Indeed, most studies to date have employed so-called coaggregation assays, in which aqueous suspensions of two strains are mixed and the degree of association is determined visually (degree of clumping) or by quantitative means (6, 7, 16, 18). This approach, while simple to perform, is difficult to observe at the electron microscope level without altering cell-cell orientation or distance. The contributions of intrastrain aggre*

gation (i.e., autoaggregation) are hard to assess in such assays. Moreover, adhesion of cells to preformed layers may be a general natural phenomenon in biofilm accumulation, whereas the ecological relevance of in vitro coaggregation of cells is not intuitively clear. Thus, several researchers have recently proposed studying coadhesion of one strain, free in suspension, to a preformed layer of the second strain adhering on a given surface. For example, Liljemark et al. used a continuous layer of Streptococcus sanguis cells affixed to plastic and enamel surfaces with a commercial adhesive (Cell-TAK) to study coadhesion with Haemophilus parainfluenzae and Streptococcus sobrinus (17). Ellen and coworkers have studied attachment of Bacteroides gingivalis to preformed layers of Actinomyces viscosus strains on salivacoated hydroxyapatite beads (7, 27). The technical complexities of preparing the substratum, coating it with the first species, and measuring coadhesion of the second species prompted us to search for a simpler solution which would also be amenable to light and electron microscopy. This was carried out by (i) immobilizing Porphyromonas (Bacteroides) gingivalis cells on hexadecane droplets by a simple mixing procedure and (ii) following coadhesion of free A. viscosus cells to the P. gingivalis-coated droplets as a function of time. MATERIALS AND METHODS A. viscosus strains and culture conditions. A. viscosus WVU627 was originally obtained from M. A. Gerencser, West Virginia University. Strain T14VJ1 (bearing both type 1 and type 2 fimbriae) and corresponding mutant strains 5519

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(lacking type 2 fimbriae), 5951 (lacking type 1 fimbriae), and 147 (lacking both fimbrial types) were obtained from J. Cisar, National Institute of Dental Research. These strains have been reported to differ in their adhesion properties (2). Strains were maintained on brain heart infusion agar slants (Difco Ltd.) and stored at 4°C. A. viscosus strains were inoculated from slants into test tubes containing 10 ml of tryptic soy broth (Difco Ltd.) and grown for 48 h at 37°C under anaerobic conditions (80% N2, 10% C02, 10% H2). For adhesion experiments, 1:1,000 dilutions were inoculated into test tubes containing the same medium and grown for 48 h as described above. Cells were harvested by low-speed centrifugation, washed three times in phosphate (P) buffer (7.26 g of KH2PO4, 22 g of K2HPO4. 3H20, distilled water to 1,000 ml [pH 7.1]), and suspended in buffer in glass test tubes (10-mm inner diameter) to obtain optical densities (corrected for nonlinearity) from ca. 1.0 to 2.0, as measured at 550 nm in a model 350 spectrophotometer (G.K. Turner Associates, Palo Alto, Calif.). P. gingivalis strains and culture conditions. P. gingivalis 2561 (ATCC 33277, type strain) was obtained from J. Slots, University of Pennsylvania. P. gingivalis W50 was obtained from J. Zambon, State University of New York at Buffalo. P. gingivalis strains were maintained on laked blood agar, comprising blood agar base no. 2 (Oxoid Ltd., Basingstoke, Hampshire, England) supplemented with 7% laked sheep blood and 1 mg each of filter-sterilized hemin and menadione per ml. Cells were inoculated into a modified Trypticasepeptone-yeast extract broth containing Trypticase-peptone (BBL Microbiology Systems, Cockeysville, Md.) supplemented with yeast extract (Difco Ltd.) (3 g), NaCl (5 g), K2HPO4 (2.5 g), glucose (2.5 g), filter-sterilized hemin (5 mg), filter-sterilized menadione (0.5 mg), and NaHCO3 (1 g) (11). All cultures were incubated at 37°C in anaerobic jars containing 80% N2, 10% C02, and 10% H2. Following 72 h of growth, cells were harvested and washed four times in P buffer. The cell suspension was passed once through a 25-gauge needle to more evenly suspend the cells and adjusted to a corrected optical density of ca. 2.0. Preparation of P. gingivalis-coated hexadecane droplets (PCHD). P. gingivalis cell suspensions (optical density, 2.0), prepared as described above, were distributed in 2.0-ml aliquots into clean borosilicate glass test tubes. n-Hexadecane (0.15 ml) was added to each tube. The test tubes were then vortexed at the highest speed setting for four consecutive 1-min periods on a Vortex-Genie (Scientific Industries, Inc., Bohemia, N.Y.). After phase separation, the percent adhesion was determined by the decrease in turibidity of the lower aqueous phase. The contents of the test tubes were then pooled into a beaker, and the lower aqueous phase, containing unbound cells, was carefully removed by using a Pasteur pipette. For every milliliter of lower aqueous phase extracted, 0.6 ml of fresh P buffer was added. The suspension was swirled gently prior to dispensing into test tubes for the coadhesion assay to ensure uniform distribution of the droplets. A. viscosus-coated hexadecane droplets were prepared in a similar manner, by employing strain WVU627. Coadhesion assay. After several preliminary experiments, the following protocol was adopted. To 1.0 ml of A. viscosus cell suspension in clean borosilicate glass test tubes (13-mm outer diameter; 11-mm inner diameter; 100-mm length) were added 0.5 ml of P buffer, with or without inhibitors, and 0.5 ml of mixed PCHD suspension. Controls included (i) test tubes containing hexadecane (62.5 ,lI) and 937.5 ,ul of P buffer rather than PCHD, (ii) test tubes containing only A. viscosus cell suspension in P buffer, with or without inhibi-

J. BACTERIOL.

tors, as indicated, and (iii) test tubes containing only PCHD and P buffer, with or without inhibitors, as indicated. All test tubes were incubated with gentle rotation at a fixed incline of 400 in a Multi-purpose Rotator (Scientific Industries, Inc., Springfield, Mass). At 2-min intervals, phases were allowed to separate and turbidity of the lower aqueous phase was measured directly in the spectrophotometer. Experiments were repeated at least two times; representative data are shown. The degree of coadhesion was calculated as the percent decrease in turbidity of the aqueous phase, compared with the initial reading. Photomicrographs of PCHD with and without adherent A. viscosus cells, stained with gentian violet (BDH Inc., Toronto, Ontario, Canada), were taken in a Leitz Orthoplan light microscope (Leitz Wetzlar, Wetzlar, Federal Republic of Germany). Hydrophobicity measurements of A. viscosus. Bacterial adhesion to hexadecane has been described previously (2326). Washed A. viscosus cell suspensions (optical densities, 1 to 2) in P buffer were distributed in 2.0-ml aliquots into clean borosilicate glass test tubes. n-Hexadecane (0.15 ml) was added to each tube. The test tubes were then vortexed as described above for P. gingivalis. The decrease in optical absorbance of the lower aqueous phase was measured as a function of mixing time. Adhesion to polystyrene microtiter plate wells (non-tissue culture treated) of 100-,ul aliquots of the washed A. viscosus cell suspensions was performed for 20 min as previously described (23). Electron microscopy. Aliquots (50 ,ul) containing suspensions of bacteria-coated hexadecane droplets, with or without coadhering strains, were mixed with 200 p.l of molten (50°C) 1% Noble agar (Difco Ltd.) and allowed to solidify at room temperature within Pasteur pipettes. After the solidified samples were ejected onto clean glass slides, they were cut into segments of 5 to 7 mm in length. The segments were subsequently prefixed in 0.1 M sodium phosphate buffer, pH 7.4, containing 1% glutaraldehyde for 1 h, which was followed by two washes and overnight incubation in P buffer. Postfixation was carried out in 2% OS04 in sodium phosphate buffer. Following three washes in P buffer (pH 7.1), the segments were dehydrated in graded ethanol solutions and then washed twice in propylene oxide (Ernest F. Fullam, Inc., Schenectady, N.Y.). The segments were then embedded in Epox 812 resin (Ernest F. Fullam, Inc.). Thin sections were cut and transferred to single-slot, Formvarcoated nickel grids. Following staining with 5% uranyl acetate and Reynold's lead citrate, the samples were examined and photographed by using a Phillips 400T transmission electron microscope. Chemicals. Except when otherwise noted, chemicals and biochemicals were obtained from Sigma Chemical Co. (St. Louis, Mo.). RESULTS Preliminary experiments were carried out to determine whether bacteria-coated hexadecane droplets could be used as a substratum for coadhesion studies. With the exception of P. gingivalis W50, the strains studied here all adhered to hexadecane in high proportions (Fig. 1). Similarly, adhesion to a hydrophobic solid surface, polystyrene, was evident for all strains which adhered well to hexadecane (data not shown). We initially envisioned measuring binding of free P. gingivalis cells to A. viscosus WVU627-coated hexadecane droplets. However, this process was difficult to follow quantitatively, since the free P. gingivalis cells in suspension tended to autoaggregate. Thus, P. gingivalis-coated hexa-

A. VISCOSUS ADHESION TO P. GINGIVALIS

VOL. 173, 1991

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34 142 MINUTES MINUTES FIG. 1. (A) Adhesion of P. gingivalis to hexadecane. Adhesion of P. gingivalis 2561 (0) and P. gingivalis W50 (O) to hexadecane was carried out as described in Materials and Methods. Results are presented as percent absorbance in the lower aqueous phase as a function of mixing time. (B) Adhesion of A. viscosus strains to hexadecane. Adhesion of A. viscosus WVU627 (0), T14VJ1 (X), 5951 (O), 5519 (A), and 147 (0) to hexadecane. 0

decane droplets were subsequently studied as a substratum for coadhesion with A. viscosus cell suspensions. P. gingivalis 2561 routinely adhered to hexadecane at levels above 80% (Fig. 1A). PCHD were stable for several days at room temperature. Light microscopic observation of cell-coated droplets was performed by using gentian violet to visualize the bound cells. In many cases, droplets were observed which were completely covered by a monolayer of adherent cells. Other droplets, however, were covered by patches or rafts of associated cells (Fig. 2a). Some droplets developed protrusions, in which the round contour of the droplet was maintained in regions covered by cells, but bare areas of hydrocarbon could be observed extruding outwards from the droplets (Fig. 2b). In contrast, vortexing hexadecane with the much less hydrophobic P. gingivalis W50 yielded unstable droplets which tended to coalesce rapidly, and these droplets bore only a few widely spaced cells when viewed by microscopy. Adhesion of P. gingivalis 2561 to hexadecane was also observed by using electron microscopy (Fig. 3). In addition to bound cells, free vesicles, probably released during growth, washing, and dispersion procedures, could be observed. Of considerable interest was the observation that the cells did not appear to penetrate into the hexadecane phase or to otherwise deform the interface. Since all the A. viscosus strains tested adhered to hexadecane following vigorous mixing (Fig. 1B), conditions were sought which would promote coadhesion to the bound P. gingivalis cells rather than adsorption directly to the hexadecane substratum. This problem was overcome by gentle mixing in tilted test tubes. Under such conditions, A. viscosus did not adhere appreciably to hexadecane, yet avid coadhesion of A. viscosus cells free in suspension to the bound P. gingivalis cells on the hexadecane droplets was observed (Fig. 4). Moreover, microscopic observation of hundreds of droplets confirmed that A. viscosus cells adhered exclusively to bound P. gingivalis cells and not to exposed areas of the hexadecane (Fig. 5). Coadhesion of A.

viscosus cells to the P. gingivalis cell layer was further corroborated by observation of thin sections in the electron microscope (Fig. 6). As an additional control of specificity, coadhesion of A. viscosus to S. sanguis-coated hexadecane droplets was also studied under the same conditions. In the calcium-free buffer employed in the present study, coadhesion was not observed. Finally, the ability of P. gingivalis cells to coaggregate when mixed with A. viscosus on glass slides remained intact following their (P. gingivalis) desorption from the hexadecane by solidification of the hexadecane phase (26). The kinetics of coadhesion was followed by turbidimetric measurement of the unbound A. viscosus cell suspension in the lower aqueous phase, as a function of mixing time (Fig. 7). In the presence of PCHD, coadhesion was rapid, attaining 80% levels within 30 min. No significant decrease in turbidity occurred in the control tube containing A. viscosus and hexadecane. Similarly, no increase in turbidity (which would suggest desorption of P. gingivalis cells) was observed in control tubes containing PCHD without A. viscosus (Fig. 7). Coadhesion of A. viscosus to PCHD followed apparent first-order kinetics. The rate of coadhesion depended on the concentration of PCHD in the assay. When one-half of the PCHD mixture was added, the coadhesion rate was approximately half that of the tube containing the higher concentration. As expected from the apparent first-order kinetics, the initial concentration of A. viscosus cells did not substantially alter the fraction of cells coadhering as a function of time. The rate of coadhesion was also increased by 73% when double the concentration of P. gingivalis was employed to prepare the PCHD, keeping the hexadecane volume constant. In contrast, a much smaller increase (24%) was observed when double the volume of hexadecane was used to form the PCHD but the P. gingivalis concentration was kept constant. This latter increase may be due to the adhesion of higher numbers of P. gingivalis cells to the higher hexadecane volume or alternatively to a more spacious distribution

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FIG. 2. Light microscopic observation of adhesion of P. gingivalis to hexadecane. After adhesion to hexadecane and phase separation, samples of the upper phase were observed by using gentian violet to enhance visualization. (a) Droplet with patches of adherent cells; (b) extrusion of hexadecane from two adjacent droplets. Bars, 10 ,um.

of P. gingivalis cells. In all these instances, when altering either the concentrations or the volumes used, the straightline linearity of the plotted curves was maintained. Since P. gingivalis cell suspensions with an optical density of 1.0 correspond to approximately 3.8 x 109 cells per ml, as opposed to approximately 1.3 x 109 cells per ml for the larger actinomyces cells (12a), the P. gingivalislA. viscosus ratio in the coadhesion assay could be calculated to be ca. 10:1. Viability of either species was not directly a factor in the assay. P. gingivalis cells were no longer viable prior to mixing with hexadecane, as determined by plating of cell suspensions. Heat-killed A. viscosus cells (75°C, 30 min) coadhered in higher proportions to the PCHD than cells that were not killed; heat-treated P. gingivalis cells (75°C, 30 min) concomitantly lost the ability to adhere to hexadecane, to coaggregate with A. viscosus, and to autoaggregate (data not shown). Coadhesion was studied by employing A. viscosus T14VJ1 and its corresponding mutants lacking type 1 fimbriae, type 2 fimbriae, or both (Fig. 8). Both A. viscosus mutants devoid of type 2 fimbriae underwent spontaneous aggregation. Nevertheless, the rapid drop in turbidity in the presence of PCHD, compared with the control, as well as microscopic observation of the droplets, demonstrated that extensive coadhesion occurred. Coadhesion rates and hydrophobicity results are compared in Table 1. No correlations were evident between fimbrial type, rate of coadhesion, and extent of adhesion to hexadecane. For example, whereas A. viscosus WVU627 adhered less avidly to hexadecane than

T14VJ1 did (76% and 97%, respectively), it coadhered with P. gingivalis at a much greater rate. Similarly, coadhesion of A. viscosus fimbrial mutants could not be correlated with either fimbrial type or hydrophobicity. Inhibition experiments were carried out by adding potential inhibitors to the aqueous phase prior to the assay. Among the potential inhibitors tested, defatted bovine serum albumin (BSA) was most potent (Fig. 9). For example, 4 mg of defatted BSA per ml inhibited adhesion by 77% following a 14-min incubation. Nondefatted BSA was also inhibitory, but much less so than the defatted protein (only 40% following a 14-min incubation). In the presence of BSA, coadhesion did not appear to follow first-order kinetics (Fig. 9). Previous studies have suggested that L-arginine, among a series of amino acids tested, may inhibit coaggregation of A. viscosus and P. gingivalis (16a). Under the assay conditions used in these experiments, coadhesion was not significantly inhibited by a variety of amino acids at final concentrations of 10 mM (L-arginine, L-asparagine, L-lysine, or L-phenylalanine [data not shown]). Tryptophan has been shown to inhibit hemagglutination by Pseudomonas aeruginosa at a 1 mM concentration (8). However, even 5 mM tryptophan (final concentration) was not significantly inhibitory in the present assay. DISCUSSION Bacterial adhesion to hydrocarbons has been extensively applied in studying hydrophobic surface properties of oral

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FIG. 3. Electron microscopic observation of adhesion of P. gingivalis to hexadecane. Ultrathin sections of cells adhering to hexadecane droplets, stabilized in agar and stained with uranyl acetate and lead citrate, were prepared as described in Materials and Methods. A small hexadecane droplet encompassed by adhering cells is shown (a). No deformation of the interface by the adhering cells is evident (b). Bars, 500 nm.

microorganisms (26). Most freshly obtained oral isolates (29), as well as laboratory strains of streptococci and other species (3, 9, 10, 20, 21, 28), are able to adhere directly to oil droplets after brief mixing periods. In the present report, we have introduced a modification of this technique to study interspecies coadhesion. This method has two stages: (i) adhesion of one of the species to hexadecane droplets under vigorous mixing conditions and (ii) measurement of coadhesion of the second species to the bound cells of the first under very mild mixing conditions. In contrast to a previous report on Bacteroides spp. from another laboratory (13), the first stage produced stable PCHD. Microscopic examinations of P. gingivalis 2561 cells adhering to the hexadecane droplets yielded several novel observations. In many cases, P. gingivalis cells adhered to hexadecane in patches. These resembled the patches of adherent oil-degrading microorganisms which are frequently observed during hydrocarbon fermentations (22a). Under microscopic examination, some droplets developed gross deformations. Hexadecane could be observed extruding outwards from the droplets in regions free of bound cells. Conversely, areas coated with a monolayer of bound cells preserved -their circular form. Such deformations, probably resulting from application of pressure on the droplets sandwiched between the microscope slide and coverslip, indicate that the adherent cells act to stabilize the hydrocarbon droplets. Indeed, as has previously been observed with other bacterial strains (23, 24, 26),

PCHD could be stored in test tubes at room temperature for days to weeks, while bare hexadecane droplets coalesce almost immediately. It is tempting to hypothesize that this stabilizing effect is due at least in part to attractive, lateral interactions among the adhering cells. Hexadecane droplets were not stabilized by P. gingivalis W50, which evidently failed to adhere to hexadecane in numbers high enough to establish cell-cell contact. Adhesion of P. gingivalis to hexadecane was also observed by using electron microscopy. To our knowledge, this is the first electron microscopic demonstration of microbial partitioning at the aqueous-hydrocarbon interface. Of considerable interest was the observation that the adhering cells did not appear to penetrate into the hexadecane phase or to substantially deform the interface (Fig. 3), in contrast to the prediction of Mudd and Mudd, which was based on interfacial tension considerations (19). After adhesion of P. gingivalis 2561 to hexadecane, the bottom aqueous phase, containing low levels of unbound cells, was replaced with fresh buffer. Suspensions of PCHD could be readily distributed into test tubes containing suspensions of A. viscosus cells. Since coadhering A. viscosus cells rose with the PCHD, the rate of coadhesion could be followed directly in the test tubes by turbidimetric readings over time. Coadhesion of A. viscosus to P. gingivalis cells followed apparent first-order kinetics, i.e., a linear correlation between the rate of coadhesion and the relative concen-

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FIG. 4. Coadhesion assay. After 30 min of incubation of A. viscosus cells with PCHD, A. viscosus-mediated turbidity in the lower aqueous phase disappeared concomitant with coadhesion of cells to PCHD (test tube on left). No significant decrease in turbidity was observed in the control tube (right), which contained A. viscosus cell suspension and hexadecane, under the mild mixing conditions employed.

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tration of unbound A. viscosus cells at any given time. The possibility that adhesion of A. viscosus cells to exposed hexadecane was responsible for the observed phenomenon can be ruled out since (i) extensive microscopic observation revealed A. viscosus cells adhering exclusively to bound P. gingivalis cells rather than to exposed areas of hexadecane, (ii) turbidity of control tubes containing A. viscosus cells and hexadecane remained almost constant throughout the experiment, and (iii) there was no correlation between adhesion to hexadecane and coadhesion to PCHD in the five A. viscosus strains tested. Furthermore, in previous coadhesion studies using A. viscosus-coated hydroxyapatite beads, free P. gingivalis cells appeared to bind almost exclusively to A. viscosus rather than in gaps between bound A. viscosus cells (27). However, the single confirmatory method of scanning electron microscopy in that study left a degree of uncertainty, which is now resolved in the current investigation. Hydrophobicity per se did not appear to be the critical factor in the coadhesion observed. Although all the A. viscosus strains tested adhered to hexadecane and polystyrene, no obvious correlations were observed between hydrophobicity and coadhesion. Furthermore, microscopic observations clearly indicated that under the mild mixing conditions employed, A. viscosus cells adhered preferentially to P. gingivalis cells rather than to the hexadecane surface. Nevertheless, the participation of hydrophobic interactions cannot be conclusively ruled out by the data.

b

FIG. 5. Adhesion of A. viscosus WVU627 to PCHD. Incubation of A. viscosus cells with PCHD was carried out as described in Materials and Methods. Following a 30-min incubation, the upper phase was removed and examined by light microscopy after being stained with gentian violet. (a) A. viscosus cells (dark rods) can be observed coadhering to patches of P. gingivalis cells (lighter coccal cells) on a hexadecane droplet. (b) A. viscosus cells can be seen coadhering to the P. gingivalis cell layer around the perimeter of the hydrocarbon droplet. Bars, 10 p.m.

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HD FIG. 6. Electron micrograph of coadhesion. Coadhesion of A. viscosus cell (Av) (longer cell) to P. gingivalis cells (Pg) adhering to hexadecane droplet (HD). Note the apparent involvement of a vesicle (bleb) associated with P. gingivalis cells (arrow) in the coadhesion process. Bar, 500 nm.

Although inhibitory proteins like BSA might interact stereochemically with enzymes or other protein-seeking adhesions on P. gingivalis, defatted BSA is known to be a potent inhibitor of microbial adhesion to hydrophobic surfaces (21, 22). It strongly inhibited coadhesion at relatively low concentrations, in contrast to the lower inhibition observed with nondefatted BSA. Such apparent contradictions in the role of hydrophobicity in oral adhesion and coaggregation have been reported previously. McIntire and coworkers found that lactose and sodium dodecyl sulfate (SDS) acted synergistically to inhibit coaggregation between A. viscosus T14V and S. sanguis 34 (18). SDS itself was 15 times more potent an inhibitor than lactose, whereas neither Tween 80 nor Triton X-100 were inhibitory. Jenkinson and Carter (14) isolated two mutants of S. sanguis, with increased and decreased hydrophobicity, compared with the parent strain. The mutant with increased adhesion to hexadecane and octyl-Sepharose coaggregated to a greater extent with A. viscosus than the wild type did. However, no differences in coaggregation were observed between the wild type and the less hydrophobic mutant. Clark et al. demonstrated positive correlations between adsorption of oral Actinomyces spp. to saliva-coated hydroxyapatite and to hydrophobic gels; however, whereas adsorption to the hydrophobic gel was inhibited by Tween 80, adhesion to saliva-coated hydroxyapatite was unaffected (3). Indeed, the common use of BSA as a blocking agent in oral adhesion studies by researchers interested in isolating the lectinlike or other complementary binding components (7, 27, 30) may underlie their tacit acknowledgment that in its absence, hydrophobic interactions are significant. Concomitant participation of hydropho-

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O3 0 TIME TIME (min.) (min.) FIG. 8. Coadhesion of A. viscosus T14VJ1 and corresponding mutants with PCHD. Coadhesion of A. viscosus T14VJ1 and mutant strains 5519 (missing type 2 fimbriae), 5951 (missing type 1 fimbriae), and 147 (missing both fimbrial types) to PCHD was performed as described in Materials and Methods. In each case, A. viscosus cells were incubated with PCHD (B), as opposed to control tubes (*), which contained hexadecane instead of PCHD. Results are expressed as the log percent decrease in turbidity [log (a,/ao) x 100] as a function of mixing time, 0

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bic interactions and stereochemical binding in adhesion of oral streptococci to saliva-coated hydroxyapatite was first proposed by Doyle and coworkers in 1982 (4). However, the mechanism by which this might occur remains unclear. In coaggregation and coadhesion of A. viscosus to P. gingivalis, the following model may be proposed. Initial attachment of individual A. viscosus cells to P. gingivalis cells or cell layers may occur primarily through complementary but not lectin-mediated (1, 7) stereochemical associations (the nature of which is presently under investigation); thus, the preferred adhesion of A. viscosus is to P. gingivalis rather than to bare hexadecane. However, subsequent atTABLE 1. Comparison of relative hydrophobicity and coadhesion ratea A. viscosus strain

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a Relative hydrophobicity was determined as percent adhesion to hexadecane following 4 min of vortexing (Fig. 1B), and coadhesion rates were derived

from the slopes presented in Fig. 7 and 8. b ND, Coadhesion rate could not be determined because of autoaggregation of cells.

tachment of additional A. viscosus cells would be greatly promoted by lateral cell-cell interactions with other, bound A. viscosus cells, in addition to attachment to the P. gingivalis cell layer. Such "like-like" associations may depend more on hydrophobic interactions. This model can account for the increase in BSA-mediated inhibition of coadhesion with time. Furthermore, the tendency of A. viscosus cell suspensions to autoaggregate in slide aggregation tests can also be blocked by BSA (data not shown). These data, taken together, strongly suggest that in coaggregation as well as coadhesion studies, possible interactions between cells of the same type should also be considered. The assay system presented here may provide an opportunity for further insight in this direction. The technique described here has several inherent advantages over other assays: (i) multiple readings over time can be made directly in the individual test tubes, (ii) coadhesion can be observed by both light and electron nmicroscopy, (iii) radiolabeling of cells is not required, (iv) no adhesive agents are required to mediate adsorption of the first species to the underlying surface, and (v) as opposed to basic coaggregation techniques, this method depends on rising, rather than sedimentation of, coadhering cells. Thus, the contribution of potential artifacts such as autoaggregation can readily be noted. One basic requirement of the proposed technique is that at least one of the species must adhere to liquid hydrocarbon, and we have demonstrated this limitation with weakly hydrophobic P. gingivalis W50. However, since

A. VISCOSUS ADHESION TO P. GINGIVALIS

VOL. 173, 1991

o^

1.9

1.8

0'

1.7

1.6 0

10

20

TIME (min.) FIG. 9. Effect of BSA and defatted BSA on coadhesion. The kinetics of coadhesion of A. viscosus WVU627 to PCHD was measured as described in Materials and Methods in the presence of BSA (U) or defatted BSA (5), at final concentrations of 0.2 mg/ml, as opposed to coadhesion in the presence of buffer alone (*). Results are expressed as the log percent decrease in turbidity [log (a/ao) x 100] as a function of mixing time.

oral (29) and nonoral (26) microbial strains tested to date share affinity for the hydrocarbon-water interface, this approach may prove a more general one, applicable in studying mechanisms of biofilm coadhesion for bacteria from diverse ecosystems. many

ACKNOWLEDGMENTS We are grateful to D. A. Grove and J. Li for helpful discussions. This investigation was supported by grant MT-5619 from the Medical Research Council of Canada. REFERENCES 1. Bourgeau, G., and D. Mayrand. 1990. Aggregation of Actinomyces strains by extracellular vesicles produced by Bacteriodes gingivalis. Can. J. Microbiol. 36:362-365. 2. Cisar, J. O., A. E. Vatter, W. B. Clark, S. H. Curl, S. Hurst-Calderone, and A. L. Sandberg. 1988. Mutants of Actinomyces viscosus T14V lacking type 1, type 2, or both types of

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Adhesion of Actinomyces viscosus to Porphyromonas (Bacteroides) gingivalis-coated hexadecane droplets.

Interbacterial adhesion (coadhesion) is considered a major determinant of dental plaque ecology. In this report, we studied several aspects of the adh...
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