Adherence of oral streptococci to an immobilized antimicrobial agent

Archs oral Bid. Vol. 42, No. 8, pp. 539-545, 1997 0 1997 Elsevim Science Ltd. All rights reserved

Printed in Great Britain PII: SOOO3-9969(97)00054-X 0003-9969/97 $17.00 + 0.00

ADHERENCE OF ORAL STREPTOCOCCI TO AN IMMOBILIZED ANTIMICROBIAL AGENT

T. SAITO,‘,*,* T. TAKATSUKA,l T. KATO,’ K. ISHIHARA’ and K. OKUDA*

‘Resea.rch and Development Department, Oral Care Business Headquarters, Sunstar Inc., 5-30-l Kamihamuro, Takatsuki, Osaka 569, Japan and 2Department of Microbiology, Tokyo Dental College,

l-2-2 Masago, Mihama, Chiba 261, Japan

(Accepted 6 May 1997)

Summary--An antimicrobial agent, 3-(trimethoxysilyl)-propyldimethyloctadecyl ammonium chloride, was immobilized on silica. Interaction between the material (termed) OAIS) and various oral bacterial species were then studied. Seven species of Streptococcus and two Actinomyces were investigated for their ability to adhere to this biomaterial. Cell-surface hydrophobicity and zeta-potential were examined as well. Analysis of extracted hydrophobic proteins which adhered to OAIS revealed that the adherence of these micro-organisms was closely related to the hydrophobicity of their cell surfaces. The results of zeta-potential assays indicated that negative charge on the cell surface inhibited adherence to OAIS. Gel electrophoresis revealed that OAIS could absorb cell-surface hydrophobic proteins from all bacterial species tested. Preadsorption of hydrophobic components on the cell surface inhibited adherence of the Strep. mutans strain to OAIS in a dose-dependent manner. The results indicate that OAIS adsorptior of these oral bacteria was dependent on the degree of hydrophobicity of their surfaces. A major component of this adherence was hydrophobic cell-surface proteins. 0 1997 Elsevier Science Ltd

Key words,: immobilized antimicrobial agent, adherence, hydrophobicity, oral streptococci.

INTRODUCTION

Immobilized antimicrobial agents became water- insoluble when combined with carriers such as water-insoluble inorganic materials (Isquith et al., 1972). Immobilization of antimicrobial agents on suitable substrates serves to lower the ability of groups on these agents to penetrate the cell membrane, thus reducing their toxicity to the host, facili- tating their eventual elimination.

Hydrolysis of a quaternary amine-containing organosilicon s,alt, 3-(trimethoxysilyl)-propyldimethyloctadecyl ammonium chloride, resulted in antimicrobial acl:ivity against Gram-positive and -negative bacteria, yeasts, algae and fungi (Isquith et al., 1972). It was discussed whether this antibac- terial activity should be attributed to phenomena involving the membrane or the cell wall. More recently, 3-(trimethoxysilyl)-propyldimethyloctadecyl ammonium chloride was immobilized on silica and this new material was termed OAIS. Its bactericidal activity was then tested against Gram-positive oral bacteria (Ta.katsuka and Mori, 1994). OAIS did indeed have bactericidal activity and a negative

*To whom all correspondence should be addressed. [Tel: 726-94-7773; Fax: 726-95-07661.

Abbreviations: c.f.u. colony-forming units, OAIS, 3-(trimethoxysilyl)-propyldi~ethyloctahecyl ammonium chloride immobilized on silica, SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electroph&esis.

correlation between this and cell-surface hydrophobicity was revealed. Also, bacteria adhere to glass slides that have various functional groups, such as amino, carboxyl, hydroxyl or hydrophobic groups, attached through covalent bonding of their carbon to the silicon of the glass (Satou et al., 1988). These data suggest that hydrophobic bonding and ionic interactions are important in bacterial adherence to such surfaces.

Exclusion of pathogenic bacteria is beneficial in controlling oral infections such as dental caries and periodontitis. Incorporating an immobilized anti- bacterial agent into toothpaste may provide a useful means of eliminating various pathogens from the mouth. Our purpose now was to characterize the adherence activity of OAIS to the surfaces of various micro-organisms.

MATERIALS AND METHODS

Cultures and culture conditions

The bacterial strains used in this study were Streptococcus mutans ATCC25175, Ingbritt, JC-2 and MT8148R, and Strep. sobrinus 6715, Strep. rattus BHT, Strep. sanguis ATCC10556, Strep. oralis ATCC10557, Strep. mitis ATCC9811, Strep. parasanguis ATCC 159 12, Actinomyces viscosus ATCCl5987 and A. naeslundii ATCC 12104. All strains were from the culture collection of our lab-

539

540 T. Saito et al.

oratory and all had been stored in 33% glycerol at -20°C. For experimental purposes, inocula from the glycerol stocks were grown in Todd- Hewitt broth (BBL, Becton Dickson Microbiology Systems, Cockeysville, MD, USA). Early station- ary-phase cells incubated anaerobically at 37°C were used in all experiments. For adhesion studies, the organisms were grown in Todd-Hewitt broth supplemented with 370 MBq of [3H]thymidine (Du Pont Company, Wilmington, DE, USA) per ml for radiolabelling, as described by Gibbons and Etherden (1983). The cells were harvested by centrifugation, washed three times, and suspended in 0.05 mol/l KC1 containing 1 mmol/l KH2P04, 1 mmol/l CaC12 and 0.1 mmol/l MgCl* at pH 6.2 (buffered KCl), also as described by Gibbons and Etherden (1983). To disperse long chains, suspensions of all streptococcal strains were passed through a 27-gauge needle. All suspensions were then adjusted to 2 x 10’ cells/ml based on a stan- dard curve of optical density to bacterial cell number determined by microscopic counting.

Preparation of immobilized antimicrobial agent

OAIS was prepared by a spray-drying procedure (Fig. 1). Spherical silica particles (3.5 pm dia.) were suspended in water (16-17 w/v%) with 3-(trimethoxysilyl)-propyldimethyloctadecyl ammonium chloride (Polon MF-50, Shin-Etsu Chemical Co., Tokyo, Japan). The suspension was pulverized at 1.3 kg/cm2 pressure in a Pulvis Basic Unit GB-21 (Yamato Scientific, Tokyo, Japan) and dried at 140°C to condense the silanol group of the silica and the hydroxyl group of the octadecylpropyldimethyl ammonium chloride.

Bacterial adherence to OAIS

Aliquots of 500 pg of OAIS particles were incubated with 2.0 ml of various concentrations of [3H]- labelled bacterial suspensions in polyallomer tubes (Beckman Instruments, Palo Alto, CA, USA). The mixtures were continuously rotated (5 rev/min) at

OH

Q OCH, CH, Cl I I

Silica OH + H,CO - Si - (CH,), - N + C,,H,,

I I OH

OCH, CH,

spray-dry

CH, CI-

I

OH CH,

Fig. 1. Condensation of 3-(trimethoxysilyl)-propyldimethyloctadecyl ammonium chloride with silica in a spray-dry-

ing procedure.

room temperature for 60 min, then left to stand for 20 min to allow the OAIS particles to settle. To wash the OAIS particles, 1.0 ml of the supernatant containing unadsorbed bacteria was discarded, 1.0 ml of buffered KC1 added to the particles, and this repeated seven times. The OAIS particles were collected by centrifugation at 15,000 g for 15 min with 1.0% Tween 80 (Kant0 Chemical Co., Tokyo, Japan). The number of bacteria that had attached to the OAIS particles was determined in a scintil- lation counter, as described by Clark et al. (1978). All values were corrected for quenching due to the OAIS particles.

Calculations of parameters for bacterial adsorption to OAIS surface

Langmuir isotherm parameters for bacterial adherence to the OAIS particles were used to calcu- late the bonding strengths, as described by Gibbons et al. (1976). The equation employed was

C/Q = 1IKN f C/N,

where C is the concentration of free cells at equili- brium, and N and Q are the maximum number of binding sites and total number of cells adsorbed per unit of OAIS, respectively. K is the affinity between the cell and the OAIS surface. The value of K x N was used to express the strength of the adsorption bond.

Determination of hydrophobicity

To determine hydrophobicity, cells grown at 37°C for 18 hr in TodddHewitt broth were washed three times and suspended in PUM buffer (22.2 g K2HP04.3H20, 7.26 g KH2P04, 1.8 g urea, 0.2 MgS04.7H20 and distilled water to 1000 ml) (Rosenberg et al., 1980) to an optical density of 0.85 and 550 nm. Aliquots of 3.0ml of bacterial suspension were placed in Pyrex round-bottom test- tubes (15 x 130 mm) and 400 ~1 of n-hexadecane (Sigma Chemical Co., St Louis, MO, USA) added. Following a lo-min incubation at 30°C the tubes were mixed twice on a vortex mixer for 30 set each time with a 5-see interval between stirrings. After waiting 15 min to allow for complete partition of the layers, the light absorbance of the aqueous phase was measured at 550 nm. The values were expressed as the percentage of bacteria that remained in the n-hexadecane phase compared with control suspensions that were not incubated with n-hexadecane but were otherwise treated similarly.

Measurement of zeta-potential

Zeta-potential is the electrical potential of the interface between a solid (bacteria) and water. For bacteria, the electrical potential generally results from negatively charged surface molecules such as lipoteichoic acid. The cell-surface zeta-potential of each bacterial strain was determined to obtain in-

Bacterial adherence to immobilized agent 541

formation about the net surface charge. Cells grown at 37°C for 18’ hr in Todd-Hewitt broth were washed three times and suspended in buffered KC1 (pH 6.2) to an optical density of 0.5 at 550 nm. Suspensions of streptococcal strains were passed through a 27-gauge needle. The zeta-potential of these bacteria was then measured with a FACE zeta-potential meter ZPOM (Kyowa Interface Science, Tokyo, Japan) to microscopically determine the electrophoretic rate at 25 V.

Antimicrobial acti:vity of OAIS

Antimicrobial activity of OAIS was demonstrated against both Strc*p. mutans ATCC25175 and Strep. sobrinus 6715. Pa’rtions of 50-500 mg of OAIS particles were incubated with 5 x lo5 c.f.u/ml of bacterial suspension, with shaking (120 strokes/min) at 37°C for 10 min. The mixture was serially diluted with phosphate-bluffered saline (pH 6.8), and then 100 ~1 of the each diluted suspension was inocu- lated on BHI agar plates. The plates were anaerobically incubated at 37°C for a 4 days, and then colony numbers were counted. Antimicrobial activity was expres,sed as the amount of OAIS particles that reduced more than 99% of the visible colony present in a similarly prepared inoculum except that OAIS was not added. As a negative control, untreated silica particles were also used in testing antimicrobial activity. The assay was repeated at least three times.

Isolation of hydrophobic surface proteins

Hydrophobic ,surface proteins were prepared according to a method described by Wibawan et al. (1992). Streptococcal strains were grown at 37°C for 18 hr in Todd--Hewitt broth, washed three times in 0.01 mol/l sodl.um phosphate buffer at pH 6.8, resuspended in 10 ml of the buffer, and incubated with 100 U mutanolysin (Sigma) for 1 hr at 37°C. After centrifugation, the supernatant was applied to phenyl-Sepharose HP column (Pharmacia Bioteck, Uppsala, Swedenjl, washed with 0.01 mol/l sodium phosphate buffer, pH 6.8, containing 4 mol/l NaC1, and eluted with 61 mol/l guanidinium chloride. The hydrophobic proteins were dialysed against distilled water with Ultrafree C3LGC (Millipore, Tokyo, Japan). The eluate fraction was subjected to SDS- PAGE as described below. The isolated hydrophobic proteins were used for the OAIS adsorption assay. First, the isolated proteins were incubated with various concentrations of OAIS suspensions; the mixtures were continuously rotated (5 rev/min) at room temperature for 30 min. After incubation, the OAIS particles were collected by centrifugation and washed three times. The particles were then incubated with 6 mol/l guanidinium chloride and subjected to SDSPAGE. The total protein (Bradford, 1976) and sugar (Dubois et al., 1956) contents were determined by previously described methods.

SDS-PAGE

The slab-gel system was as described by Laemmli (1970). The isolated proteins and the proteins that had been detached from the OAIS particles were each suspended in 60 mmol/l Tris-HCl (pH 6.8) containing 2% SDS, 10% glycerol and 5% 2-mer- captoethanol and boiled for 3 min. Samples were then separated by one-dimensional SDS-PAGE using Multi Gel lo/20 (Daiichi Pure Chemical Co., Tokyo, Japan). Gels were stained with 2D- Silverstain. (Daiichi). Molecular-weight markers ranging from 6.5 to 200 kDa (BioRad Laboratories, Hercules, CA, USA) were also run on the gels.

Inhibition of bacterial adhesion to OAIS by cell- surface components

The isolated hydrophobic components- were used for assaying the inhibition of bacterial adhesion to OAIS. These particles were preincubated with 2-20 mg/ml of the hydrophobic proteins for 30 min at room temperature and subsequently used in the bacterial adherence assay.

RESULTS

Adherence and surface properties

The adherence activity, cell-surface hydrophobicity and zeta-potential of the tested strains are sum- marized in Table 1. Adherence was expressed at the value of K x N of the Langmuir isotherm. As negative control, untreated silica particles were also used in testing adherence activity and the results are shown in Table 1.

Adherence of bacteria to the OAIS surface appeared to parallel the hydrophobicity of each strain; the correlation coefficient was 0.602 (Fig. 2a). The highly hydrophobic Actinomyces strains, Strep. mitis, Strep. sanguis, Strep. oralis and Strep. parasanguis, were all strongly adherent to this material. Weakly hydrophobic strains of Strep. mutans adhered to OAIS less tightly.

The correlation between adherence activity and zeta-potential is shown in Fig. 2(b). The correlation coefficient in this case was 0.606. Figure 3 shows the adherence activity of three groups with differing hydrophobicity. The first group with high hydrophobicity included Strep. mitis ATCC98 11, Strep. oralis ATCC10557 and A. naeslundii ATCC12104 (Fig. 3a); the next group with less hydrophobicity included Strep. sanguis ATCC10556 and Strep. parasanguis ATCC15912 (Fig. 3b); and the least hydrophobic group consisted of Strep. mutans ATCC25175 and BHT (Fig. 3~). These data indicate that the adherence of strains with a high negative charge was reduced. No clear relation based on ionic interaction was found between OAIS and the anionic components of the bacterial surfaces.

542 T. Saito et al.

Table 1. Adherence to OAIS, hydrophobicity and zeta-potential of Streptococcus and Actinomyces strains

Strains Adherence activity Hydrophobicity (X f SD) Zeta-potential (mV k SD

S. mutans ATCC25175 S. mutnns Ingbritt S. mutans MT8148R S. mutans JC-2 S. sobrinus 67 15 S. rattus BHT S. sanguis ATCC10556 S. oralis ATCC10557 S. mitis ATCC9811 S. parasanguis ATCC15912 A. viscosus ATCC15987 A. naeslundii ATCC12104

1.87 (1.02)* 0.76 (0.19) 1.60 (0.44) 1.21 (0.28) 0.81 (0.37) 1.38 (0.11) 2.68 (0.60) 3.28 (0.79) 5.24 (0.28) 1.71 (0.42) 1.61 (0.46) 1.61 (0.23)

65.89 k 2.87 21.25 + 0.35 54.81+ 2.57 48.12 + 1.60 70.25 f 3.06 65.44 + 10.5 89.26 f 4.01 97.82 k 1.71 96.39 k 1.28 91.53 + 0.76 85.82 k 2.68 98.74 + 1.01

-75.52 +_ 13.60 -88.00 + 28.04 -84.47 _t 11.05 -51.50 + 3.23 -76.34 + 17.70 -95.44 + 30.50 -69.37 f 18.74 -58.13 + 11.22 -50.83 $ 14.72 -73.10+ 15.14 -72.51 + 16.92 -65.51 ) 18.52

*As a negative control, untreated silica particles were also used in testing adherence activity.

The findings indicate that the cell-surface hydrophobicity was the most important factor in the interaction between OAIS and these micro-organisms.

Antimicrobial activity of OAIS

OAIS particles (60 mg) reduced the viable cells of both Strep. mutans ATCC25175 and Strep. sobrinus 6715 by more than 99% over 10 min. Untreated silica as a negative control did not reduce either Strep. mutans ATCC25175 or Strep. sobrinus 6715 with any amount of silica tested.

Adherence of hydrophobic cell-surface components to OAIS

The cell-surface components of Strep. mutans Ingbritt and ATCC25175, and Strep. mitis ATCC9811, were solubilized by mutanolysin and 21.8, 68.9 and 362.7 pg/ml of proteins were isolated, respectively. Their hydrophobic components were subsequently isolated by chromatography on a phenyl-Sepharose column. These components contained numerous copies of identical proteins (Fig. 4). The amount of the extracted protein was 47.1 g/

A

.

. . .*

/

. . .

0 2.5 50 75 loo Cell surface hydrophobicity (%)

100 g dry wt and the amount of extracted hexose was 5.4 g/100 g dry wt. As shown in Fig. 4, adsorption of the extracted hydrophobic proteins to OAK particles was examined by SDSPAGE, and the types of hydrophobic proteins and their affinity to OAIS were found to vary from strain to strain.

Inhibition of bacterial adhesion to OAIS by cell- surface hydrophobic components

Competitive inhibition tests were carried out with each strain in the presence of homologous extracted cell-surface hydrophobic components or other cell- surface components. Preadsorption with the hydrophobic components inhibited bacteria-OAIS interactions by 17.3-63.9% (Table 2). On the other hand, although the other cell-surface components also inhibited the interaction, the extent of inhibition was not as high as that obtained with the hydrophobic proteins. Furthermore, cell-surface components that did not bind to the phenyl- Sepharose column, including hydrophilic-rich proteins and other components, exhibited little or no inhibition of bacteria-OAIS interactions (-7.33

I3

.

-100 -75 -50 -25 0 Zeta-potential (mv)

Fig. 2. Relation between: (a) cell-surface hydrophobicity of Streptococcus and Actinomyces strains and adherence activity (Y = 3.13 x IO-’ X-0.33, r = 0.602); and (b) cell-surface zeta-potential of

Streptococcus and Actinomyces strains and adherence activity (Y = 5.47 x IO-* X + 5.90, r = 0.606).


B 6-

5 -

4 -

S. sangti 10556 3 - (89.3).

C 6

5

-70 -60 -50 -75 -70 -65 -100 -90 -80 -70

Fig. 3. Relation between cell-surface hydrophobicity of Streptococcus strains and cell-surface zeta- potential of: (a) highly hydrophobic group 1; (b) less highly hydrophobic group 2; and (c) least hydro-

phobic group 3; as well as their adherence activity. Hydrophobicity values are shown in parentheses.

15.2%). Cell-surface hydrophobic components inhibited the adhesion of all strains tested. Inhibition of Strep. mutans Ingbritt adhesion to

OAIS by the hydrophobic proteins increased in a

dose-dependent manner (Fig. 5).

S. mutans

kDa 25175

I I E A

200

116.25 97.4

66.2

45.0

DISCUSSION

Bacterial adherence to several materials, including hydroxyapatite, epithelial cells and dental materials, has been extensively studied (Ofek and Doyle, 1994a; Naito and Gibbons, 1988). Electrostatic,

S. mutans S. m&is Ingbritt 9811

nn E A E A

Fig. 4. SDlj%PAGE of hydrophobic proteins. E, extracted surface hydrophobic proteins from Strep. mutans ATCC25175, Strep. mutans Ingbritt and Strep. mitis ATCC9811, respectively; A, the OAIS- adsorbed proteins from Strep. mutans ATCC25175, Strep. mutans Ingbritt and Strep. mitis ATCC9811,

respectively.

544 T. Saito et al.

Table 2. Inhibition of bacterial adhesion to OAIS by extracted cell-surface hydrophobic proteins

Inhibition (% &- SD)

Hydrophobic Whole surface Strains components* extracts*

S. mutans ATCC25 175 30.1 * 3.9 13.2 k 4.8 S. mutans Ingbritt 63.9 * 9.3 62.1 + 6.2 S. mutans MT8 148R 43.5 + 3.6 19.1 k 6.4 S. mitis ATCC9811 17.3 + 18.6 14.4 * 2.8 S. sanguis ATCC10556 58.9 L 9.2 44.3 * 1.1 S. oralis ATCC10557 39.1 + 11.3 23.8 k 9.3

*OAIS was incubated with 4 ng of surface proteins, and the bacterial adherence assay was carried out with 1 .O x 10’ cells/ml of bacterial suspension.

hydrophobic and complementary interactions have been proposed as factors in the attachment of bacteria to target surfaces (Naito et al., 1993; Gibbons, 1988; Ofek and Doyle, 1994b). Isquith et al. (1972) have noted interactions between several surface- bonded organosilicon quaternary ammonium chlor- ides and certain micro-organisms. The hydrolysis product of a quaternary amine-containing organosilicon salt, 3-(trimethoxysilyl)-propyldimethyloctadecyl ammonium chloride, exhibited antimicrobial activity against a broad range of micro-organisms when chemically bonded to a variety of substrates, including glass and cotton fabric (Walters et al., 1973). This antimicrobial activity might be attributed to phenomena involving the membrane or cell wall, such as membrane lysis, membrane enzyme in- activation or interference with ion transport. To confirm whether lysis had occurred during our experimental procedures, we incubated radiolabelled Strep. mutans MT8148R with OAIS for 60 min. The radioactivities of the suspension and the filtered solution (0.22 pm) were then determined in a scintil- lation counter, and almost no radioactivity was detected in the filtrate (data not shown). This result

E 100 g 90

S 80 ? 70 ‘3 m a 60 a 50 z 40 = Y 30 2 z

20 8 10 z P-l 0

I 0 12 3 4 5 6 7 8 9

Added hydrophobic protein (fl g)

Fig. 5. Effect of extracted hydrophobic proteins on Strep. mutans Ingbritt adhesion to OAK

indicated that membrane lysis had not occurred during the experimental procedure. Our findings indicate that the adherence of several oral bacteria to OAIS was due to cell-surface hydrophobicity. Highly hydrophobic bacterial strains of Strep. sanguis, Strep. oralis, and A. viscosus were all found to strongly adhere to OAIS, whereas less hydrophobic Strep. mutans strains adhered less strongly. Satou et al. (1988) examined bacterial adherence mechanisms using glass slides with various functional groups, such as amino, carboxyl, hydroxyl or hydrophobic groups, attached to their surfaces through covalent bonds between their carbons and the silicon of the glass. They concluded that adherence to the glass was related to the physico-chemical surface properties of individual strains; hydrophobic bonding seemed to be the most important factor for Strep. sanguis ATCC10556, whereas ionic interactions made the highest contribution to adhesion by Strep. mutans 0MZ176.

We show that a negative charge on the bacterial surface affected interactions with OAIS as well as hydrophobicity. However, contrary to our predic- tion, this negative charge seemed to inhibit adherence. When the zeta-potential of OAIS was evaluated, we found that the material used had a negative charge (data not shown). It is probable that the total amount of octadecylpropyldimethyl ammonium chloride immobilized on the silica was not sufficient to change the negative charge imparted by the silanol group of the silica to a net positive charge. There is apparently a negative correlation between bactericidal activity of OAIS and cell-surface hydrophobicity (Takatsuka and Mori, 1994); we found a positive correlation between the adherence ability of OAIS and cell-surface hydrophobicity. This discrepancy may come from the difference in the quantity of immobilized antimicrobial agent on silica. Takatsuka and Mori (1994) used 1.4 pmol of the antimicrobial agent per 1 g of silica in their OAIS. We used OAIS that consisted of 0.005 pmol of the antimicrobial agent per 1 g of silica. Because preliminary experiments revealed that adherence properties were affected by the quantity of the immobilized antimicrobial agent (data not shown), another factor, such as electrostatic force, rather than hydrophobicity, may affect both adherence properties and bactericidal activity.

Comparing OAIS adherence activities of strains that are similarly hydrophobic allowed us to exclude the effect of hydrophobicity on adherence. The data indicate that the strength of the negative charge is related to inhibition of adherence of OAIS (Fig. 3). From the present data, it is not clear which of the two properties, hydrophobicity or zeta-potential, contributes more to bacteria-OAIS interactions. The extent of the contribution may vary among bacterial strains. Hydrophobic proteins on the cells may contribute to overall surface


hydrophobicity (Jenkinson, 1986; McNab and Jenkinson, 1992).

Analysis of the extracted cell-surface components by chromatography on phenyllsepharose revealed that the component-rich fraction contained 47.1 g of protein and 5.4 g of sugar per 100 g dry wt. SDS-PAGE showed that the OAIS-adsorbed, cell- surface hydrophobic proteins had a wide range of molecular weight. Interactions between OAIS and these hydropho‘bic proteins did not appear to be due to any specific reaction. This non-specificity and the wide range of adherence activity of these proteins indicate that OAIS might adsorb many other micro-organisms with high hydrophobicity. A competitive assay revealed that the cell-surface components inhibited bacterial adherence to this material. The hydrophobic components in particular exhibited a strong inhibitory effect on adherence to OAIS that had adsorbed hydrophobic proteins, indicating that these types of proteins are major factor in adherence.

In conclusion, interactions between bacteria and OAIS were due to hydrophobicity. Moreover, although the eflect on adherence of other hydrophobic components on the cell surface, such as lipids, was not investigated, the role of surface hydrophobic proteins seems to be a major one. The mouth is populated by several hundred different kinds of bacteria, fungi and protozoa which together compris,e a microbial ecosystem. It is also known that micro-organisms which invade estab- lished plaque masses are often capable of causing dental disease (Schonfeld, 1992). Discouraging the build-up of excessive plaque is therefore advan- tageous in preventing various forms of dental disease such as dental caries. We suggest that using OAIS in a toothpaste preparation might help exclude various bacterial species from tooth surfaces.

Acknowledgements--We thank Y. Saeki for his suggestions on technical procedures.

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Adherence of oral streptococci to an immobilized antimicrobial agent