The Influence of Biosurfactants Released by S. mitis BMS on the Adhesion of Pioneer Strains and Cariogenic Bacteria

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1 Biofouling, December 2004 VOL 20 (6), pp The Influence of Biosurfactants Released by S. mitis BMS on the Adhesion of Pioneer Strains and Cariogenic Bacteria CHRIS G VAN HOOGMOED*, HENNY C VAN DER MEI and HENK J BUSSCHER Department of Biomedical Engineering, University of Groningen, Groningen, The Netherlands (Received 21 July 2004; in final form 11 November 2004) The influence of Streptococcus mitis BMS biosurfactants on the adhesion of eight pioneer and four cariogenic oral bacterial strains was, for a first screening, examined in a microtiter plate assay. The adhesion to pellicle-coated wells of three cariogenic strains was inhibited 4 70% by the biosurfactants, while only one pioneer strain showed 4 70% reduction. The reduction for the other strains did not exceed 50%. Subsequently, adhesion of Streptococcus mutans ATCC and Streptococcus sobrinus HG 1025, both cariogenic strains, and Actinomyces naeslundii T14V-J1 and Streptococcus oralis J22, two pioneer strains, to biosurfactants-coated enamel with and without a salivary pellicle was studied in a parallel plate flow chamber. A biosurfactants coating to enamel with or without a pellicle caused a reduction in the number of adhering cariogenic organisms, although no such reduction was observed for the pioneer strains. Consequently, it is concluded that S. mitis BMS biosurfactants may play a protective role against adhesion of cariogenic bacteria. Keywords: enamel; microorganisms; adhesion; biosurfactants INTRODUCTION Microbial populations colonize different habitats in man, such as the nasal and oropharyngeal cavities, the digestive tract (including the esophagus, the stomach, and the small and large intestines), the vagina, and the skin. Mostly, these populations live in a harmonious relationship with the host and both parties benefit from the association. In the oral cavity, hard and soft tissues are available for colonization. The formation of the microbial community associated with the teeth, known as dental plaque, starts immediately after tooth cleaning with the adsorption of salivary proteins, i.e., the acquired enamel pellicle (Socransky et al., 1977; Busscher et al., 1989). Within 2 h after cleaning, Actinomyces spp., coccal bacteria like Neisseria, and streptococci (predominantly Streptococcus mitis, Streptococcus sanguis and Streptococcus oralis), attach to the enamel pellicle (Theilade et al., 1982; Nyvad & Kilian, 1987; Busscher et al., 1989; Nyvad & Kilian, 1990). These members of the pioneer population and their metabolic products form a base to which their progeny and secondary colonizers adhere (Syed & Loesche, 1978). An important function of the indigenous dental flora is to prevent colonization by exogenous pathogens. To this end, indigenous bacteria rapidly attach to salivary ligands and receptors, compete for nutrients present in the oral cavity, change the local circumstances such as ph and redox potential, and produce inhibitory substances, such as H 2 O 2, bacteriocins or acidic products (Marsh & Martin, 1999). Previous studies from the authors laboratory have shown that S. mitis BMS, a pioneer strain, inhibits adhesion of Streptococcus mutans NS to glass (Pratt- Terpstra et al., 1989). S. mutans and also Streptococcus sobrinus strains both belong to the most cariogenic group of oral bacteria, namely the mutans streptococci (Loesche et al., 1975; Loesche, 1986). A sequel study showed that the presence of biosurfactants releasing S. mitis BMS and S. mitis BA or isolated S. mitis biosurfactants reduced the adhesion of S. mutans NS to glass with and without a salivary conditioning film (Van Hoogmoed et al., 2000). This suggests a role for S. mitis BMS in the prevention of dental caries, requiring further exploration. *Corresponding author; fax: ; C.G.van.Hoogmoed@med.rug.nl ISSN print/issn online # 2004 Taylor & Francis Ltd DOI: /

2 262 C G VAN HOOGMOED et al. Oral bacteria on surfaces in the oral cavity are subject to a number of different shear-off forces as, for instance, those arising from salivary flow. The influence of flow on adhesion of oral bacteria can be studied in vitro using a parallel plate flow chamber with in situ observation and image analysis options (Sjollema et al., 1989). Moreover, the parallel plate flow chamber allows control of mass transport conditions and air-liquid interface passages are avoided, yielding accurate enumeration of the number of bacteria that attach under the flow conditions of an experiment. Salivary pellicles can be easily applied and anti-adhesives can be introduced before or during an experiment (Busscher & Van der Mei, 1995). The drawback, however, to the use of the parallel plate flow chamber is that it is time-consuming, which makes the device unsuitable for screening of a large number of bacterial strains. The aim of this study was to examine the influence of biosurfactants released by S. mitis BMS on the adhesion of a large number of pioneer and cariogenic strains. For a first screening, a dose-response study on the effects of biosurfactants on adhesion of 12 oral bacterial strains to pellicles was determined in a microtiter plate assay under static conditions. Subsequently, adhesion of two cariogenic and two pioneer strains, to biosurfactant-coated enamel with and without a salivary pellicle was studied in a parallel plate flow chamber. MATERIAL AND METHODS Microorganisms Actinomyces naeslundii T14V-J1, A. naeslundii PK 333 and A. naeslundii PK 213 were precultured from a frozen stock in a 10 ml batch culture of Schaedler s broth supplemented with 0.01 g l 71 hemin for 24 h at 378C in 10% H 2, 80% N 2, 10% CO 2. S. oralis 34, S. oralis J22, S. oralis ATCC 35037, S. sanguis PK 1889, S. sanguis ATCC 10556, S. sobrinus ATCC 33478, S. sobrinus HG 1025, S. mutans ATCC and S. mutans NS, were precultured from a frozen stock in a 10 ml Todd Hewitt Broth (THB) (Oxoid, Basingstoke, England) batch culture for 24 h at 378C in ambient air. S. mitis BMS, originally isolated from the human oral cavity, was precultured from a frozen stock in a 10 ml THB batch culture supplemented with 0.5% (w/v) sucrose for 24 h at 378C in ambient air. With the exception of S. mitis BMS, all precultures were used to inoculate 200 ml cultures of the appropriate broth. All bacterial strains were harvested in their stationary phase by centrifugation at 4000 g, washed twice with adhesion buffer (2 mm potassium phosphate, 50 mm potassium chloride and 1 mm calcium chloride, ph 6.8) and resuspended in adhesion buffer. To break bacterial chains, the bacterial suspensions were sonicated for s at 30 W (Vibra Cell model 375, Sonics and Materials Incorporated, Danbury, CT, USA). Sonication was done intermittently while cooling in an ice/water bath. The cell concentrations were fixed with the aid of a Bürker-Türk counting chamber to ml 71 adhesion buffer for bacterial adhesion to microtiter plates and to ml 71 for parallel plate flow chamber experiments. The S. mitis BMS preculture was used to inoculate 1400 ml of a corresponding second culture. After growth for 18 h, centrifugation at 4000 g and washing twice with adhesion buffer, the S. mitis BMS cells were resuspended in 200 ml demineralized water. Biosurfactants Release Crude biosurfactants were produced by gently stirring the S. mitis BMS suspension for 2 h at room temperature. Subsequently, the organisms were separated from the released biosurfactants by centrifugation at 10,000 g for 10 min. To ensure complete removal of all bacterial remnants, the supernatant was centrifuged again at 10,000 g for 10 min. After the final centrifugation, the crude biosurfactants were freeze-dried and stored at 208C. The collected freeze-dried crude biosurfactants samples were pooled and then resuspended in demineralized water. Subsequently, a purification step by acid precipitation was carried out using concentrated HCl down to ph 2.0. After decanting the supernatant, the precipitate was washed twice with acidic water (ph 2) and collected by centrifugation at 4000 g. After redissolving in water, the acid precipitate was freeze-dried and stored at 208C until use. Adhesion Assays Microtiter plate assay The anti-adhesive activity of S. mitis BMS biosurfactants (1500, 250, and mg purified biosurfactants per ml adhesion buffer) was evaluated in a polystyrene microtiter plate (Cellstar 1 Microplaten, TC, Greiner) with and without a salivary conditioning film. In order to create a salivary conditioning film, wells were filled with 200 ml of reconstituted human whole saliva (for preparation, see below) for a 2 h exposure period. Thereafter, wells were washed three times with adhesion buffer. Wells with and without a salivary conditioning film then were filled with 50 ml of the biosurfactants solution, left for 18 h at 48C, and subsequently washed three times with adhesion buffer. Control wells contained only adhesion buffer. After washing the microtiter plate wells, 200 ml of

3 BIOSURFACTANTS AND ORAL BACTERIAL ADHESION 263 washed suspensions of A. naeslundii T14V-J1, A. naeslundii PK 333, A. naeslundii PK 213, S. oralis 34, S. oralis J22, S. oralis ATCC 35037, S. sanguis PK 1889, S. sanguis ATCC 10556, S. sobrinus ATCC 33478, S. sobrinus HG 1025, S. mutans ATCC and S. mutans NS were added and incubated in the wells for 4 h at room temperature. Then, the wells were washed three times with adhesion buffer and the bacteria retained in the wells were stained with crystal violet, after which optical density readings were taken at 595 nm (Biorad, Microplate Reader, Model 3550-UV). The changes in optical densities, between the biosurfactant-coated wells and the control well, were used to calculate the percentage inhibition of adhesion for each strain and biosurfactants concentration. This procedure was done in duplicate with separately cultured microorganisms. Parallel Plate Flow Chamber In situ observation of bacterial adhesion to enamel was done in a parallel plate flow chamber. The flow chamber (dimensions: l x w x h = 7.6 x 3.8 x 0.06 cm) and image analysis system have been described in detail elsewhere (Sjollema et al., 1989). The bottom and top plates (5.8 x 3.8 cm) were made of Perspex 1 and glass, respectively. Both materials were cleaned by sonication for 5 min in 0.5% (v/v) RBS 35 (Societé des Traitements Chimiques de Surface, Lambersat, France), rinsed thoroughly with tap water, dipped in methanol, and rinsed with demineralized water. For the experiments, enamel chips (for preparation, see below) were affixed to the Perspex 1 bottom plate of the flow chamber by a small droplet of nail polish. For samples selected to have a salivary pellicle, a droplet of reconstituted human whole saliva (see below) was placed on the enamel chips at room temperature. After 2 h, the saliva was aspirated carefully and the enamel was dipped once in adhesion buffer. When a biosurfactants coating was required on bare or pellicle-coated enamel, a droplet of biosurfactants solution (1.5 mg ml 71 in adhesion buffer) was placed on the enamel overnight. Subsequently, biosurfactants were aspirated and the enamel dipped once in adhesion buffer. Following salivary protein and/or biosurfactants adsorption, the flow chamber was assembled and mounted on the stage of a phase contrast microscope. An image analysis system connected to the microscope allowed the monitoring of the bacterial deposition in real time. Flasks, containing a bacterial suspension and buffer, were connected with the flow chamber and placed at a height above the flow chamber to create a flow of ml s 71, well within the limits of laminar flow, and corresponding with a moderate salivary flow at a shear rate of 10 s 71 (Dawes et al., 1989). For the first 15 min, only adhesion buffer flowed through the chamber, to remove remnants of saliva and biosurfactants from the enamel chips. Thereafter, flow was switched to a bacterial suspension. Using the image analysis software, images were taken from the enamel chips at defined time points during the 4 h flow period for enumeration of the number of attached bacteria. The initial increase in the number of bacteria over time, was expressed in a so-called initial deposition rate (cm 72 s 71 ), i.e., the number of organisms initially attached per unit area and time. The number of bacteria attached per unit area after 4 h (cm 72 ), was taken as an estimate of bacterial adhesion in a more advanced state of the adhesion process. All flow chamber experiments were done at room temperature, in triplicate, with separately cultured organisms. Contact Angle Measurements Water contact angles were measured using the sessile drop technique on enamel and polystyrene surfaces with and without a salivary pellicle film, and in the absence and presence of adsorbed S. mitis BMS biosurfactants. Enamel surfaces were prepared by grinding and polishing the labial surfaces of bovine incisors (for details see below). Polystyrene surfaces were cut from the bottom of Petri dishes (Greiner). The coating procedures for saliva and biosurfactants were done as described above. Subsequently, coated and uncoated surfaces were left to air dry in ambient air until so-called plateau contact angles could be measured. Enamel Preparation Enamel chips were prepared from bovine dental incisors. The labial surface of an incisor was ground under running tap water with abrasive paper (1200 grit) and polished with Al 2 O 3 (successive particle diameters 5, 1 and 0.05 mm). The polished enamel surface was cleaned in an ultrasonic bath filled with demineralized water for min. Then, the polished enamel surface was cut off the tooth and, subsequently, the back of the enamel surface was ground with abrasive paper (1200 grit) to a thickness of mm and polished with a slurry of Al 2 O 3,as described above. After polishing, the enamel chip was cleaned ultrasonically for a few seconds. Saliva Human whole saliva from healthy volunteers of both sexes was collected into ice-chilled cups after stimulation of the salivary flow by chewing Parafilm 1 at least 1.5 h after eating, drinking, or tooth cleaning. After the saliva was pooled and centrifuged at 12,000 g for 15 min at 48C, phenylmethylsulfonylfluoride was added to a final concentration of 1 mm

4 264 C G VAN HOOGMOED et al. as a protease inhibitor. The solution was again centrifuged, dialyzed (MW cutoff 6,000 8,000, Spectra/por, Spectrum Medical Industries Incorporated, LA, California, USA) for 48 h at 48C against demineralized water, and freeze-dried for storage. Reconstituted, human whole saliva was prepared from the lyophilized stock by dissolution of 1.5 mg ml 71 in adhesion buffer, a buffer approximating the ionic composition of saliva (Gibbons & Etherden, 1985). RESULTS Figure 1 shows the percentage reduction in bacterial adhesion to polystyrene wells coated with S. mitis BMS biosurfactants in the absence of a salivary pellicle, as adsorbed from solutions with different biosurfactants concentrations. For most strains, inhibition varied from 80% to almost 100% and inhibition was less, ranging from 40% to 70%, for the A. naeslundii strains, S. sanguis PK 1889, and S. mutans NS. The presence of a salivary conditioning film on the polystyrene wells in the absence of adsorbed biosurfactants reduced the adhesion of the strains compared to bare polystyrene (data not shown), except for S. oralis ATCC The reduction in adhesion by S. mitis BMS biosurfactants adsorbed to pelliclecoated polystyrene wells is presented in Figure 2 and shows more diverse effects. For the highest biosurfactant concentration (1500 mg ml 71 ), adhesion of A. naeslundii PK 333, S. sobrinus ATCC and S. mutans NS was totally inhibited and for S. sobrinus HG % reduction was seen. Adhesion reduction was 5 50% for all other strains and, in some cases at lower biosurfactant concentrations, a small increase in adhesion was observed. Based on the results obtained in the microtiter plates, it was decided to apply a 1500 mg ml 71 biosurfactants solution to coat the enamel chips for the parallel plate flow chamber experiments. The resulting reductions in adhesion are presented in Figure 3 for the pioneer strains S. oralis J22, and A. naeslundii T14V-J1 and the cariogenic strains S. mutans ATCC and S. sobrinus HG No reductions in initial deposition rates (Figure 3A) and numbers of bacteria retained after 4 h (Figure 3B) were found for the pioneer strains, but a biosurfactants coating on bare enamel caused a reduction in adhesion of the cariogenic strains. In the presence of a salivary pellicle, however, the S. mitis BMS biosurfactants had a substantial effect on the adhesion of S. sobrinus HG 1025 to enamel, while the effect of the biosurfactants coating on the adhesion of S. mutans ATCC to saliva-conditioned enamel was small. The effects of adsorbed S. mitis BMS biosurfactants on the contact angles of water on bare and salivacoated enamel and polystyrene are presented in Table I. Adsorption of biosurfactants hardly affected FIGURE 1 The percentage reduction in adhesion of different oral bacterial strains by S. mitis BMS biosurfactants, as carried out in polystyrene microtiter wells. All percentages are expressed relative to adhesion of the organisms to bare polystyrene wells, in the absence of adsorbed biosurfactants. Biosurfactants were adsorbed from solutions with concentrations of (&), 250 ( ) and 1500 ( ) mg ml 71 ; nd = not detected. Results are averages of duplicate experiments with separately grown cultures, varying within 15%. FIGURE 2 The percentage reduction in adhesion of different oral bacterial strains by S. mitis BMS biosurfactants, as carried out in polystyrene microtiter wells in the presence of a salivary pellicle. All percentages are expressed relative to adhesion of the organisms to pellicle-coated wells, in the absence of adsorbed biosurfactants. Biosurfactants were adsorbed from solutions with concentrations of (&), 250 ( ) and 1500 ( ) mg ml 71. Results are averages of duplicate experiments with separately grown cultures varying within 10%.

5 BIOSURFACTANTS AND ORAL BACTERIAL ADHESION 265 the hydrophobicity of bare enamel and polystyrene, as water contact angles averaged 40 and 708, respectively, both before and after biosurfactant adsorption. However, contact angles of water on saliva-coated enamel and polystyrene increased as a result of adsorption of biosurfactants (from 32 and 248 to 53 and 488, respectively). FIGURE 3 Initial deposition rates (a) and numbers of bacteria retained after 4h (b) for different oral bacterial strains on enamel with and without a salivary pellicle and in the presence and absence of adsorbed S. mitis BMS biosurfactants. & = bare enamel; = biosurfactants-coated enamel; = saliva-coated enamel; = saliva + biosurfactants-coated enamel; bars represent the SD over three experiments with separately cultured bacteria. TABLE I Water contact angles (y w ) on enamel and polystyrene with and without a pellicle in the presence and absence of a S. mitis BMS biosurfactants coating. All data are the results of at least three droplets placed over different substratum surfaces, yielding an average SD of approximately 28 y w (degrees) Coating Enamel Polystyrene No coating Biosurfactants Pellicle Pellicle + Biosurfactants DISCUSSION Biosurfactants are, in the majority of cases, released by microorganisms. The main role described for biosurfactants is the solubilization of hydrocarbons for nutrient uptake and utilization. Recently, however, more and more biosurfactants have been described that are released by microorganisms and adsorbed to a surface to negatively influence the adhesion of other competing microbial strains to the same surface. Also, S. mitis BMS has been characterized as a biosurfactants-releasing strain (Van der Vegt et al., 1991). Crude S. mitis BMS biosurfactants dissolved in water (1 mg ml 71 ) reduce the surface tension of an aqueous solution to approximately 48 mj m 72. Purification of the crude biosurfactants has resulted in extremely surface active biosurfactants compared with the crude biosurfactants. An aqueous solution of the purified biosurfactants containing 1mgml 71 has a surface tension of 35 mj m 72. Chemical characterization revealed that the protein content of the biosurfactants is extremely low ( 5 1%) and that the main component is glycolipid (Van Hoogmoed et al., 2000). Due to the release of these biosurfactants, S. mitis BMS can interfere in the adhesion of the cariogenic S. mutans NS to glass in the presence and absence of a salivary conditioning film (Van Hoogmoed et al., 2000). In the present investigation, the study of S. mitis BMS biosurfactants was extended to 12 oral bacterial strains, eight pioneer and four cariogenic strains (mutans streptococci). It should be noted, however, that pioneer strains also can be acidogenic and aciduric and, as a consequence, they contribute to the development of caries by developing suitable growth conditions (De Soet et al., 2000) for the more acidogenic and aciduric mutans streptococci (Loesche, 1986). Bacterial adhesion to the saliva-coated and bare enamel was reduced for the cariogenic strains S. mutans ATCC and S. sobrinus HG 1025 as a result of the S. mitis BMS biosurfactants coating, although no such reduction was seen for the pioneer strains S. oralis J22 and A. naeslundii T14V-J1 (Figure 3). Examples of other biosurfactants-releasing strains are Streptococcus thermophilus (Busscher et al., 1994) and Lactobacillus (Velraeds et al., 1996). S. thermophilus negatively interferes with the adhesion of yeast to silicone rubber voice prostheses (Busscher et al., 1997) while lactobacilli hinder the adhesion of uropathogens (Velraeds et al., 1996). This study demonstrated that adsorbed S. mitis BMS biosurfactants can influence the adhesion of most pioneer and cariogenic strains to polystyrene wells in the presence and absence of a pellicle under static conditions, while biosurfactants on bare and pellicle-coated enamel only showed decreased adhesion of cariogenic strains when studied in a parallel plate flow chamber.

6 266 C G VAN HOOGMOED et al. In many cases, bacterial adhesion to dental enamel in vitro has been studied under static conditions (Weerkamp et al., 1988; Gurgan et al., 1997; Steinberg et al., 2003). In the present study, the use of thin enamel slabs and the parallel plate flow chamber was combined, a method which has hitherto only been used incidentally (Landa et al., 1996; Van der Mei et al., 2002; Decker et al., 2003a; 2003b). This combination offers the possibility of studying the adhesion of oral bacteria to enamel with in situ observation under dynamic conditions. The use of enamel as a substratum is not only relevant for understanding bacterial interactions with hydroxyapatite, the predominant crystalline component in tooth enamel, but also pellicle-bacteria interactions are more factual, as the composition of the pellicle is determined by the physical and chemical nature of the solid (Fine et al., 1984; Ruan et al., 1986). As a drawback, it is difficult and time-consuming to fabricate enamel slabs, sufficiently large and thin for use in the flow chamber. For this reason, a first screening of the effects of adsorbed biosurfactants was done in a microtiter assay and only a selected number of strains were studied in the parallel plate flow chamber. The major organic constituents of saliva are proteins and glycoproteins. In the experiments reported here, these proteins and glycoproteins with different molecular compositions adsorbed to the enamel and polystyrene surfaces to form a salivary conditioning film. When bare and saliva-conditioned enamel and polystyrene surfaces are coated with biosurfactants, their surface properties change. More specifically, because of their amphiphatic character, adsorbed biosurfactants can invert hydrophobic domains into hydrophilic ones and vice versa (Neu, 1996) with an impact on bacterial adhesion. In this study, S. mitis BMS biosurfactants hardly changed the hydrophobicity of bare enamel and polystyrene, but the hydrophobicity of saliva-coated enamel and polystyrene increased upon application of S. mitis BMS biosurfactants (see Table I). However, the ability of the S. mitis BMS biosurfactants to reduce bacterial adhesion was most pronounced on bare enamel and polystyrene when compared to saliva-coated enamel and polystyrene surfaces, despite the fact that biosurfactants adsorption did not influence their hydrophobicity. In contrast, for pellicle-coated surfaces, a decrease in the adhesion of cariogenic strains was concurrent with an increase in surface hydrophobicity. Nevertheless, it is proposed that the reduction in bacterial adhesion by S. mitis BMS biosurfactants was not brought about solely by a change of the hydrophobicity of the substratum surfaces. Other mechanisms due to the S. mitis BMS biosurfactants coating, such as a reduction in the number of receptors for bacterial adhesion and/or electrostatic repulsion, probably actively contribute to the suppression of bacterial adhesion. It is important to notice that, besides S. mitis BMS, other S. mitis strains also are known to release biosurfactants with a possible potential to interfere with adhesion of pathogenic mutans streptococci (Van Hoogmoed et al., 2000). The adhesion of mutans streptococci to the tooth surface is an essential step in the development of dental caries. It has been pointed out that a reduction or exclusion of these bacteria at the tooth surface could be beneficial in the prevention of dental caries (Loesche, 1986; Saeki et al., 1996; Ooshima et al., 2000). In the absence of animal model data, prudence is required for the interpretation of the in vivo significance of the observations presented here. 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