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Influence of Biosurfactant on Interactive Forces between Mutans Streptococci and Enamel Measured by Atomic Force MicroscopyDepartment of Biomedical Engineering, University Medical Centre Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands Correspondence: * corresponding author, C.G.van.Hoogmoed{at}med.rug.nl
Although interactive forces, influenced by environmental conditions, between oral bacteria and tooth surfaces are important for the development of plaque, they have never been estimated. It is hypothesized that interactive forces, as measured by atomic force microscopy, between enamel with or without a pellicle and two strains of mutans streptococci become less attractive by the application of a Streptococcus mitis BMS biosurfactant coating. Upon approach of each of the strains toward bare and pellicle-coated enamel, adsorbed biosurfactant increased the range of the repulsive forces. Upon retraction of the enamel surface, small adhesion forces (0.8–0.9 nN) were measured for bare enamel that almost disappeared after biosurfactant coating. The prevalence and magnitude of the adhesion forces also decreased upon pellicle-coating of the enamel, with a minor effect of adsorbed biosurfactant. These findings indicate that adsorbed S. mitis BMS biosurfactant changes the interactive forces between the mutans streptococci studied and enamel, explaining the effects of biosurfactant on adhesion.
Key Words: enamel • pellicle • biosurfactant • microbial adhesion forces • atomic force microscopy
Dental plaque can develop and maintain its position on the tooth surface only if the interactive forces between the organisms and the tooth surface are sufficiently strong to withstand oral shear forces. The interactive energies by which micro-organisms adhere to surfaces have been only roughly estimated and reportedly range from 2 kT up to a few tens of kT (Rutter and Vincent, 1980; Van Loosdrecht et al., 1989; Rijnaarts et al., 1993), but with regard to the interactive forces between oral bacteria and the acquired enamel pellicle, not even rough estimates exist. Interactive forces involved in bacterial adhesion to surfaces can be directly measured through atomic force microscopy (AFM). Hitherto, these studies have been carried out to measure interactive forces between the silicon nitride tip of the AFM and different microbial strains (Fang et al., 2000; Abu-Lail and Camesano, 2003), or between an AFM cantilever coated with a confluent layer of bacteria and surfaces (Ong et al., 1999; Lower et al., 2000). The interactive forces between oral bacteria and dental enamel have not yet been measured by AFM, but can be measured by the mechanical trapping of bacteria in membrane filters, and by the gluing of a small enamel particle to the AFM cantilever. Thus, the enamel particle acts as the AFM tip, yielding a relatively poor image of the bacterium under study, followed by accurate measurement of the interactive forces. In the oral cavity, these interactive forces are influenced by a variety of environmental conditions, such as the presence of saliva, oral detergents, or biosurfactants released by the indigenous oral flora interfering with the adhesion of competitor strains. Streptococcus mitis biosurfactant, for instance, is known to interfere with the adhesion of cariogenic Streptococcus mutans (Van Hoogmoed et al., 2000) and Streptococcus sobrinus strains, although biosurfactant effects on S. sobrinus adhesion were more pronounced than those on S. mutans adhesion (Van Hoogmoed et al., 2004). Chemical characterization of the S. mitis biosurfactant revealed that the active component is glycolipid-like (Van Hoogmoed et al., 2000). The aim of the present work was, first, to measure the interactive forces between enamel with and without a salivary pelicle and S. sobrinus HG 1025 and S. mutans ATCC 25175 and, second, to determine whether S. mitis biosurfactant affected the interactive forces between the two mutans streptococcal strains and the enamel.
Bacterial Strains, Growth Media, and Biosurfactant Collection S. sobrinus HG1025, S. mutans ATCC 25175, and S. mitis BMS were cultured aerobically from a blood agar plate in 10 mL Todd-Hewitt Broth (THB, Oxoid, Basingstoke, UK) for 24 hrs at 37°C. For the S. mitis BMS, the THB was supplemented with 0.5% sucrose. The mutans streptococcal pre-cultures were used to inoculate second 10-mL cultures, which were grown for 16 hrs. Subsequently, these suspensions were centrifuged at 4000 g, washed twice with adhesion buffer (2 mmol/L potassium phosphate, 50 mmol/L potassium chloride, 1 mmol/L calcium chloride; pH 6.8), and re-suspended in adhesion buffer to a concentration of 105 cells per mL. We used the S. mitis BMS pre-culture to inoculate a 1400-mL culture. After 18 hrs of growth, cells were harvested by centrifugation at 4000 g, washed twice with adhesion buffer, and re-suspended in 200 mL demineralized water. Crude biosurfactant was produced by gentle stirring of the S. mitis BMS suspension for 2 hrs at room temperature. Subsequently, the organisms and the biosurfactant released were separated by centrifugation at 10,000 g. To ensure complete removal of all bacteria, we centrifuged the supernatant twice at 10,000 g. The crude biosurfactant was re-suspended in demineralized water and purified by acid precipitation with concentrated HCl down to pH 2.0. After the supernatant was decanted, the precipitate was washed twice with acidic water (pH 2) and collected by centrifugation at 4000 g. After being redissolved in water, the acid precipitate was freeze-dried and stored at –20°C.
Preparation of Enamel Particles
Saliva
Atomic Force Microscopy (AFM) We used a Nanoscope III AFM (Digital Instruments, Santa Barbara, CA, USA), operating in the contact mode, to measure interactive forces. A silicon nitride V-shaped cantilever (Veeco Instruments, Inc., Woodbury, NY, USA), with a probe curvature of ~ 50 nm, was used for measurements at room temperature in Millipore water. We experimentally determined spring constants by measuring the resonance frequency of each tip used, from which the spring constant could be calculated according to
where k is the spring constant, f the true resonance frequency, and a is a proportionality constant provided by Veeco. Spring constants measured were similar to those provided by the manufacturer (0.06 Nm–1) and were assumed not to be influenced by the attachment of an enamel particle. Force-distance curves were subsequently taken with a z-displacement of 2000 nm and a scan rate of 1.99 Hz. Integral and proportional gains of the feedback loop were about 2 and 3, respectively. The slopes of the retraction force curves in the region where probe and sample are in contact were used to convert the voltage into a cantilever deflection. The conversion of deflection into force was carried out as has been previously described (Dufrêne et al., 2001). The point of zero separation was defined as the onset of the constant-compliance regime for the retraction curve. When the interacting surfaces approach one another, an electrosteric repulsive force F is always generated (Vadillo-Rodríguez et al., 2004); it decays exponentially with distance D according to
where F0 is the repulsive force at zero separation distance, and Results were presented as an average of 50 force-distance curves, taken over 5 different organisms on 10 randomly selected locations per organism, and including 4 different enamel particles.
In their interaction with streptococcal surfaces, enamel particles attached to a silicon nitride AFM cantilever present a surface with clear crystalline features (Fig. 1  ). Note that the enamel surface for interaction is large enough to be classified as macroscopic in comparison with streptococci.
Upon approach of the enamel particles toward each of the mutans streptococcal strains, a repulsive force was observed (Fig. 2 ). For bare enamel, the repulsive force at contact (Fig. 2A; Table) was significantly (p < 0.01, Student’s t test) larger for S. sobrinus (3.9 nN) than for S. mutans (0.9 nN). For both bacteria, this repulsive force decayed rapidly within 30–53 nm. After biosurfactant-coating of bare enamel particles, the range and magnitude of this repulsive force for S. sobrinus increased significantly, with p < 0.01 (Fig. 2B; Table). A biosurfactant coating on bare enamel also significantly (p < 0.01, Student’s t test) increased the range of the repulsive force for the S. mutans, although the magnitude of the repulsive force at contact was not affected (Table). The repulsive force at contact for S. sobrinus (4.6 nN) and S. mutans (5.5 nN) with pellicle-coated enamel was similar (Fig. 2C; Table), although ranging over significantly (p < 0.01) longer distances for S. mutans than for S. sobrinus. Upon adsorption of biosurfactant to pellicle-coated enamel, the range of the repulsive forces increased significantly (p < 0.01) for both strains, in line with the observations for bare enamel (Fig. 2D; Table).
From the summary of quantitative features of the retraction curves (Table  ), it can be seen that adhesive forces were found in about 75% of the force-distance curves measured upon retraction of bare enamel away from S. sobrinus and S. mutans surfaces. The maximum adhesive force averaged 0.8–0.9 nN, occurring at a distance of 39–80 nm between the interacting surfaces. For bare enamel, the percentage of the force-distance curves showing adhesion with S. mutans decreased to 10% upon biosurfactant adsorption, while with S. sobrinus no adhesive forces were observed upon retraction. Also, in the presence of a salivary pellicle, only a small percentage of the force-distance curves with S. sobrinus showed adhesion, while no adhesive forces were observed upon retraction for S. mutans. A biosurfactant coating on the salivary pellicle had only a minor effect on the characteristics of the retraction curves.
In the present study, we prepared an AFM probe by gluing enamel particles onto silicon nitride cantilevers and subsequently using it to produce force-distance curves between enamel and two strains of mutans streptococci: S. sobrinus HG 1025 and S. mutans ATCC 25175. To our knowledge, this is the first time that the interactive force between enamel and oral bacteria has been measured. The approach curves have been described to be most pertinent to bacterial adhesion (Vadillo-Rodríguez et al., 2004), and adhesion of micro-organisms is more difficult if the repulsive energy between two interacting surfaces increases. Since the range of this repulsive force far exceeded that of the electrostatic forces distinguished in the DLVO (Derjaguin, Landau, Verwey, and Overbeek) theory (Rutter and Vincent, 1980) for the interaction of colloidal particles with a surface, the nature of this repulsion has been suggested to be electrosteric (Camesano and Logan, 2000). The most important effect of a salivary pellicle on the force-distance curves measured is an increase in the range of this electrosteric repulsion. In line with this increased range after pellicle-coating of an enamel surface, both strains of mutans streptococci involved in this study adhered less to a salivary pellicle than to bare enamel, as established in a parallel-plate flow chamber (Van Hoogmoed et al., 2004). Moreover, S. mutans ATCC 25175 adhered in fewer numbers to a salivary pellicle than did S. sobrinus HG 1025, which, again, is in agreement with the range of the electrosteric repulsion for S. mutans. Pellicle compositions vary intra- and inter-individually, owing to, respectively, site-specific differences in composition and secretion of saliva (Carlén et al., 1998) and differences in salivary flow rates and genetic polymorphism among proline-rich proteins (PRPs) and amylase. When saliva donated by a group of volunteers was pooled, these differences were averaged out. As a consequence of centrifugation, the reconstituted saliva might be deficient in high-molecular-weight mucins, which is probably of minor relevance, since these proteins are hardly present in 30-minute pellicles as applied here, though valuable for Streptococcus mutans adhesion (Carlén and Olsson, 1995). Biosurfactants are, in the majority of cases, released by micro-organisms. Well-known actions of biosurfactants are the solubilization of hydrocarbons for nutrient uptake and metabolism, and their antibiotic activities on several micro-organisms. In the last decade, biosurfactants have also been increasingly recognized as substances that alter surface properties, thereby preventing the adhesion of other harmful micro-organisms to that surface (Neu, 1996). In an earlier study (Van Hoogmoed et al., 2004), it was demonstrated that S. mitis biosurfactant inhibited adhesion of S. mutans ATCC 25175 and S. sobrinus HG 1025 to bare enamel. S. mitis biosurfactant could inhibit adhesion of S. sobrinus HG 1025 only to salivary pellicles, but less so the adhesion of S. mutans ATCC 25175. Here, these reductions can be attributed to an increased range of the electrosteric repulsion upon approach between the bacteria and the biosurfactant-coated pellicles. In conclusion, this paper presents direct AFM measurements of interactive forces between enamel with and enamel without a salivary pellicle and two strains of mutans streptococci. In addition, we demonstrated that the major effects of applying an S. mitis BMS biosurfactant coating to the enamel was an increased range of the electrosteric repulsion upon approach of the interacting surfaces, explaining the reductions observed in adhesion of these mutans streptococci brought about by S. mitis biosurfactant coatings.
This study was supported by the University Medical Centre Groningen. Thanks are extended to I. Stokroos for making the electron micrographs. Received for publication July 14, 2004. Revision received August 19, 2005. Accepted for publication September 11, 2005.
Journal of Dental Research, Vol. 85, No. 1,
54-58 (2006) This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
is the decay length of the repulsive force. The retraction curves can show local maxima in adhesion forces, and since these did not occur in all curves, we recorded the percentage of curves displaying adhesion forces. Furthermore, the largest adhesion force (Fmax), as well as the maximum distance (Dmax) at which an adhesion force occurred, were registered. 
