Ah Weerkamp

Effect of Zeta Potential and Surface Energy on Bacterial Adhesion to Uncoated and Saliva-coated Human Enamel and Dentin

Physicochemical surface characteristics of early plaque-forming bacteria and of human tooth surfaces were measured to establish their role in bacterial adhesion to intact dental tissue slabs. In addition, the influence of an experimental salivary pellicle was evaluated. Strains of S. mutans, S. sanguis, S. salivarius, A. viscosus, and A. odontolyticus showed relatively high surface free energies (range, 99-128 mJ.m-2) and carried a negative surface charge, at both physiological (μ = 0.057) and low (μ = 0.020) ionic strengths of the medium. Very large differences in hydrophobicity were detected when the hexadecane adsorption test was used for measurement. Powdered enamel and dentin were also negatively charged at low ionic strength but were slightly positively charged in the physiological buffer. The surface free energy of enamel and dentin increased upon saliva coating, whereas the surface charge was always negative. The adhesion experiments showed: (1) large differences in the binding of various bacteria to the same surface; (2) an up to 20-fold difference in the binding of the same bacterium to different surfaces, although the binding of some strains was relatively independent of the type of surface or presence of a salivary pellicle; (3) a significant decrease in adhesion when the ionic strength of the medium was lowered, due to increased electrostatic repulsion (however, the adhesion of some bacteria was independent of the ionic strength of the medium); (4) different time-dependent adherence kinetics, depending on both the bacteria and nature of the solid surface; and (5) a propensity for plaque streptococci to bind to uncoated dentin.

A COMPARISON OF VARIOUS METHODS TO DETERMINE HYDROPHOBIC PROPERTIES OF STREPTOCOCCAL CELL-SURFACES

Abstract In order to determine whether various methods which are commonly applied to determine the hydrophobicity of bacterial cell surfaces yield similar results, the hydrophobicity of a range of oral streptococcal isolates was measured using various methods including adsorption to hexadecane, hydrophobic interaction chromatography, salt-aggregation and contact angle measurements. In addition the zela potential and the adhesion to saliva-coated hydroxyapatite were determined. To compare the results of the different methods, linear and rank correlation coefficients were calculated. The resulting correlations were weak when applied to the whole range of strains even in those cases where the tests are expected to probe the same surface characteristics. However, generally good correlations were obtained when a set of strains was examined which were derived from a single parent strain and which only differed in defined surface structures. Accordingly it must be concluded from the methods applied here, that if is not possible to define the surface ‘hydrophobicity’ of a bacterium other than on a comparative level with closely related strains. No clear correlation was found between any hydrophobicity test and the adhesion to saliva-coated hydroxyapatite.

Properties of oral streptococci relevant for adherence: Zeta potential, surface free energy and elemental composition

Abstract In order to characterize the functional properties of the cell surface relevant for adherence, the zeta potentials, surface free energies and elemental surface compositions of oral streptococci were determined. Zeta potentials of fully hydrated cells were measured as a function of pH in dilute potassium phosphate solutions, yielding isoelectric points (IEP) for the different strains. Surface free energies were calculated according to the geometric mean equation from contact angles, measured on bacterial cells deposited on a membrane filter. The elemental analyses were carried out on freeze-dried cells by X-ray photoelectron spectroscopy. A decrease in the N/C concentration ratio among the different strains from 0.124 (S. mitisBMS) to 0.053 (S. salivarius HBC12) was found to be concurrent with an increase in the O/C concentration ratio from 0.312 to 0.495. Simultaneously, the IEP shifted from 3.7 to 1.3 and the surface free energy changed from 37 to 125 mJ m−2. The zeta potentials, the surface free energies and the elemental compositions of the bacterial cell surface showed clear relationships despite the fact that these parameters were measured in different states of hydration of the surface.

The Surface Free Energy of Oral Streptococci after being Coated with Saliva and its Relation to Adhesion in the Mouth

Contact angle measurements on layers of bacteria were used to determine the bacterial surface free energy (γb) of a variety of oral streptococcal strains, both without and after being coated with human whole saliva. At least four isolates of each species, either freshly isolated or laboratory strains, were used. The species Streptococcus mutans, S. sanguis, and S. salivarius were homogeneous, having high surface free energies, and were not affected by saliva treatment (γb = 106 ± 12 and 107 ± 10 erg·cm-2 in the absence and presence of saliva coating, respectively; n = 20). S. mitis had a very low surface free energy (46 ± 15; n = 5), which was significantly increased after salivary adsorption (71 ± 14 erg·cm-2; p<0.002). The species S. milleri contained strains with both high and low γ b. Calculation of the interfacial free energy of adhesion (ΔF adh) for bacteria from a saliva suspension to solid surfaces with various arbitrary surface free energies (γs) showed that, theoretically, most strains will encounter thermodynamically favorable conditions for adhesion to surfaces with a γs above 62 erg·cm-2. However, S. mitis strains not coated with saliva would only be able to adhere to surfaces with γs lower than this value. Saliva-coating reverses the calculated relationship with γs for these strains. The results indicate that an enamel surface with a low γs value would be thermodynamically unfavorable for adhesion of most oral streptococci.

EXPERIMENTAL APPROACHES FOR STUDYING ADHESION OF MICROORGANISMS TO SOLID SUBSTRATA - APPLICATIONS AND MASS-TRANSPORT

Abstract Various experimental systems have been developed which enable the study of adhesion of microorganisms to solid substrata. These systems can be distinguished in flow systems and static systems; in addition, static systems can be divided into systems with relatively small and large suspension volumes. It will be argued that a proper knowledge of the mechanisms of mass transport is required in order to fully understand the deposition process, a fact that has been largely negelected in the current literature. Furthermore, it should be noted that the adsorption kinetics are different for systems with relatively small and large suspension volumes. Therefore two different but analogous phenomenological description of adhesion kinetics are presented. Finally, it is concluded that flow systemscan be advantageously employed over static systems whenever qunatitative results are required, and especially since correlations with data from other types of flow systems can be made more easily. Static systems may be preferred when specific types of interactions are studied.

Monobacterial and Mixed Bacterial Plaques of Streptococcus mutans and Veillonella alcalescens in an Artificial Mouth: Development, Metabolism, and Effect on Human Dental Enamel

To gain greater understanding of the role of Streptococcus mutans and Veillonella in the caries process, studies of both aerobically and anaerobically grown plaques of S. mutans C67-1 and V. alcalescens V-1 on human enamel slabs were carried out in an artificial mouth. Plaque development, acid production, and demineralization were measured. Early plaque development of monobacterial and mixed bacterial plaques started from randomly adhering cells on day 1 to confluent multilayered microcolonies on day 4. Differences were observed in viable cell counts, total cell mass, and in acid production. In most cases CFU, DNA and acid production were higher in the mixed bacterial plaque, especially in the anaerobic mixed plaque. Lactic acid was the predominant acid in all cases following the supply of sucrose to the plaque. No decisive role could be found for acetic, formic, and propionic acid. No inhibition of demineralization was observed in the enamel slabs inoculated with both aerobic and anaerobic mixed plaques. Demineralization ranged from the more classical picture of lesion development in the aerobic monobacterial plaque-treated samples to an aggressive etching of the enamel surface in the anaerobically mixed treated slabs.

A Comparison Between the Elemental Surface Compositions and Electrokinetic Properties of Oral Streptococci With and Without Adsorbed Salivary Constituents

In order to characterize the functional cell surface, isoelectric points and elemental surface concentration, the ratios of nitrogen, oxygen and phosphorus to carbon of saliva-coated strains were determined by pH-dependent zeta potential measurements and X-ray photoelectron spectroscopy and compared with those of uncoated strains. The measurements of potential were carried out on completely hydrated cells, whereas the spectroscopy was on freeze-dried micro-organisms. The small increase in the nitrogen:carbon surface concentration ratio of saliva-coated streptococci in comparison to uncoated strains varied from 0.001 (Streptococcus mitis BA) to 0.029 (Streptococcus sanguis CH3) and was concurrent with an increase of the isoelectric point, ranging from 0.0 to 0.9. Increases in the oxygen:carbon ratio ranged from 0.006 (Strep. mitis BA) to 0.041 (Streptococcus mutans NS), whereas the phosphorus:carbon surface concentration ratio was unchanged after saliva treatment. Despite the fact that isoelectric and compositional measurements were made in different states of surface hydration, a decrease in the nitrogen:carbon ratio accompanied by an increase in oxygen:carbon ratio, was related to a decrease of the isoelectric point of the saliva-coated strains, and so analogous with previous observations for uncoated strains. Although there were changes in the physico-chemical properties of the strains upon saliva coating, all more or less kept their own surface identity despite adsorption of salivary constituents, possibly indicating some capacity to protect their own physico-chemical identity.

Physico-chemical properties of oral streptococcal cell surfaces and their relation with adhesion to solid substrata in vitro and in vivo

Abstract Thermodynamic approaches to bacterial adhesion emphasizing macroscopic properties of interacting bacterial cell and solid substratum surfaces are often criticized for not sufficiently accounting for microscopic, stereochemically complementary molecular interactions between the surfaces. Yet, as is demonstrated in this paper for a set of oral streptococci, three completely different overall macroscopic bacterial cell surface properties (elemental composition by XPS, surface free energy by contact angles and isoelectric point) are related in a comprehensible way, even though bacteria are analyzed at different states of hydration. Moreover, streptococcal adhesion from a moderate flow (σ=21 s−1) to inert solid substrata was related to both the interfacial free energy of adhesion and a strain-dependent microbial factor. This empirical factor measures the effect of cell surface appendages or excretion of (anti-) adhesives which assist or impede adhesion in excess of strictly thermodynamic considerations. Although bacterial adhesion strongly decreased after precoating substrata with bovine serum albumin or salivary proteins, negative slopes between the number of adhering streptococci and the interfacial free energy of adhesion (calculated on basis of the bare solid substratum surface free energies) suggest that surface free energy effects are transferred through adsorbed protein films. Much stronger evidence for such modification of adsorbed protein films by substratum surface free energies is obtained from in vivo experiments in the human oral cavity which demonstrated that the amount of plaque collected on various materials after nine days exposure to oral fluids was approximately five times smaller on a low surface free energy material as PTFE-teflon than on a high surface free energy material such as enamel.

ZETA-POTENTIAL AND THE ADHESION OF ORAL STREPTOCOCCI TO POLYMETHYLMETHACRYLATE

Adhesion of Candida albicans to the acrylic fitting surface of dentures can lead to the development of denture induced stomatitis and is suggested to be preceeded by the adhesion of indegeneous oral bacteria. Bacterial adhesion can be considered as a result of attractive van der Waals and repulsive electrostatic forces. In this paper we investigate the role of electrostatic interactions in the adhesion of oral streptococci to polymethylmethacrylate (PMMA), the most commonly employed denture-base material. Zeta potentials of the bacterial strains as well as of PMMA fillings were reduced by increasing the ionic strength in the buffer suspension, causing an elevated number of adhering S. mitis, S. sanguis and S. mutans. As streptococcal adhesion is suggested to be a prerequisite for the subsequent adhesion of Candida albicans, the use of more negatively charged denture-base materials can be recommended in order to prevent streptococcal adhesion and therewith adhesion of candidae, and the occurrence of denture-induced stomatitis.

DEVELOPMENTAL AND METABOLIC ASPECTS OF A MONOBACTERIAL PLAQUE OF STREPTOCOCCUS-MUTANS C 67-1 GROWN ON HUMAN-ENAMEL SLABS IN AN ARTIFICIAL MOUTH MODEL .1. PLAQUE DATA

Development and metabolic aspects of a monobacterial plaque of Streptococcus mutans C 67-1, inoculated on flattened human enamel slabs, were studied using an artificial mouth model.