Physico-chemical interactions in initial microbial adhesion and relevance for biofilm formation

This paper summarizes initial microbial adhesion events in dental plaque formation, including the physico-chemistry of the interaction between micro-organisms and solid substrata, detachment phenomena under the fluctuating shear of the oral cavity, co-adhesion between pairs of microbial strains, and biosurfactant release. A hypothesis is forwarded on how these initial events might influence the final microbial composition and structure of the plaque, although it is simultaneously emphasized that the necessary techniques for verification of the hypothesis have only recently become available, and supporting evidence is still to be collected.</p

The use of X-ray photoelectron spectroscopy for the study of oral streptococcal cell surfaces.

Physicochemical and structural properties of microbial cell surfaces play an important role in their adhesion to surfaces and are determined by the chemical composition of the outermost cell surface. Many traditional methods used to determine microbial cell wall composition require fractionation of the organisms and consequently do not yield information about the composition of the outermost cell surface. X-ray photoelectron spectroscopy (XPS) measures the elemental composition of the outermost cell surfaces of micro-organisms. The technique requires freeze-drying of the organisms, but, nevertheless, elemental surface concentration ratios of oral streptococcal cell surfaces with peritrichously arranged surface structures showed good relationships with physicochemical properties measured under physiological conditions, such as zeta potentials. Isoelectric points appeared to be governed by the relative abundance of oxygen- and nitrogen-containing groups on the cell surfaces. Also, the intrinsic microbial cell-surface hydrophobicity by water contact angles related to the cell-surface composition as by XPS and was highest for strains with an elevated isoelectric point. Inclusion of elemental surface compositions for tufted streptococcal strains caused deterioration of the relationships found. Interestingly, hierarchical cluster analysis on the basis of the elemental surface compositions revealed that, of 36 different streptococcal strains, only four S. rattus as well as nine S. mitis strains were located in distinct groups, well separated from the other streptococcal strains, which were all more or less mixed in one group.</p

Two Dimensional, Spatial Arrangement of Fibronectin Adsorbed to Biomaterials with Different Wettabilities

The effects of concentration, pH and substratum wettability on the two dimensional, spatial arrangement of adsorbed fibronectin are determined. Substrata with different wettability were exposed to a well defined flow of a fibronectin solution (0.1 or 1 mg/ml) in potassium phosphate buffer (pH 7.0 or 4.8) during 120 minutes at a shear rate of 20 s1. Rotary shadowed replicas of the surfaces were examined by transmission electron microscopy. Well defined structures, island-like in character on the low wettability substrata and knotted, reticulated on the high wettability substrata, could be seen in case of adsorption from pH 7.0 and the high concentration solution. Structures became more blurred upon lowering the solution pH and fibronectin concentration. Compared with bovine serum albumin, fibronectin shows smaller island-like structures, but the reticulated structure is thicker than for albumin

A comparison of bacterial growth inhibiting effects of six commercially available mouthrinses

In this study the bacterial growth inhibiting effects of six commercially available mouthrinses (Hibident® Prodent® Merocet® Listerine® Veadent® and Meridol® were determined. Hibident® was used as a positive control. Five strains were tested (Streptococcus mutans C67, Streptococcus sanguis CH3, Veillonella alcalescens V1, Lactobacillus acidophilus JP and Actinomyces viscosus C74), as representatives of the supragingival human microflora. The Maximal Inhibiting Dilution (MID) was measured in batch cultures for each product and strain. With respect to the positive control, Hibident® (containing 0.2 per cent chlorhexidine), the most effective product was Meridol® (containing 125 ppm aminefluoride 297 and 125 ppm stannous fluoride) followed by Merocet® (containing 0.05 per cent cetylpyridinium chloride), Veadent® (containing 0.03 per cent sanguinarine), Listerine® (containing phenolic compounds) and Prodent® (containing 0.5 per cent sodium fluoride). Although all products have been separately reported to yield a plaque reduction in vivo, this study provides a firm basis for a comparison between products, as they were all evaluated in a similar way.</p

On the relative importance of specific and non-specific approaches to oral microbial adhesion

In this paper, it is suggested that specificity and non-specificity in (oral) microbial adhesion are different expressions for the same phenomena. It is argued that the same basic, physico-chemical forces are responsible for so-called 'non-specific' and 'specific' binding and that from a physico-chemical point of view the distinction between the two is an artificial one. Non-specific interactions arise from Van der Waals and electrostatic forces and hydrogen bonding, and originate from the entire cell. A specific bond consists of a combination of the same type of Van der Waals and electrostatic forces and hydrogen bonding, now originating from highly localized chemical groups, which together form a stereo-chemical combination. The absence or presence of specific receptor sites on microbial cell surfaces must therefore be reflected in the overall, non-specific surface properties of cells as well. This point is illustrated by showing that glucanbinding lectins on mutans streptococcal strains may determine the pH dependence of the zeta potentials of these cells. When studying microbial adhesion, a non-specific approach may be better suited to explain adhesion to inert substrata, whereas a specific approach may be preferred in case of adhesion to adsorbed protein films. Adhesion is, however, not as important in plaque formation in the human oral cavity as is retention, because low shear force periods. during which adhesion presumably occurs, are followed by high shear force periods, during which adhering cells must withstand these detachment forces. Evidence is provided that such detachment will be through cohesive failure in the pellicle mass, the properties of which are conditioned by the overall, non-specific substratum properties. Therefore, in vivo plaque formation may be more readily explained by a non-specific approach.</p

Depth profiling of the elemental surface composition of the oral microorganism S. salivarius HB and fibrillar mutants by X-ray photoelectron spectroscopy

X-ray photoelectron spectroscopy (XPS) on microbial cell surfaces requires freeze-drying of cells, and as a result, the cell surface appendages flatten out on the cell surface and form a collapsed fibrillar mass. At present, it is unclear how the density, length and composition of these fibrils influence the elemental surface composition as probed by XPS. The sampling depth of XPS can be varied by changing the electron take-off angle. In this article, we made a depth profiling of the collapsed fibrillar mass of Streptococcus salivarius HB and fibril-deficient mutants by angle-dependent XPS. Methylamine tungstate negative staining and ruthenium red staining followed by sectioning revealed distinct classes of fibrils with various lengths on each of the strains. Interpretation of the angle dependence of the oxygen/carbon (O/C) and phosphorus/carbon (P/C) surface concentration ratios of these strains was difficult. However, the angle dependence of the nitrogen/carbon (N/C) surface concentration ratio could be fully interpreted: N/C did not vary with sampling depth on a bald strain, S. salivarius HBC12 and on S. salivarius HB7, a strain with a dense array of fibrils of uniform length. N/C decreased with sampling depth in case of a sparsely fibrillated strain, S. salivarius HBV51 and eventually reached the value observed for the bald strain, HBC12. A high N/C at small sampling depth was observed for S. salivarius HB with protruding, protein rich fibrils. We conclude that elemental depth profiling of microbial cell surfaces by XPS can be interpreted to coincide with structural and biochemical information on the cell surface as obtained by electron microscopy and can therefore be considered as a useful technique to study structural features of cell surfaces in combination with electron microscopy.</p

Detachment of linking film bacteria from enamel surfaces by oral rinses and penetration of sodium lauryl sulphate through an artificial oral biofilm.

The biofilm mode of growth protects plaque micro-organisms against environmental attacks, such as from antimicrobials or detergents. Dental plaque is linked to enamel through the adhesion of initial colonizers. Once this link is disrupted, the entire plaque mass adhering to it detaches. Experiments in a parallel-plate flow chamber demonstrated that bacteria adhering to saliva-coated enamel could not be stimulated to detach by perfusion of the flow chamber with two traditional mouthrinses (Corsodyl and Scope), whereas perfusion with a prebrushing rinse (Plax) or its detergent components stimulated detachment from saliva-coated enamel of a wide variety of bacterial strains. Following perfusion of the flow chamber with the mouthrinses, little additional detachment of adhering bacteria by the passage of a liquid-air interface occurred. After perfusion with the prebrushing rinse, however, significant numbers of still-adhering bacteria could be stimulated to detach by passage of a liquid-air interface, indicating that Plax had weakened their adhesive bond. The ability of Plax or its detergent components to detach plaque bacteria is not always obvious from in vivo experiments, and reports on its clinical efficacy are inconsistent. Likely, antimicrobials or detergents are unable to penetrate the plaque and reach the linking film bacteria, as demonstrated here by Fourier transform infrared spectroscopy.</p

Characterization of eukaryotic cell surfaces prior to and after serum protein adsorption by X-ray photoelectron spectroscopy - Fibroblasts, HELA epithelial, and smooth muscle cells

Elemental surface concentration ratios N/C, O/C, and P/C of fibroblasts, HELA epithelial cells, and smooth muscle cells, prior to and after washing in the absence or presence of serum proteins, were determined by X-ray photoelectron spectroscopy. Cell surfaces appeared to adsorb hardly any serum proteins, and the relatively high P/C, as compared to N/C and O/C, elemental surface concentration ratio indicated that the cell surfaces consisted mainly of the phospholipid bilayer, with little or no proteins present. The lack of adsorption of serum proteins to the cell surfaces seems at odds with the common notion that cells require adhesive proteins in order to adhere and spread. However, the adsorption behavior of cellularly produced proteins may be completely different, particularly since they seem to be able to displace adsorbed serum proteins from biomaterials surfaces. Interestingly, only HELA epithelial cells (a tumor cell line) appeared to adsorb a very small amount of proteins.</p

Influence of glutaraldehyde fixation of cells adherent to solid substrata on their detachment during exposure to shear stress

In order to determine the response of fixed and nonfixed cells adherent to a solid substratum to shear stress, human fibroblasts were allowed to adhere and spread on either hydrophilic glass or hydrophobic Fluoroethylene-propylene (FEP-Teflon) and fixed with glutaraldehyde. Then, the cells were exposed to an incrementally loaded shear stress in a parallel plate flow chamber up to shear stresses of about 500 dynes/cm2, followed by exposure to a liquid-air interface passage. The cellular detachment was compared with the one of nonfixed cells. In case of fixed cells, 50% of the adhering cells detached from FEP-Teflon at a shear stress of 350 dynes/cm2, whereas 50% of the adhering, nonfixed cells detached already at a shear stress of 20 dynes/cm2. No fixed cells detached from glass for shear stresses up to at least 500 dynes/cm2. More than 50% of the nonfixed cells were detached from glass at a shear stress of 350 dynes/cm2. Furthermore, the shape and morphology of fixed cells did not change during the incrementally loaded flow, in contrast to the ones of nonfixed cells, which clearly rounded up prior to detachment.</p

Physicochemical characterization of Escherichia coli:A comparison with gram-positive bacteria

Eight Escherichia coli strains were characterized by determining their adhesion to xylene, surface free energy, zeta potential, relative surface charge, and their chemical composition. The latter was done by applying X-ray photoelectron spectroscopy (XPS) and infrared spectroscopy (IR). No relationship between the adhesion to xylene and the water contact angles of these strains was found. Three strains had significantly lower surface free energies than the other strains. Surface free energies were either obtained from polar and dispersion parts or from Lifshitz-van der Waals and acid/base parts of the surface free energy. A correlation (r=0.97) between the polar parts and the electron-donor contributions to the acid/base part of the surface free energy was found. The zeta potentials of all strains, measured as a function of pH (2–11), were negative. Depending on the zeta potential as a function of pH, three groups were recognized among the strains tested. A relationship (r=0.84) was found between the acid/base component of the surface free energy and the zeta potential measured at pH=7.4. There was no correlation between results of XPS and IR studies. Data from the literature of XPS and IR studies of the gram-positive staphylococci and streptococci were compared with data from the gram-negativeE. coli used in this study. It appeared that in these three groups of bacteria, the polysaccharide content detected by IR corresponded well with the oxygen-to-carbon ratio detected by XPS