Department of Biomedical Engineering, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands1
Author for correspondence: Henk J. Busscher. Tel: +31 50 3633140. Fax: +31 50 3633159. e-mail: H.J.Busscher{at}med.rug.nl
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ABSTRACT |
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Keywords: electrophoretic mobility, staphylococci, capsules, slime
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INTRODUCTION |
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For S. epidermidis strains, two distinct extracellular entities have been identified with a potential impact on their adhesion: a capsule, which is intimately associated with the cell wall, and a slime layer, which can be removed by washing (Christensen et al., 1982 ; Matthews et al., 1991
). Capsular material and slime can be regarded as soft, polyelectrolyte layers surrounding the organism. Bacterial cell surfaces most frequently carry a net negative surface charge, and negative zeta potentials are reported for physiological pH values (Jucker et al., 1996
), as for nearly all surfaces in nature. Staphylococci are thus expected to experience a strong electrostatic repulsion in their adhesion to substratum surfaces, which is opposite to many experimental findings (Bos et al., 1999
). Bacterial zeta potentials are derived by particulate microelectrophoresis (Hiemenz, 1977
; James, 1979
). During particulate microelectrophoresis, bacteria are suspended in a solution of a given ionic strength and composition and the velocity (mobility) that bacteria acquire under the influence of an applied electric field is measured. For so-called rigid particles (see Fig. 1
), the potential decreases exponentially with distance from the particle surface, while a thin, stagnant layer separates the movable part of the ionic double layer and the particle surface. The potential at this slip plane is the zeta potential (Lyklema, 1994
). The outer layer of biological cell surfaces differs substantially from the surface of a rigid particle and is, depending upon the strains and species under consideration, covered with an ion- and fluid-penetrable layer of charged polymers, which may be associated, for instance, with bacterial fimbriae, fibrils, capsular material or a slime layer. Consequently, during particulate microelectrophoresis, a fluid flow will develop through this charged layer. The zeta potential at the slip plane will be determined by a combination of the electrolyte charge density, as for rigid particles, and the fixed charge density of surface polymers (see Fig. 1
). From Fig. 1
, it can be seen that, due to the fixed charge density, the zeta potential at the slip plane of a soft particle is usually considerably more negative than the potential at its outer surface, which interacts with the environment and plays a role in adhesion.
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Recently, we studied the softness of two oral streptococcal strains, Streptococcus salivarius HB and HBC12, by particulate microelectrophoresis in KCl solutions of varying ionic strengths (Bos et al., 1998 ). Electron microscopy of negatively stained organisms, and X-ray photoelectron spectroscopy, confirmed that strain HB had several classes of proteinaceous fibrils with lengths up to 178 nm on its outermost surface (Weerkamp et al., 1986
), while variant HBC12 had a bald, peptidoglycan-rich outer surface. The fibrillated strain HB appeared as relatively soft (1/
=1·4 nm) from analysis of its electrophoretic mobility according to Ohshima, while the bald variant HBC12 was hard (1/
=0·7 nm) because of its comparatively rigid, peptidoglycan-rich outer surface, characteristic of Gram-positive bacteria. When the electrophoretic softness and ion-penetrability of polyelectrolyte layers on bacterial cell surfaces is properly accounted for (Morisaki et al., 1999
; Poortinga et al., 2001
), the energy barrier in adhesion of negatively charged organisms to negatively charged substratum surfaces is calculated to be far lower than usually estimated. Accurate, predictive models based on the DLVO theory for bacterial deposition to substratum surfaces can then be prepared (Poortinga et al., 2001
).
The aim of the present study was to compare the cell surface softness and fixed charge densities as derived from microelectrophoresis of 20 Staphylococcus epidermidis strains grown in liquid medium or on solid blood agar.
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METHODS |
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Particulate microelectrophoresis.
Electrophoretic mobilities were measured at 25 °C with a Lazer Zee Meter 501 (PenKem, Bedford Hills, NY, USA) equipped with an image-analysis option for tracking and zeta sizing (Wit et al., 1997 ). Measurements were carried out in KCl solutions of various ionic strengths (pH 6). Immediately prior to each measurement, an aliquot of the bacterial suspension was added to the appropriate KCl solution at a density of approximately 1x108 cells ml-1. The pH of the solutions did not change upon addition of the bacterial cells. The Lazer Zee Meter was set at 150 V for determination of the electrophoretic mobilities of the bacteria.
The electrophoretic mobilities, measured as a function of ionic strength, were fitted to equation (1) using a least-squares curve-fitting routine kindly provided by Dr H. Ohshima, Tokyo, Japan:
![]() | (1) |
in which µ is the electrophoretic mobility, r the relative permittivity,
0 the permittivity of vacuum,
the viscosity of the solution, 1/
m the DebyeHückel length in the polymer layer, 1/
the softness of the polyelectrolyte layer, z the valence of charged groups in the polyelectrolyte, e the electrical unit charge, N the density of charged groups in the polyelectrolyte layer,
0 the potential at the boundary between the polyelectrolyte layer and the surrounding solution, and
DON the Donnan potential within the polyelectrolyte layer (Ohshima, 1995
; Ohshima & Kondo, 1991
). By taking 1/
, the softness of the polyelectrolyte layer, and zN, the density of charged groups in the polyelectrolyte layer, as parameters of the fit, both 1/
and zN can be calculated from electrophoretic mobilities measured as a function of ionic strength. Note that 1/
m, the DebyeHückel length, is also a function of ionic strength and ranges from 3 nm in 0·01 M to 1 nm in 0·1 M KCl (Hiemenz, 1977
).
All electrophoretic mobilities and softness values reported are the mean values of three different measurements with separately cultured bacteria.
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RESULTS |
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The amount of fixed charge zN in the outer cell surface layer (see Table 1) varied from -16 to -56 mM for staphylococci grown in liquid medium; a similar range in amount of fixed negative charge was observed for cells grown on blood agar. On average over all strains, staphylococci grown in liquid medium had a fixed charge of -28±9 mM, while for bacteria grown on a solid agar the fixed charge was -24±12 mM. No relation existed between the amount of fixed charge and the cell surface softness.
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DISCUSSION |
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Staphylococcal cell surfaces are very different from streptococcal cell surfaces and physico-chemical models to explain staphylococcal adhesion to substratum surfaces have generally been less successful than models of streptococcal adhesion (Bos et al., 1999 ; Van der Mei et al., 1997
; Van Pelt et al., 1985
). This is probably due to a strong overestimation of the staphylococcal zeta potentials as derived from particulate microelectrophoresis neglecting the cell surface softness (see also Fig. 1
). Note that the average staphylococcal cell surface after growth in liquid medium (1/
=1·7 nm) is softer than that of a heavily fibrillated streptococcal strain grown in liquid culture (1/
=1·4 nm), while staphylococci grown on a solid agar are even softer (1/
=2·8 nm). As another comparison, the amount of fixed outer layer charges for the proteinaceous streptococci ranged between -13 and -15 mM, while the fixed charge densities of the staphylococcal cell surface layers were between -24 and -28 mM on average, indicative of the presence of highly negatively charged polysaccharides.
Morisaki et al. (1999) described that Vibrio alginolyticus had a cell surface softness of between 5·1 nm and 6·4 nm, with highly negative zeta potentials calculated. As a consequence the interaction energy barrier between V. alginolyticus and negatively charged glass was highly repulsive, with an energy barrier of around 150 kT. When the cell surface softness was accounted for, surface potentials were far lower and the energy barrier disappeared completely. Analogously, Poortinga et al. (2001)
failed to obtain theoretical deposition rates for staphylococci to glass in a parallel-plate flow chamber by solving the convective-diffusion for mass transport. Theoretically obtained deposition rates did not correspond with the high experimentally observed deposition rates, but a perfect match was calculated when the staphylococcal cell surface softness and ion-penetrability were taken into account.
The present study shows that staphylococcal cell surfaces are relatively soft, and that their softness increases when the organisms are grown on a solid agar. However, there appears to be no relation between the physico-chemical softness measured in this study and previous reports on the possession of a capsule or slime layer by the strains. This is consistent with previous attempts to relate physico-chemical cell surface properties of the staphylococci like charge, chemical composition or hydrophobicity (Van der Mei et al., 1997 ) with encapsulation or slime layer. The most likely explanation for this is still the hypothesis that all staphylococci are indeed encapsulated, with an effect on the physico-chemistry of their cell surfaces, but that in many cases the traditional India ink staining methods (Matthews et al., 1991
) to visualize capsules are inadequate to detect thin capsules, which may potentially have a thickness in the nanometer range.
Summarizing, we have demonstrated that the outermost layers on S. epidermidis cell surfaces are electrophoretically soft with a high density of fixed, negative charge, especially when compared with streptococcal cell surfaces. The staphylococcal cell surface softness increases after growth on a solid agar rather than in a liquid culture, although the difference in nutrient medium employed may contribute to this conclusion. Accounting for the softness of staphylococcal cell surfaces allows us to deal adequately with electrostatic interactions in the adhesion of the cells to negatively charged substratum surfaces.
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REFERENCES |
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Received 20 June 2000;
revised 27 October 2000;
accepted 10 November 2000.