The electrophoretic softness of the surface of Staphylococcus epidermidis cells grown in a liquid medium and on a solid agar

Paskal J. M. Kiers1, Rolf Bos1, Henny C. van der Mei1 and Henk J. Busscher1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Many Staphylococcus epidermidis strains possess capsule or slime layers and consequently the staphylococcal cell surface should be regarded as a soft, polyelectrolyte layer allowing electrophoretic fluid flow through a layer of fixed charges. The presence of such a soft layer decreases the energy barrier due to electrostatic repulsion in the interaction of the organisms with negatively charged substrata [Morisaki, H., Nagai, S., Ohshima, H., Ikemoto, E. & Kogure, K. (1999), Microbiology 145, 2797–2802] and hence plays an important role in their adhesion. In this paper, the authors compare the electrophoretic softness and amount of fixed charge in the outer cell surface layers of 20 S. epidermidis strains, grown in a liquid medium or on a solid agar, as determined from the dependencies of their electrophoretic mobilities upon the ionic strength of a suspending fluid. Most of the staphylococcal cell surfaces were relatively soft, with a mean cell surface softness (1/{lambda}) for strains grown in liquid medium of 1·7±0·6 nm (standard deviation over all 20 strains) which is soft by comparison with a completely bald, peptidoglycan-rich streptococcal cell surface (1/{lambda}=0·7 nm). When the staphylococcal strains were grown on solid agar, the cell surface softness of 17 of the 20 strains increased, sometimes by a factor of two. On average for 20 strains, the cell surface softness increased significantly (P<0·05, Student’s t-test) to 2·8±1·8 nm. The amount of fixed charge in the outer cell surface layer was -28±9 mM for bacteria grown in liquid medium and -24±12 mM for bacteria grown on agar. A soft, highly negatively charged polyelectrolyte layer was inferred by microelectrophoresis for all the staphylococcal cell surfaces, regardless of whether staining had indicated the presence of a capsule or slime layer.

Keywords: electrophoretic mobility, staphylococci, capsules, slime


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Staphylococcus epidermidis, a coagulase-negative bacterium, is a common cause of infections associated with prosthetic devices and indwelling catheters, such as heart valves, pacemakers, orthopaedic implants, Hickman catheters, and cerebrospinal fluid shunts. It is also a frequent infectious agent in many non-biomaterial-related infections, as for example peritonitis, neonatal sepsis, and native valve endocarditis (Dankert et al., 1986 ). An understanding of the fundamental mechanisms of microbial adhesion is imperative for effective preventive and eradication strategies against all bacterial infections (Bos et al., 1999 ).

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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Fig. 1. Schematic representation of the surfaces of an electrophoretically rigid and an electrophoretically soft particle. The electrokinetic potential decreases exponentially with distance from the rigid particle surface and the slip plane, determining its zeta potential, is close to the surface. The soft particle is covered by a polyelectrolyte layer with fixed negative charges, through which electrophoretic fluid flow is possible. Consequently, the slip plane remains at approximately the same position as for the rigid particle and is inside the soft layer, and the zeta potential is much more negative than the potential {Psi}0 at the outside of the soft layer.

 
H. Ohshima and colleagues (Ohshima, 1995 ; Ohshima & Kondo, 1991 ) have proposed a new theory to interpret the electrophoretic mobilities of soft, polyelectrolyte-covered particles as opposed to rigid particles, when measured as a function of ionic strength. This theory describes the electrophoretic mobility of soft particles, of which micro-organisms are typical examples, as originating from a weighted average of potentials over the surface charge layer and that of the volume density of fixed charges. The polyelectrolyte layer around microbial cell surfaces allows electrophoretic fluid flow, and the ease by which fluid can flow through the soft layer is expressed as a so-called electrophoretic softness, 1/{lambda}. The theory of Ohshima enables quantitative determination of the volumetric charge density and the softness (1/{lambda}) of this polyelectrolyte layer from the dependence of the electrophoretic mobility upon ionic strength, and it has been successfully applied to describe the surfaces of human erythrocytes (Kawahata et al., 1990 ) and rat lymphocytes (Morita et al., 1991 ).

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/{lambda}=1·4 nm) from analysis of its electrophoretic mobility according to Ohshima, while the bald variant HBC12 was hard (1/{lambda}=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.


   METHODS
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INTRODUCTION
METHODS
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DISCUSSION
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Bacterial strains and culture conditions.
The Staphylococcus epidermidis strains used in this study (see Table 1) were cultured in Trypticase soy broth (TSB, Oxoid). Strains were inoculated from blood agar (Blood agar no. 2, Oxoid) into broth, grown in batch culture for 24 h at 37 °C, and this culture was used to inoculate a second culture that was grown for 16 h at 37 °C. Bacteria were harvested by centrifugation (5 min at 10000 g), washed twice with demineralized water and suspended in water. To prepare agar-grown cells for experiments, bacteria were grown for 24 h on blood agar at 37 °C, harvested from the plates and resuspended in water.


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Table 1. Cell surface softness 1/{lambda} and charge density zN of 20 S. epidermidis isolates, as derived from particulate microelectrophoresis on three separate cultures of each strain

 
All staphylococci cultured, both in TSB medium and on blood agar, were examined microscopically for the presence of a capsule by India ink staining or slime layer by methylene blue staining as described by Christensen et al. (1982) and Matthews et al. (1991) .

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, {epsilon}r the relative permittivity, {epsilon}0 the permittivity of vacuum, {eta} the viscosity of the solution, 1/{kappa}m the Debye–Hückel length in the polymer layer, 1/{lambda} 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, {Psi}0 the potential at the boundary between the polyelectrolyte layer and the surrounding solution, and {Psi}DON the Donnan potential within the polyelectrolyte layer (Ohshima, 1995 ; Ohshima & Kondo, 1991 ). By taking 1/{lambda}, the softness of the polyelectrolyte layer, and zN, the density of charged groups in the polyelectrolyte layer, as parameters of the fit, both 1/{lambda} and zN can be calculated from electrophoretic mobilities measured as a function of ionic strength. Note that 1/{kappa}m, the Debye–Hückel 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.


   RESULTS
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DISCUSSION
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As an example, Fig. 2 shows the electrophoretic mobility of S. epidermidis ATCC 35983 as a function of ionic strength. The electrophoretic mobility of this strain levels off at an ionic strength of between 0·05 and 0·10 M, and the electrophoretic mobility at high ionic strength is more negative for bacteria grown on solid agar than for organisms grown in liquid medium. For rigid particles, electrophoretic mobilities are essentially zero in 0·10 M KCl (Ohshima & Kondo, 1991 ) and according to equation (1) the residual electrophoretic mobilities of the staphylococci at 0·10 M are a result of fixed charges in the outer cell surface layer. The relatively minor change in electrophoretic mobility over the range of ionic strengths in Fig. 2 is, in combination with the residual mobility at high ionic strength, indicative of a soft cell surface.



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Fig. 2. Electrophoretic mobilities in KCl solutions (pH 6) of S. epidermidis ATCC 35983 as a function of ionic strength. The solid lines represent the best fit to equation (1). Error bars indicate the standard deviation over three experiments with separately cultured bacteria. {bullet}, Staphylococci grown in liquid medium; {circ}, staphylococci grown on a solid agar.

 
The cell surface softness (1/{lambda}) and charge density (zN) of all 20 staphylococcal strains, as derived by fitting the dependence of the electrophoretic mobilities upon ionic strength as presented in Fig. 2 to equation (1), are compiled in Table 1. All strains were also assessed for presence of a capsule or slime layer (Table 1). Strain ATCC 35984 had a relatively hard cell surface (1/{lambda}=0·5 nm) when grown in liquid medium, while the softest strain grown in liquid medium, 3399, had a cell surface softness of 2·9 nm. On average over all 20 staphylococcal strains grown in liquid medium, the cell surface softness was 1·7±0·6 nm, showing that staphylococcal cell surfaces were soft. For comparison, a completely bald, peptidoglycan-rich streptococcal cell surface had 1/{lambda}=0·7 nm (Bos et al., 1998 ). Upon growth on agar instead of in liquid medium, 17 out of the 20 strains become softer. For some strains, e.g. ATCC 35983, ATCC 35984, HBH 171 and HBH2 3, the cell surface softness more than doubled. On average over all 20 strains, the cell surface softness increased to 2·8±1·8 nm, which was significant by comparison with the softness of staphylococci grown in liquid medium (P<0·05, Student’s t-test). Conclusively, a soft polyelectrolyte layer was inferred from microelectrophoresis for all staphylococcal cell surfaces. The softness of the staphylococcal cell surface does not correlate with microscopic evidence for the absence or presence of a capsule or slime layer, as can also be seen in Table 1.

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.


   DISCUSSION
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Over the past decade, several authors have argued that bacterial cell surfaces should be considered much more as a gel-like layer rather than as a rigid surface. The negative cell wall charge at neutral pH of most bacterial strains results from high concentrations of anionic carboxyl, phosphate and amino groups that may associate at reduced pH to cause a positive cell wall charge. This charge is compensated in part by counterions penetrating into the gel-like layer (Van der Wal et al., 1997 ). Van der Wal et al. (1997) concluded that for their collection of bacterial species (including corynebacteria, rhodococci and Bacillus, all organisms virtually devoid of any structural surface appendages), most of the mobile charges were located in a hydrodynamically stagnant layer in the gel, envisaged to form the cell surface. In the Ohshima model an electrophoretic fluid flow is thought to be possible through this gel-like, soft layer around microbial cell surfaces. The ease with which fluid can flow through this layer is expressed as an electrophoretic softness. For two streptococcal strains, Bos et al. (1998) showed that an electrophoretically hard strain was devoid of structural surface appendages, while an electrophoretically soft strain had surface fibrils with lengths up to 178 nm. Recently, we applied atomic-force microscopy to these two streptococcal strains and found that an electrophoretically soft, fibrillated strain is also mechanically soft when probed by the tip of an atomic force microscope (Van der Mei et al., 2000 ), providing substantial support for the Ohshima model from independent experimentation. Moreover, this finding suggests that a layer allowing fluid flow is more open and consequently also mechanically softer than a more closed structure and thereby simplifies the rather abstract concept of ‘electrophoretic softness’.

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/{lambda}=1·7 nm) is softer than that of a heavily fibrillated streptococcal strain grown in liquid culture (1/{lambda}=1·4 nm), while staphylococci grown on a solid agar are even softer (1/{lambda}=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.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bos, R., Van der Mei, H. C. & Busscher, H. J.(1998). ‘‘Soft particle’’ analysis of the electrophoretic mobility of a fibrillated and non-fibrillated oral streptococcal strain: Streptococcus salivarius. Biophys Chem 74, 251-255.[Medline]

Bos, R., Van der Mei, H. C. & Busscher, H. J.(1999). Physico-chemistry of initial microbial adhesive interactions – its mechanisms and methods to study. FEMS Microbiol Rev 23, 179-230.[Medline]

Christensen, G. D., Simpson, W. A., Bisno, A. L. & Beachy, E. H.(1982). Adherence of slime-producing strains of Staphylococcus epidermidis to smooth surfaces. Infect Immun 37, 318-326.[Medline]

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Morisaki, H., Nagai, S., Ohshima, H., Ikemoto, E. & Kogure, K.(1999). The effect of mobility and cell-surface polymers on bacterial attachment. Microbiology 145, 2797-2802.[Abstract/Free Full Text]

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Van der Mei, H. C., Van de Belt-Gritter, B., Reid, G., Bialkowska-Hobrzanska, H. & Busscher, H. J.(1997). Adhesion of coagulase-negative staphylococci grouped according to physico-chemical properties. Microbiology 143, 3861-3870.[Abstract]

Van der Mei, H. C., Busscher, H. J., Bos, R., De Vries, J., Boonaert, C. J. P. & Dufrene, Y. F.(2000). Direct probing by atomic force microscopy of the cell surface softness of a fibrillated and nonfibrillated oral streptococcal strain. Biophys J 78, 2668-2674.[Abstract/Free Full Text]

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Received 20 June 2000; revised 27 October 2000; accepted 10 November 2000.