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On Relations between Microscopic and Macroscopic Physicochemical Properties of Bacterial Cell Surfaces: An AFM Study on Streptococcus mitis Strains Virginia Vadillo-Rodrı´guez,† Henk J. Busscher,† Willem Norde,†,‡ Joop de Vries,† and Henny C. van der Mei*,† Department of Biomedical Engineering, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands, and Laboratory of Physical Chemistry and Colloid Science, Wageningen University, Dreijenplein 6, 6703 HB Wageningen, The Netherlands Received July 22, 2002. In Final Form: December 9, 2002 Bacterial adhesion to substratum surfaces is determined by the combined action of a large number of different interactions, which have hitherto been inferred from macroscopic cell surface properties, such as electrostatic double-layer forces, hydrophobic interactions, hydrogen bonding, and steric interactions. The origin of these interactions arises from a molecular level, but nevertheless they have always been estimated from a macroscopic point of view. The macroscopic bacterial cell surface hydrophobicity, for instance, is commonly inferred from water contact angles on bacterial lawns, and cell surface charge is macroscopically determined by electrophoresis or titration methods. Although these macroscopic properties of bacteria have been demonstrated to correlate with bacterial adhesion of certain strains and species to substratum surfaces, a generalized physicochemical understanding of bacterial adhesion to surfaces does not yet exist. The introduction of the atomic force microscope (AFM) and its application to biological surfaces has offered new possibilities to obtain microscopic, physicochemical properties of bacterial cell surfaces. In this paper, a detailed analysis of the interaction forces between a silicon nitride AFM tip and the surface of nine different oral bacterial strains, Streptococcus mitis, was carried out. Interestingly, microscopic features of force-distance curves could be amalgamated in such a way that relations between microscopic cell surface properties and macroscopic cell surface properties were obtained, even though these relations were not fully understood.
Introduction The bacterial cell wall is a critical structure, allowing organisms to selectively interact with their environment. Moreover, the cell wall imparts shape to the organisms, supports internal turgor pressure, and acts as a selective barrier for nutrients and metabolites. Chemically, the cell wall is constituted of a variety of spatially organized molecular structures, each with a specific function. All biological membranes, including those of the bacterial cell wall, are composed of a thin film of (glyco)lipids and proteins, held together mainly by noncovalent interactions. The composition of the outer cell wall varies considerably between Gram-positive and Gram-negative bacteria and may contain a variety of external structures, such as S-layers, capsules, fibrils, fimbriae, and pili.1 The combination of these structures determines the physicochemical cell surface properties of a particular bacterial strain. These properties, however, are not fixed in space or time but vary with environmental changes and as a result of mutations and various mechanisms of gene transfer between organisms.2 Bacterial cell surface hydrophobicity and charge are commonly accepted as influential on bacterial interactions with their environment, but a generalized physicochemical theory accounting for bacterial adhesion to substratum * Corresponding author. Tel: 31-50-3633140. Fax: 31-503633159. E-mail:
[email protected]. † University of Groningen. ‡ Wageningen University. (1) Hancock, I. C. In Microbial cell surface analysis: Structural and Physicochemical Methods; Mozes, N., Handley, P. S., Busscher, H. J., Rouxhet, P. G., Eds.; VCH Publishers: New York, 1991; p 21. (2) Savage, D. B.; Fletcher, M. Bacterial Adhesion; Plenum Press: New York, 1985.
surfaces is still lacking.3 Cell surface hydropbobicity can be inferred from water contact angles on bacterial lawns.4 For instance, water contact angles on oral streptococci can be as high as 103° for Streptococcus mitis strains but as low as 19° for Streptococcus rattus.5 Extensive evaluations of a large number of bacterial strains and species have demonstrated that cell surface hydrophobicity is conveyed mostly to the cell surface through nitrogencontaining groups, especially for oral streptococci.6 Nearly all bacterial cell surfaces found in nature are negatively charged.7 Hence, from an overall physicochemical point of view, they are expected to be repelled by negatively charged substratum surfaces. However, Hayashi et al.8 and Poortinga et al.9 have both described that this electrostatic repulsion is often overestimated due to the neglect of bacterial cell surface softness. Soft, ionpenetrable cell surfaces experience less electrostatic repulsion than similarly charged, hard, ion-impenetrable surfaces, since their diffuse double layer charges are driven into the ion-penetrable cell walls causing an effective decrease in surface potential and, hence, electrostatic repulsion. Recently, Morisaki et al. explained adhesion of (3) Bos, R.; Van der Mei, H. C.; Busscher, H. J. FEMS Microbiol. Rev. 1999, 23, 179. (4) Busscher, H. J.; Bialkowska-Hobrzanska, H.; Reid, G.; Kuijl-Booij, M.; Van der Mei, H. C. Colloids Surf., B 1994, 2, 73. (5) Van der Mei, H. C.; Bos, R.; Busscher, H. J. Colloids Surf., B 1998, 11, 213. (6) Van der Mei, H. C.; De Vries, J.; Busscher, H. J. Surf. Sci. Rep. 2000, 39, 1. (7) Wilson, W. W.; Wade, M. M.; Holman, C.; Champlin, R. F. J. Microbiol. Methods 2001, 43, 153. (8) Hayashi, H.; Tsuneda, S.; Hirata, A.; Sakasi, H. Colloids Surf., B 2001, 22, 149. (9) Poortinga, A. T.; Bos, R.; Busscher, H. J. Colloids Surf., B 2001, 20, 105.
10.1021/la020658l CCC: $25.00 © 2003 American Chemical Society Published on Web 02/07/2003
Micro- and Macroscopic Cell Surface Properties
a negatively charged marine bacterium, Vibrio alginolyticus, onto a negatively charged substratum by considering the softness of the strains.10 Although physicochemical approaches based on overall cell surface hydrophobicity and charge density have explained adhesion to substrata of many bacterial strains and species, generalization is still impossible. Probably, this must be attributed in part to the fact that in such physicochemical approaches bacterial cell surfaces have been considered as smooth, rigid, and chemically homogeneous. Contact angles and surface charge densities are both macroscopic properties reflecting the cell surface chemistry and structure. Calculation of the interaction energy between a negatively charged bacterial cell surface and a negatively charged substratum surface using the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory showed at low electrolyte concentration a shallow secondary minimum and a large potential energy barrier. If it was accounted for that only a minor number of positively charged sites existed on the cell surface, the energy barrier disappeared and highly adhesive conditions were revealed.11 In fact, the interaction between spirochetes and human erythrocytes has been described in terms of interactions between oppositely charged sites on the interacting surfaces, although both surfaces carry an overall negative charge on a macroscopic scale.12 Obviously, a minor number of positively charged sites, while instrumental for adhesion, hardly affects the macroscopic cell surface charge density. Although macroscopic cell surface characterization adds to our understanding of bacterial adhesion, it has provided relatively little information at the molecular level of the forces governing the adhesion process. The introduction of the atomic force microscope (AFM) and its application to biological surfaces13-16 has offered new possibilities to obtain microscopic physicochemical properties of bacterial cell surfaces. Recently, the turgor pressure of a spherical bacterium, Enterococcus hirae, in deionized water was derived from the indentation depth caused by an AFM tip and found to be between 4 and 6 × 105 Pa.17 Razatos et al. showed that the adhesion force between a silicon nitride AFM tip and Escherichia coli was affected by the length of lipopolysaccharide molecules on the cell surface and by the production of a capsular polysaccharide.18 Furthermore, it was discovered using AFM that E. coli JM109 and K12J62 have different surface morphologies dependent on environmental conditions, while lysozyme treatment led to the loss of surface rigidity and eventually to dramatic changes in bacterial shape.19 S. mitis is a Gram-positive oral bacterial strain that belongs to the primary colonizers of dental hard tissues and also adheres to mucous membranes, most notably the cheek and the tongue.20 By comparison with other oral streptococci, S. mitis strains usually carry sparsely (10) Morisaki, H.; Nagai, S.; Ohshima, H.; Ikemoto, E.; Kogure, K. Microbiology 1999, 145, 2797. (11) Van Loosdrecht, M. C.; Norde, W.; Zehnder, A. J. J. Biomater. Appl. 1990, 5, 91. (12) Dong, H.; Onstott, T. C.; Ko, C.-H.; Hollingsworth, A. D.; Brown, D. G.; Mailloux, B. J. Colloids Surf., B 2002, 24, 229. (13) Cowan, M. M.; Mikx, F. H. M.; Busscher, H. J. Colloids Surf., B 1994, 2, 407. (14) Dufreˆne, Y. F. Biophys. J. 2000, 78, 3286. (15) Meagher, L.; Griesser, H. J. Colloids Surf., B 2002, 23, 125. (16) Kidoaki, S.; Matsuda, T. Colloids Surf., B 2002, 23, 153. (17) Takahara, A.; Hara, Y.; Kojio, K.; Kajiyama, T. Colloids Surf., B 2002, 23, 141. (18) Yao, X.; Walter, S.; Burke, S.; Steward, S.; Jericho, M. H.; Pink, D.; Hunter, R.; Beveridge, T. J. Colloids Surf., B 2002, 23, 213. (19) Razatos, A.; Ong, Y.-L.; Sharma, M. M.; Georgiou, G. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 11059.
Langmuir, Vol. 19, No. 6, 2003 2373 Table 1. Macroscopic, Physicochemical Cell Surface Properties of Nine S. mitis Strains, Including Their Electrophoretic Cell Surface Softness 1/λ and Fixed Charge Density G,a Their Cell Surface Hydrophobicity by Water Contact Angle θw,b and Elemental Surface Concentration Ratio N/C by X-ray Photoelectron Spectroscopyc S. mitis
1/λ (nm)
r (106 C m-3)
θw (deg)
N/C
ATCC9811 ATCC33399 244 272 357 398 BMS BA T9
1.7 1.1 1.8 2.1 1.0 2.0 1.2 2.5 1.7
-2.2 -3.5 -2.3 -1.6 -4.3 -2.7 -3.9 -1.2 -2.2
68 56 60 54 53 59 100 103 91
0.110 0.106 0.097 0.131 0.119 0.116 0.124 0.129 0.125
a
Reference 23. b Reference 22. c Reference 6.
distributed but long fibrils.21 A collection of nine S. mitis strains has been macroscopically characterized with regard to their cell surface hydrophobicities by water contact angles22 and their surface charge properties using the soft-layer model as proposed by Ohshima.23 X-ray photoelectron spectroscopy has been carried out to determine the chemical composition of the cell surface.6 A summary of the results obtained from these studies is presented in Table 1. The aim of the present paper is to microscopically characterize the cell surfaces of these strains by atomic force microscopy and to find out if and how microscopic cell surface properties can be amalgamated into the macroscopic cell surface properties previously determined. Materials and Methods Bacterial Strains, Growth Conditions and Harvesting. The nine S. mitis strains used in this study were all cultured in Todd Hewitt Broth (Oxoid, Basingstoke, U.K.). For each experiment, the strains were inoculated from blood agar in a batch culture. This culture was grown overnight and used to inoculate a second culture that was grown for 16 h in ambient air prior to harvesting. Bacteria were harvested by centrifugation (5 min at 10 000g), washed twice with demineralized water, and resuspended in water. To break up bacterial chains, cells were sonicated for 30 s at 30 W (Vibra Cell model 375, Sonics and Materials Inc., Danbury, CT). Sonication was done intermittently while cooling in an ice/water bath. These conditions were found not to cause cell lysis in any strain. Atomic Force Microscope. Sample Preparation. Bacterial cells were suspended in water to a concentration of 105 per mL, after which 10 mL of the suspension was filtered through an Isopore polycarbonate membrane (Millipore) with a pore size of 0.8 µm.24 The pore size is chosen slightly smaller than the streptococcal dimensions to immobilize the bacteria by mechanical trapping. After filtration, the filter was carefully fixed with double-sided sticky tape on a sample glass and transferred to the AFM. AFM Imaging and Force-Distance Measurements. A Nanoscope III AFM (Digital Instruments, Santa Barbara, CA) operating in contact mode was used to image cells and to measure interaction forces. Measurements were taken at room temperature, either in deionized water or in a 0.1 M KCl solution. V-Shaped silicon nitride cantilevers from Park Scientific Instru(20) Bolshakova, A. V.; Kiselyovaa, O. I.; Filonova, A. S.; Frolova, O. Y.; Lyubchenkoc, Y. L.; Yaminskya, I. V. Ultramicroscopy 2001, 86, 121. (21) Marsh, P.; Martin, M. Oral Microbiology, 3rd ed.; Chapman and Hall: London, 1992. (22) Cowan, M. M.; Van der Mei, H. C.; Rouxhet, P. G.; Busscher, H. J. J. Gen. Microbiol. 1992, 138, 2707. (23) Van der Mei, H. C.; Busscher, H. J. Eur. J. Oral Sci. 1996, 104, 48. (24) Vadillo Rodrı´guez, V.; Busscher, H. J.; Norde, W.; Van der Mei, H. C. Electrophoresis, in press.
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Figure 2. Force distribution histogram for S. mitis 357 in water (a) and 0.1 M KCl (b), as derived from Figure 1, with the relative prevalence of each force indicated.
Figure 1. Force-distance curves for S. mitis 357 in water (a) and 0.1 M KCl (b). The solid lines represent the approach curve, while the dashed lines indicate the retraction curve. Minimum and maximum local maxima in adhesion force during retraction are marked by arrows. ments (Mountain View, CA) with a spring constant of 0.06 N m-1 and a probe curvature radius of ∼50 nm, according to manufacturer specifications, were used. Contact mode topographic images in deflection, height, and friction modes were taken with an applied force maintained below 1 nN at a scan rate of ∼2 Hz. Integral and proportional gains of the feedback loop were about two and three, respectively. Individual force curves with z-displacements of 100-200 nm were collected at z-scan rates of e1 Hz. The slope of the retraction force versus distance curves in the region where probe and sample are in contact was used to convert the position sensitive detector voltage into cantilever deflection. The direct result of such a force measurement is the cantilever deflection d versus position of the piezo z normal to the surface. To obtain a force-distance curve, d and z have to be converted into force and distance. The force F is obtained by multiplying the deflection d of the cantilever with its spring constant k, while the tip-sample separation D is calculated by subtracting the deflection d from the position z of the piezo. Topographic images were recorded for at least 10 bacterial cells of each S. mitis strain (either in deionized water or in a 0.1 M KCl solution). To this end, the tip was positioned over the top of a trapped bacterium, scanning was stopped, and 10 force measurements were performed at randomly selected locations around the top for each bacterial cell studied. The selected forcedistance curves (see Figure 1 for an example) were analyzed in order to determine various characteristic parameters. First, the approach part of a force curve was fitted to a negative exponential
F ) F0 exp-D/Λ
(1)
in which F is the measured force at separation distance D, F0 is the force at zero separation distance, and Λ is the characteristic decay length (separation distance over which F decays from F0
to F0/e). Second, the retraction part of a force curve usually showed various local attractive maxima. The magnitude of the adhesion forces, represented by these maxima, and the distances at which they occurred were quantitatively registered in histograms (see Figure 2 for an example), as well as the minimum Dmin and maximum Dmax distances at which local maxima in adhesion forces occurred. On the basis of the relative prevalence of the local maxima in adhesion forces (expressed in percentages in the histograms, Figure 2), an average attractive force Fadh between the AFM tip and the cell surface of each S. mitis strain was calculated too. Forces with a prevalence of less than 2% were neglected in this averaging process. Statistical Analysis. To determine possible relationships between the microscopic properties obtained by force curve analysis and the macroscopic physicochemical surface properties of the S. mitis strains, both sets of properties were submitted to a Pearson correlation test. Pearson’s correlation coefficient r reflects the strength and direction of the linear relationship between two variables. The outcome ranges from +1 to -1, where -1 is a perfect (inverse) correlation, 0 is no correlation, and +1 is a perfect positive correlation. In addition, to help the reader visualize the tendencies observed between our parameters, linear regression analyses between those parameters, showing high correlations in the Pearson analysis, were performed. Note, however, that strictly speaking the Pearson correlation test is different from linear regression analysis, as it constitutes only a rank testing.
Results Figure 1 presents an example of the interaction force between S. mitis 357 and a silicon nitride tip immersed in water and 0.1 M KCl solution, respectively, as a function of the separation distance between the tip and the bacterium. Interestingly, the approach curve is always repulsive and does not show attraction, not even at a short separation distance. Instead, the approach curve shows a gradually increasing repulsion between the tip and the cell surface that increases exponentially at close approach until contact at F0. Repulsive forces at contact and decay lengths are summarized in Table 2 for all nine strains. Repulsive forces at contact in water can either be larger (S. mitis ATCC9811, 357, and BA) or smaller (S. mitis 244, 398, and BMS) than in 0.1 M KCl, while for the strains S. mitis ATCC33399, 272, and T9 there was no statistical difference between them. The decay lengths in water
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Table 2. Summary of Quantitative Data Derived from Force-Distance Curves in Water and 0.1 M KCl of Nine S. mitis Strains, Including the Force at Zero Separation Distance F0 and Its Decay Length Λ (Both from Approach Curves) and Minimum Dmin and Maximum Dmax Distances at Which Local Maxima in Adhesion Forces Are Registered, Together with the Number of Local Maxima N and the Average Adhesion Force Fadh Calculated (All from Retraction Curves) approach water
retraction 0.1 M KCl
water
0.1 M KCl
S. mitis
F0 (nN)
Λ (nm)
F0 (nN)
Λ (nm)
Dmin (nm)
Dmax (nm)
Fadh (nN)
N
Dmin (nm)
Dmax (nm)
Fadh (nN)
N
ATCC9811 ATCC33399 244 272 357 398 BMS BA T9
8.0 ( 1.0 3.4 ( 1.4 1.0 ( 0.4 4.7 ( 1.8 2.9 ( 0.5 3.2 ( 0.8 4.1 ( 1.5 4.5 ( 1.2 2.7 ( 0.7
33 ( 5 12 ( 2 10 ( 2 11 ( 2 20 ( 7 11 ( 3 33 ( 11 33 ( 8 25 ( 4
2.1 ( 0.8 3.5 ( 0.7 2.8 ( 0.7 3.6 ( 1.4 1.1 ( 0.2 7.7 ( 1.0 1.6 ( 0.8 2.7 ( 0.6 4.8 ( 2.0
50 ( 6 9(2 13 ( 3 13 ( 6 10 ( 2 8(1 10 ( 3 8(1 11 ( 3
64 ( 34 21 ( 11 17 ( 2 10 ( 0 37 ( 12 38 ( 25 50 ( 16 106 ( 24 43 ( 16
585 ( 40 98 ( 39 280 ( 31 977 ( 25 235 ( 28 167 ( 30 459 ( 32 642 ( 24 910 ( 30
-1.3 ( 0.6 -1.3 ( 0.8 -0.9 ( 0.4 -1.3 ( 0.7 -1.3 ( 0.3 -1.8 ( 1.0 -1.5 ( 0.8 -2.9 ( 1.2 -1.4 ( 0.8
6 2 4 6 3 2 5 4 9
78 ( 28 14 ( 4 59 ( 28 29 ( 11 13 ( 0 36 ( 14 43 ( 10 36 ( 2 53 ( 19
167 ( 33 170 ( 30 426 ( 29 534 ( 29 196 ( 43 120 ( 35 867 ( 40 588 ( 23 1186 ( 38
-0.5 ( 0.4 -1.4 ( 0.9 -1.8 ( 1.1 -1.0 ( 0.5 -0.7 ( 0.3 -1.5 ( 0.7 -1.1 ( 0.4 -1.4 ( 0.3 -2.2 ( 1.0
2 3 4 6 3 2 9 5 11
Table 3. Pearson Correlation Coefficients between Microscopic Cell Surface Characteristics from AFM and Macroscopic Cell Surface Characteristics in Water or in 0.1 M KCl for Nine S. mitis Strainsa
a
Correlation coefficients below 0.5 have been omitted, while correlations marked in bold have been graphically presented in this paper.
(“group average” of 21 ( 10 nm) tend to be larger than in 0.1 M KCl (“group average” of 15 ( 13 nm) for four out of the nine strains studied. Upon retraction, multiple local maxima in adhesion forces can be observed. The separation distances at which these adhesion forces occur, their magnitude, and their relative prevalence as averaged per strain can be summarized in so-called distribution force histograms (see Figure 2, showing the distribution force histogram for S. mitis 357). The minimal and maximal distances at which local adhesion forces occur are presented in Table 2, together with the average adhesion force arising from a bacterial cell surface as calculated from the relative prevalence of all local adhesion forces. Adhesion forces tend to be slightly stronger in water than in 0.1 M KCl, but not for all S. mitis strains involved in this study. Some strains, such as S. mitis ATCC33399 and 398, possess only two maxima in local adhesion forces at small separation distances, whereas other strains such as T9 express nine local adhesion forces at separation distances up to 910 nm. To determine possible correlations between the microscopic cell surface characteristics revealed by the AFM force curves and previously published macroscopic cell surface properties (see Table 1), Pearson’s correlation coefficients r between all parameters were calculated (Table 3). In water, the minimum distance Dmin at which adhesion forces occur correlates well with the average
Figure 3. Nitrogen surface concentration ratio of S. mitis strains obtained by XPS as a function of the maximum distance Dmax at which a local maximum in adhesion force was registered by the AFM tip upon retraction in water.
adhesion force Fadh (r ) -0.85). The maximum distance Dmax at which adhesion occurs correlates with the amount of surface nitrogen as detected by X-ray photoelectron spectroscopy (XPS) (r ) 0.67) and also with the amount of fixed cell surface charge determined by electrophoresis analysis (r ) 0.64). Cell surface hydrophobicity by water contact angles correlates with Dmin and the average adhesion force Fadh (r ) 0.73 and -0.63, respectively).
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Figure 5. Fixed charge density of the bacterial cell wall of S. mitis strains as a function of the minimum distance Dmax at which a local maximum in adhesion force was registered by the AFM probe tip upon retraction in water.
angle, the fixed charge density correlates best with Dmax, as can be seen in Figure 5. Discussion
Figure 4. Water contact angle on lawns of S. mitis strains as a function of (a) the minimum distance Dmin at which a local maximum in adhesion force was registered by the AFM tip upon retraction in water, (b) the average attractive force Fadh between the AFM tip and the cell surface of each S. mitis strain in water, and (c) the maximum distance Dmax at which a local maximum in adhesion force was registered by the AFM tip upon retraction in 0.1 M KCl.
Remarkably, while cell surface hydrophobicity correlates best with Dmin in water, it correlates better with Dmax in 0.1 M KCl. Figures 3-5 present some of the correlations between different properties as revealed by the Pearson correlation test. Figure 3 shows that the amount of macroscopically measured surface nitrogen influences, at least in water, the maximum distance at which adhesion forces occur in the retracting mode. In Figure 4a, it can be seen that when the affinity of the cell surface for water is smallest (larger contact angle), the minimum distance over which adhesion forces in water were observed by the hydrophilic tip of the AFM is largest. Interestingly, in 0.1 M KCl, the water contact angle correlates best with Dmax (see Figure 4c). Although the water contact angle reflects the affinity of the surface for a hydrophilic medium such as water, the average adhesion force experienced by the AFM tip upon retracting from the surface correlates inversely with the contact angle (see Figure 4b). Similar to the water contact
The frequency of occurrence of surface structures with variable lengths on S. mitis strains is high and differs considerably between different isolates. In general, the density of fibrils on S. mitis strains has been described as sparse. Macroscopic cell surface properties of these strains have been extensively studied, but it is unknown how the microscopic properties of the cell surface, as determined for example by atomic force microscopy, make up the macroscopic properties of the cell surfaces. Force-distance curves for the S. mitis isolates involved in this study show two common features, regardless of the environmental conditions: a repulsive force upon approach and multiple adhesion forces upon retraction. Thus, the interaction is attractive but has to overcome an energy barrier upon approach. The repulsive interaction upon approach has a decay length that varies between 11 and 33 nm in water with associated contact forces of between 1.0 and 8.0 nN, dependent on the strain considered (see Table 2), while in 0.1 M KCl these ranges are between 8 and 50 nm and 1.1 and 7.7 nN, respectively. Note that although an electrostatic interaction between the silicon nitride tip and the bacterial cell surface was expected, the decay lengths observed are considerably larger than the Debye-Hu¨ckel lengths (9.6 and 0.96 nm in water and 0.1 M KCl, respectively). Electrosteric forces resulting from the presence of surface structures, with different length and charge, probably increase the range and magnitude of these repulsive forces. Previously, also Camesano and Logan25 concluded that the interaction between negatively charged bacteria and the silicon nitride tip of an atomic force microscope was dominated by electrosteric repulsion. Moreover, the energy that the AFM tip has to overcome in order to reach the bacterial surface, that is, the area enclosed below the approach curve toward zero separation distance (contact point), can be calculated to be 2 times as high in water (∼20 000 kT) as in 0.1 M KCl (∼10 000 kT). For a sphere-flat plate configuration, the DVLO theory also predicts a higher energy barrier in water than in 0.1 M KCl, although the energy barriers calculated in water (∼400 kT) and in 0.1 M KCl (∼40 kT) do not correspond numerically with those calculated from (25) Kasas, S.; Ikai, A. Biophys. J. 1995, 68, 1678.
Micro- and Macroscopic Cell Surface Properties
the AFM approach curves. Recently, Ong et al.26 compared forces of interaction measured with the AFM to those of model predictions based on an extended-DVLO approach. They concluded that the inclusion of steric interactions in the theoretical models leads to an additional repulsive component for the wild-type E. coli strain, expressing long lipopolysaccharide molecules on its cell surface. Accounting for the long appendages carried by S. mitis strains, this discrepancy between theory and experimental data maybe be due as well to steric interactions. Upon retraction, multiple adhesion forces between the tip and the various S. mitis cell surfaces were observed. Hypothetically, these adhesion forces might be due to interactions between the tip and fibrils of different lengths on the cell surfaces. Moreover, the different lengths may represent different molecules and are not necessarily associated with a single polymer. Adhesion forces have been found to extend up to 1186 nm for S. mitis T9 in buffer solution. While these distances may appear large, they are of the order of magnitude of fibrillar lengths observed with electron microscopy after negative staining.27 Also, Frank and Belfort28 found adhesion forces between an AFM tip and Pseudomonas atlantica extending over 1200 nm, which they ascribed to the presence of extracellular polysaccharides. The above interpretation of local adhesion forces is supported by the observation that the number of local maxima in adhesion forces N (see Table 2) is not very sensitive to ionic strength, although in general the adhesion forces tend to be slightly stronger in water than in 0.1 M KCl. The relations between macroscopic and microscopic cell surface properties reported in Table 3 are not always evident. Amounts of macroscopically measured surface nitrogen clearly increase with the maximum distance over which an adhesion force occurs in the retracting mode in water (Figure 3), as does the fixed charge density (Figure 5). Assuming that the nitrogen content reflects the length and density of the fibrils present on these strains and the charge is associated with components in these fibrils, the above relationships merely demonstrate that a thicker extracellular layer is required to accommodate those (26) Camesano, T. A.; Logan, B. E. Environ. Sci. Technol. 2000, 34, 3354. (27) Handley, P. S. Biofouling 1990, 2, 239. (28) Frank, B. P.; Belfort, G. Langmuir 1999, 13, 66234.
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components. The water contact angle, however, results from short-range molecular interactions: the more hydrophobic the inner dense layer of the cell surface (higher water contact angle), the stronger the adhesion force (Figure 4b) and the larger the minimum separation distance at which maximum adhesion is observed in water (Figure 4a). We hypothesize that the forces controlling the affinity of the bacterial cell surfaces for water arise largely from the presumably more dense inner layer of the cell surface, represented by its thickness Dmin. In 0.1 M KCl, however, the water contact angle correlates best with the maximum separation distance over which an adhesion force occurs (Figure 4c), which is opposite to our expectations. Further, it was seen that even though the water contact angle reflects the affinity of the surface for a hydrophilic medium such as water, the average adhesion force experienced by the AFM tip upon retracting from the surface correlates inversely with the contact angle. The high hydrophobicity of S. mitis surfaces in water could stimulate dehydration to account for the stronger adhesion forces probed by the hydrophilic AFM tip on more hydrophobic strains. This phenomena of hydrophobic dehydration of one of the two interacting surfaces has also been reported to be responsible for protein adsorption on solid surfaces.29 If the surfaces of the protein molecule and the sorbent are polar (hydrophilic), their hydration is favorable. In that case, it is probable that some hydration water is retained between the sorbent surface and the adsorbed protein molecule. However, if (one of) the surfaces are (is) apolar, that is, hydrophobic, dehydration would be a driving force for adhesion.29 In summary, this study is the first attempt to correlate microscopic and macroscopic bacterial cell surface properties. Although some of the relations between macroscopic and microscopic cell surface properties can be readily understood, full understanding of all relations observed requires a better theory describing short-range molecular interactions than is hitherto available. Moreover, this problem is more complicated, as the force-distance curves confirm that the cell surface is not homogeneous and that by consequence, water contact angles and charge densities reflect only average properties. LA020658L (29) Haynes, C. A.; Norde, W. Colloids Surf., B 1994, 2, 517.
