1 Department of Biomedical Engineering, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
2 Laboratory of Physical Chemistry and Colloid Science, Wageningen University, Dreijenplein 6, 6703 HB Wageningen, The Netherlands
Correspondence
Henny C. van der Mei
H.C.van.der.Mei{at}med.rug.nl
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ABSTRACT |
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INTRODUCTION |
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Until recently, bacterial adhesion has been evaluated mainly by enumeration of the number of bacteria adhering to a surface (An & Friedman, 1997). The parallel-plate flow chamber has turned out to be an effective tool for studying bacterial adhesion to surfaces (McClaine & Ford, 2002
). With the aid of phase-contrast microscopy, ultra-long-working-distance objectives and image analysis, bacterial adhesion can be monitored in real time and enumeration is instantaneous under the shear conditions of the actual experiments. The use of flow devices, however, is time-consuming and results provide no quantitative information on the magnitude of the interaction forces between bacteria and a surface.
Atomic force microscopy (AFM) provides new possibilities for probing the structural and physical properties of bacterial cells, as well as information on interaction forces involved in adhesion (Dufrêne, 2000, 2002
). Using topographic imaging, cell surface nanostructures (e.g. appendages and flagella) have been visualized, including changes of cell surface morphology during physiological processes. E. coli JM109 and K12J169, for instance, have been found to possess different morphologies depending on whether topographic images were taken in liquid or in air. Imaging in air revealed many topographic features that were missing for the cells imaged in liquid. The loss of topographic features in liquid was attributed to the dynamics of cellular filaments, which may collapse onto the wall surface, creating a strain-specific topography of the surface. Furthermore, it was observed that lysozyme treatment led to the loss of surface rigidity and eventually to dramatic changes in bacterial shapes (Bolshakova et al., 2001
). Forcedistance curves provide complementary information on surface properties, including viscoelasticity and localized surface charge density and hydrophobicity. Interestingly, forces measured between the AFM tip and sulfate-reducing bacteria showed that the adhesion forces at both the cellsubstratum periphery and the cellcell interface were higher than those measured in the centre top of the bacterial cell. This has been suggested to be due to the accumulation of extracellular polymeric substances in interfacial regions (Fang et al., 2000
). The role of hydrophobic interactions in bacterial adhesion at a microscopic level has been pointed out by Ong et al. (1999)
, measuring the interaction forces between E. coli-coated AFM probes and solids of different surface hydrophobicity. It was shown that both attractive forces and cell adhesion were promoted by the hydrophobicity of the substratum surfaces.
Streptococcus mitis is one of the primary colonizers of surfaces in the oral cavity and it colonizes both dental hard tissues and mucous membranes, most notably the cheeks and the tongue (Marsh & Martin, 1992). By comparison with other oral streptococci, S. mitis usually carries sparsely distributed but extremely long fibrils, as demonstrated by electron microscopy (Cowan et al., 1992
). It is thought that bacterial surface appendages could exert localized attraction and consequently function to bridge a gap between substratum and the bacterial cell during adhesion (Smit et al., 1986
).
The aim of the present study was to relate microscopic adhesion properties of S. mitis strains as derived from forcedistance curves obtained using AFM to their macroscopic adhesion to surfaces in a parallel-plate flow chamber.
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METHODS |
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Parallel-plate flow chamber and data analysis.
The flow chamber (internal dimensions: 76x38x0·6 mm) and image analysis system have been described in detail previously (Busscher & Van der Mei, 1995). Images were taken from the bottom glass plate (55x38 mm) of the parallel-flow chamber. The top plate of the chamber was also made of glass. Glass plates were cleaned by sonicating for 2 min in 2 % RBS35 surfactant solution in water (Omnilabo International), rinsed thoroughly with tap water, dipped in methanol, and again rinsed with demineralized water. The flow chamber was cleaned with Extran (Merck) and thoroughly rinsed with water and demineralized water. Prior to each experiment, all tubes and the flow chamber were filled with 0·1 M KCl solution, taking care to remove all air bubbles from the system. Once the system was filled, a bacterial suspension of 3x108 cells ml1 in 0·1 M KCl was allowed to flow through the system at a flow rate of 1·44 ml min1, corresponding to a Reynolds number of 0·6 and a shear rate at the wall of the flow chamber of 10·6 s1. Deposition was observed with a CCD-MXRi camera (High technology) mounted on a phase-contrast microscope (Olympus BH-2) equipped with a x40 ultra-long-working-distance lens (Olympus ULWD-CD Plan 40 PL). The camera was coupled to an image analyser (TEA; Difa). The bacterial suspension was perfused through the system for 4 h with recirculation at room temperature.
The total number of adhering bacteria per unit area n(t) was recorded as a function of time by image sequence analysis during 4 h and the affinity of an organism for the glass surface was expressed as the initial deposition rate j0, representing the initial increase of n(t) with time (see also Fig. 1). Note that since the initial deposition rate is extracted only from the first, initial adhesion data, it represents the affinity of the organisms for the substratum surface without intervening influences of interactions between adhering bacteria. From the total number of adhering bacteria per unit area as function of time n(t) and the number of particles adhering over the full duration of an experiment n
, i.e. saturation adhesion, the so-called characteristic adhesion time
was calculated using
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Atomic force microscopy.
Bacterial cells were suspended in water to a concentration of 105 ml1, after which 10 ml of the suspension was filtered through an Isopore polycarbonate membrane (Millipore) with a pore size of 0·8 µm (Dufrêne et al., 1999). The pore size was 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 measurements were made at room temperature under 0·1 M KCl solution, using an optical lever microscope (Nanoscope III, Digital Instruments) with an applied force maintained below 1 nN and a scan rate of
2 Hz. V-shaped silicon nitride cantilevers from Park Scientific Instruments with a spring constant of 0·06 N m1 and a probe curvature radius of
50 nm, according to the manufacturer's specifications, were used. Individual force curves with z-displacements of 100200 nm were collected at z-scan rates
1 Hz. The slope of the retraction force curves in the region where probe and sample are in contact was used to convert the voltage into cantilever deflection. The conversion of deflection data to force data was carried out as has been previously described by others (Dufrêne, 2000
).
Topographic images were recorded for at least ten bacterial cells per culture of each S. mitis strain. To this end, the tip was positioned over the top of a trapped bacterium, scanning was stopped and ten force measurements were performed at randomly selected locations around the top for each bacterial cell studied. Forcedistance curves for both approach and retract interactions were analysed as follows.
Approach.
To quantify steric interactions between the AFM tip and cell surface polymers upon approach, a model developed for grafted polymers at relatively high surface coverage was used. The force per unit area between two parallel flat surfaces (Fst) with only one coated with polymer has been modelled following the work of Alexander (1977) and De Gennes (1987)
. To describe the force between a spherical AFM tip and a flat surface, the model was modified by Butt et al. (1999)
by integrating the force per unit area over the tip surface to produce the interaction force
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Retraction.
Retraction curves of all nine S. mitis strains showed various local attractive maxima. These attractive maxima and the distances at which they occurred were quantitatively registered in histograms (see Fig. 3). Based on the relative prevalence of the local maxima in adhesion forces (expressed in percentages in the histograms), a mean attractive force Fadh, mean between the AFM tip and the cell surface of each S. mitis strain was calculated. Forces with prevalence less than 2 % were neglected in this averaging process.
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Fig. 4 shows the result of this modelling, displaying the interaction forces as divided by the effective radius of the tip.
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RESULTS |
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Relations found between microscopic adhesion properties of S. mitis strains as derived from forcedistance curves analysis and macroscopic adhesion in the parallel-plate flow chamber are depicted in Figs 57. Fig. 5
shows that the initial deposition rates j0 decrease as the force needed to achieve contact between the AFM tip and bacterial surface, i.e. Fst at zero separation distance, increases, indicating that the bacteria have to overcome a barrier before they can adhere. Note that S. mitis 398 constituted an exception to this behaviour for reasons unknown and the strain has been omitted from this analysis. The radius of the area blocked rblocked by an adhering organism decreases in an almost linear fashion with the maximum distance Dmax over which the adhesion forces detected by the AFM tip upon retraction are operative. Note, in addition, that the order of magnitude of the radii of these blocked areas and the distance over which the adhesion forces act are roughly similar (see Fig. 6
), while here S. mitis ATCC 9811 constituted an exception. In Fig. 7
, it can be seen that, unexpectedly, no relation could be found between the desorption rate coefficients
and the adhesion forces Fadh, mean.
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DISCUSSION |
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The approach curves in the AFM measurements were used to assess steric interactions in adhesion of the organisms. The interaction forces given in Fig. 4 demonstrate the relative importance of LifshitzVan der Waals, electrostatic, acidbase and steric terms for our collection of strains. X-DLVO interactions decay over a very short distance (
10 nm) and are therefore less influential on bacterial deposition to a substratum surface than steric forces that extend over much longer distances. Even though the X-DLVO does not show any energy barrier for adhesion, forcedistance curves for all nine S. mitis strains always presented a repulsive force upon approach. Based on the theoretical predictions and on the distance observed at which this repulsive force becomes effective, we conclude that the forcedistance curves for approach could only be described in terms of steric repulsion.
The steric interactions as obtained from AFM play an important role in relating microscopic cell surface properties and macroscopic adhesion in the parallel-plate flow chamber. According to X-DLVO theory and based on the analogy between the surface properties of the tip and the glass surface, macroscopic adhesion of S. mitis to the glass substratum in the parallel-plate flow chamber occurs under barrierless conditions (see Fig. 4). However, the AFM tip detected a steric energy barrier that must be overcome in order to achieve close contact with the bacterial surface. This energy barrier could be interpreted as an activation energy for the organisms to successfully deposit, as by the relation shown in Fig. 5
: the initial deposition rate increases as the force required to overcome the energy barrier by the AFM tip decreases. These forces range between 1·1 and 7·7 nN for the nine S. mitis isolates employed in this study, and integrating equation 3 over the entire interaction range yields activation energies of
2700 kT and 15 000 kT, respectively. These activation energies are prohibitively high for spontaneous adhesion to occur for the entire collection of strains studied. Therefore, even though steric repulsion forces can be used to predict adhesion according to Fig. 5
, they are not fully responsible for adhesion at the separation distances at which they become operative.
The steric model is not only useful in explaining aspects of bacterial adhesion to macroscopic surfaces, but also in better understanding the cell surface itself. L0 defines the equilibrium length of the bacterial surface polymers and under our specific ionic strength conditions amounts to several tens of nanometers, except for S. mitis ATCC 9811. Other studies, also on fibrillated strains, reported equilibrium lengths L0 in the order of several hundreds of nanometers (Camesano & Logan, 2000). The present study was done, however, in a relatively high ionic strength solution, which may condense the electrically charged polymers on the cell surface and yield a relatively thin layer. Macroscopic as well as microscopic measurements at various ionic strengths have provided evidence of a collapse of the fibrillated material at high ionic strength (Van der Mei et al., 1994
, 2000
).
The initial stages of macroscopic adhesion are governed mainly by interactions between the bacterial cell surfaces and the substratum surface, but in the more advanced stages, adhesion is in essence an interplay between interactions occurring between the substratum surface, a depositing bacterium and an already adhering one. It is interesting that this mechanism is confirmed by the combination of microscopic and macroscopic data shown in Fig. 6. As the distance over which the bacterium exerts adhesive forces extends, for instance through the extension of fibrils of different lengths (Van der Mei et al., 2000
), bacteria are brought closer together and the distance between adhering bacteria is reduced (Smit et al., 1986
), yielding smaller blocked areas.
Desorption rate coefficients are generally small for all S. mitis strains, on average in the order of
105 s1. The adhesion force measured by the AFM tip upon retraction was expected to be indicative of the desorption of bacteria in the parallel-plate flow chamber. Fig. 7
shows, however, that contrary to our expectation, no clear relation was found. Possibly, this has to do with the nature of the desorption process. Desorption in the parallel-plate flow chamber takes place as a spontaneous process under the prevailing shear conditions, while in AFM the contact between bacterium and surface substratum is forced to break by application of an external force.
Summarizing, this study demonstrates that the repulsive force probed by AFM upon approach of the tip to a bacterial cell surface corresponds to a steric activation energy barrier, governing the rate of initial, macroscopic adhesion of the organisms to a glass surface. Moreover, the maximum distance at which attractive forces are probed by AFM upon retraction of the tip is related to the area blocked by an adhering bacterium (i.e. the distance kept between adhering bacteria), but bacterial desorption could not be related to adhesive forces as probed by the AFM.
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Received 13 October 2003;
revised 1 December 2003;
accepted 19 December 2003.
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