University of Groningen
Bacterial interaction forces in adhesion dynamics
Boks, Niels
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to
cite from it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date:
2009
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Boks, N. P. (2009). Bacterial interaction forces in adhesion dynamics Groningen: s.n.
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the
author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately
and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the
number of authors shown on this cover page is limited to 10 maximum.
Download date: 17-06-2017
CHAPTER 7
GENERAL DISCUSSION
Chapter 7
Introduction
Microbial adhesion onto surfaces is a problem occurring in many fields and is
therefore widely studied [1-3]. Adhesion takes place when there is sufficient
affinity of the microbial cell surface for a substratum surface. To determine
these affinities, several experimental techniques as well as theoretical
predictions are available [4-8].
In this thesis we performed experiments in the parallel plate flow
chamber, as well as force measurements with an atomic force microscope
(AFM) to determine bacterial adhesion parameters. The extended DLVO-theory
was used as a theoretical reference.
Adhesion in the parallel plate flow chamber
In the parallel plate flow chamber, the deposition process occurs without
imposing forced contact and allows to determine several interaction parameters,
like the hydrodynamic force to prevent adhesion (Fprev), the hydrodynamic force
to detach adhering bacteria (Fdet) and desorption rate coefficients. Initial
interaction parameters, i.e. Fprev and initial desorption rate coefficient (β0)
showed no clear relation with the hydrophobicity of the substratum surfaces
used. Furthermore, regardless of the substratum used some general trends were
observed. The final desorption rate coefficient (β∞) was always lower than β0
and Fdet was always larger than Fprev, although no correlation between them was
observed. These parameters together indicate that over time the adhesive bond
between bacteria and surfaces strengthens and that adhesion and desorption are
independent processes. However, a clear influence of substratum surface
hydrophobicity was observed when comparing Fdet and β∞ on hydrophilic glass
and hydrophobic DDS-coated glass. As a rule, Fdet was larger and β∞ was lower
122
General discussion
on the hydrophobic substratum surface. Interestingly, also the adhesion
dynamics differed on both surfaces. On hydrophilic glass, a larger fraction of the
adhering bacteria slid along the surface, while on DDS-coated glass bacteria
tended to stick to the surface. All these observations lead to the conclusion that
adhesion of bacterial strains on hydrophobic DDS-coated glass was more
favourable than on hydrophilic glass.
Adhesion in Atomic Force Microscopy
In contrast to experiments in the parallel plate flow chamber, in AFM
experiments bacteria and surfaces are forced into contact. After 0 s of contact
between the bacterial probe and the substratum surface, retract curves showed
that adhesion forces (F0s) were hardly affected by substratum surface
hydrophobicity. However, upon prolonged contact between bacterium and the
hydrophilic substratum surface, adhesion forces gradually became stronger,
whereas this effect was virtually absent on the hydrophobic surface. This lead to
the conclusion that hydrophilic glass was a more favourable substratum surface
to adhere to, contradicting the findings in the parallel plate flow chamber.
Combining experimental data with theoretical predictions
Initial adhesion is governed by macroscopic interaction forces as described in
the extended-DLVO-theory for colloid stability [8]. In this theory, the total free
energy of interaction is described as the sum of Lifshitz-Van der Waals (LW)-,
electrostatic (EL)- and Lewis acid-base (AB) interactions as a function of
separation distance. Figure 1 gives an example of an extended-DLVO
interaction energy curve for a bacterium on hydrophilic glass and hydrophobic
DDS-coated glass. As can be seen from Figure 1, the major difference in
123
Chapter 7
predicted interaction energies is the AB-component, which also constitutes the
hydrogen bonding component of the free energy of cohesion of water [9], and
can directly be related to the substratum surface hydrophobicity.
Figure 1. Example of extended-DLVO profile (ΔGTOT) for Staphylococcus
epidermidis HBH2 169 as well as contributions of the Lifshitz-Van der Waals (ΔGLW),
electrostatic (ΔGEL) and Lewis acid-base (ΔGAB) interactions on hydrophilic glass and
hydrophobic DDS-coated glass. Positive values denote repulsion, negative values
denote attraction.
On hydrophilic glass, hydrogen bonds between the surface and surrounding
water molecules can occur easily, which need to be broken first before a
bacterium can make contact with the surface. As a consequence, the ABinteraction on hydrophilic glass is repulsive (Figure 1). Conversely, on
124
General discussion
hydrophobic DDS-coated glass surrounding water molecules cannot bind
through hydrogen bonding and are restricted in their rotational freedom. As a
consequence their release from the surface is entropically more favourable upon
approach of a bacterium, resulting in attractive AB-interactions (Figure 1). It is
clear that, if there is any influence of surface hydrophobicity on adhesion, this is
only effective at close contact between bacteria and substratum surfaces.
Initially, bacteria will approach the surface towards a shallow secondary
minimum of interaction according to LW- and EL-interaction forces. As can be
seen from Figure 1, this minimum does not differ much on both surfaces.
Interestingly, none of the parameters concerning initial adhesion (i.e. Fprev, β0
and F0s), although different in magnitude and nature, show an unambiguous
influence of the substratum surface. It is therefore very likely that initial
adhesion is governed by long-range (LW) interaction forces in combination with
electrostatic interactions and that time and close approach are needed for a
significant effect of surface hydrophobicity on the observed adhesion
parameters.
When, in case of free, non-forced adhesion, as occurs in the flow
chamber, contact times were prolonged, surface appendages (like, for example,
extracellular polymeric substances, pili and fimbriae) present on the adhering
bacterium might be able to bridge with the hydrophobic DDS-coated glass due
to a favourable short-range AB-interaction. On hydrophilic glass these
interactions are repulsive which does not only cause less favourable final
adhesion parameters Fdet and β∞, but also in a higher percentage of mobile
adhering bacteria. At this point it is interesting to note that in case of a more
biologically relevant adhesion system (i.e. adhesion of Staphylococcus aureus to
fibronectin or bovine serum albumin in PBS), mobile adhesion was virtually
absent, regardless of the possibility of specific interaction. Here, a combined
125
Chapter 7
effect of ligand-receptor binding and high ionic strength prevented a rolling or
sliding mode of adhesion.
In the case of forced adhesion in AFM, the situation is different. Lewis
acid-base interactions are mechanically overcome by pressing the bacterium to
the surface. Once the water molecules adjacent to the substratum surface, as well
as those associated with the outside of the bacterial cell wall are removed into
the bulk, sites for hydrogen bond formation become available. However, DDScoated glass is apolar and therefore not capable to facilitate hydrogen binding
with the bacterium. Conversely, on hydrophilic glass numerous hydrogen
binding sites are present facilitating a steady increase in adhesion strength with
prolonged time.
At this point it should be noted that the forces measured in the flow
chamber (Fprev and Fdet) are orders in magnitude lower than adhesion forces
measured with AFM. A probable cause is the fact that AFM measurements
probe the force normal to the substratum surface, whereas Fprev and Fdet are
indicative force strengths in the tangential direction. Furthermore, by pressing
the bacteria towards the surface in AFM, deformation of the outer layer of the
bacterial cell wall may occur. This increases the contact area between bacterium
and substratum surface and therewith also the number of binding sites and as a
consequence the measured adhesion force.
Conclusions
Direct correlation of adhesion forces measured in a parallel plate flow chamber
and an atomic force microscope, is obscured by the nature of both experimental
techniques. Force values measured in a flow chamber, resemble the natural
process best and give the most accurate approximation of bioadhesion in vivo. In
this situation, attractive AB-interactions, due to release water molecules, cause a
126
General discussion
stronger attachment to a hydrophobic substratum surface, probably caused by
bridging of extracellular polymeric substances. On hydrophilic glass, ABinteractions are repulsive, making bridging more difficult. In AFM experiments,
interaction forces are measured that are, because of the forced contact, very
often not reached in naturally occurring adhesion. After the initial release of
water molecules into the bulk solution upon close approach of a bacterium, the
apolar groups present on the hydrophobic substratum surface cannot form
hydrogen bridges directly with the rather hydrophilic bacterial cell surface,
preventing strengthening of the initial adhesion force. The opposite is true for
adhesion to hydrophilic glass, where numerous sites for hydrogen bonds are
present, facilitating a gradual increase of the adhesion force and contradicting
the findings in the flow chamber.
This means that researchers have to be careful in using AFM to prove that
microbial adhesion on one surface is more favourable as compared to another,
especially when these surfaces are very different in nature. However, both
experimental techniques indicate the importance of hydrogen bonding capability
in bacterial adhesion to non-specifically binding (inert) surfaces, especially with
respect to bond strength and associated adhesion dynamics.
Future research
To gain a better insight in the influence of substratum surface hydrophobicity on
adhesion dynamics, more experiments with substratum surfaces with
intermediate hydrophobicities should be performed. Also, in elucidating
microbial adhesion dynamics in biologically relevant adhesion systems (i.e. via
specific ligand-receptor binding), it is advisable to perform such experiments in
suspending media containing biopolymers to better mimic natural conditions.
However, it should be noted that in systems where ligand-receptor binding does
127
Chapter 7
not play a role, high ionic strength influences the dynamics significantly [10].
Therefore in elucidating adhesion dynamics in biologically more relevant
systems, experiments should be performed with media of different ionic
strengths, even though this implies that experiments may lose some of their
biological relevance.
Furthermore, a suggestion can be made with respect to direct force
measurements. AFM was, and may still be, a golden standard for direct
determination of interaction forces. But nowadays also optical tweezers can be
used to trap a single cell and probe its interactions with a surface [11]. The
advantage is that contact is not forced and perpendicular forces might be probed
more naturally, as compared to AFM. Interestingly, forces obtained using
optical tweezers are generally also in a similar range as obtained in a flow
chamber (i.e. 10-12 N [11,12]) and may lead to better correlations between
tangential and perpendicular forces. Therefore, this technique should be
optimized for measurements that allow direct force measurements of whole cells
on surfaces.
References
1. Cooksey, K.E. and Wigglesworth-Cooksey, B. (1995), Adhesion of bacteria and
diatoms to surfaces in the sea - A review, Aquat Microb Ecol 9, 87 - 96.
2. Costerton, J.W., Stewart, P.S. and Greenberg, E.P. (1999), Bacterial biofilms: A
common cause of persistent infections, Science 284, 1318 - 1322.
3. Flemming, H.C. (2002), Biofouling in water systems - Cases, causes and
countermeasures, Appl Microbiol Biot 59, 629 - 640.
4. Azeredo, J., Visser, J. and Oliveira, R. (1999), Exopolymers in bacterial adhesion:
Interpretation in terms of DLVO and XDLVO theories, Colloid Surface B 14, 141 - 148.
128
General discussion
5. Fang, H.H.P., Chan, K.Y. and Xu, L.C. (2000), Quantification of bacterial adhesion
forces using atomic force microscopy (AFM), J Microbiol Meth 40, 89 - 97.
6. Mendez-Vilas, A., Gallardo-Moreno, A.M., Gonzalez-Martin, M.L., CalzadoMontero, R., Nuevo, M.J., Bruque, J.M. and Perez-Giraldo, C. (2004), Surface
characterisation of two strains of Staphylococcus epidermidis with different slimeproduction by AFM, Appl Surf Sci 238, 18 - 23.
7. Owens, N.F., Gingell, D. and Rutter, P.R. (1987), Inhibition of cell-adhesion by a
synthetic polymer adsorbed to glass shown under defined hydrodynamic stress, J Cell
Sci 87, 667 - 675.
8. Hermansson, M. (1999), The DLVO theory in microbial adhesion, Colloid Surface B
14, 105 - 119.
9. Van Oss, C.J. (2003), Long-range and short-range mechanisms of hydrophobic
attraction and hydrophilic repulsion in specific and aspecific interactions, J Mol
Recognit 16, 177 - 190.
10. Castelain, M., Pignon, F., Piau, J.M. and Magnin, A. (2008), The initial single yeast
cell adhesion on glass via optical trapping and Derjaguin-Landau-Verwey-Overbeek
predictions, J Chem Phys 128, 135101-1 - 135101-14
11. Sharp, J.M., Clapp, A.R. and Dickinson, R.B. (2003), Measurement of long-range
forces on a single yeast cell using a gradient optical trap and evanescent wave light
scattering, Colloid Surface B 27, 355 - 364.
12. Fallman, E., Schedin, S., Jass, J., Andersson, M., Uhlin, B.E. and Axner, O. (2004),
Optical tweezers based force measurement system for quantitating binding interactions:
system design and application for the study of bacterial adhesion, Biosens Bioelectron
19, 1429 - 1437.
129
Chapter 7
130