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Bacterial interaction forces in adhesion dynamicsBoks, Niels

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Bacterial interaction forces in adhesion dynamics

Copyright © 2008 by N.P. Boks All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means without permission of the author and the publisher holding the copyright of the published articles. Printed by Gildeprint drukkerijen b.v., Enschede ISBN: 978-90-367-3638-1 (printed version) ISBN: 978-90-367-3667-1 (electronic version) Cover: Fluorescence image of staphylococci attached to a tipless AFM-cantilever. Using a LIVE/DEAD Baclight viability stain (Molecular Probes Europe BV, Leiden, The Netherlands), bacteria appear red when they are dead and green when they are still alive. Omslag: Fluorescentie opname van staphylococci die aan een tiploze AFM-cantilever zijn geplakt. Met behulp van een LIVE/DEAD Baclight viability kleuring (Molecular Probes Europe BV, Leiden, The Netherlands) kleuren dode bacteriën rood en levende bacteriën groen.

Bacterial interaction forces in adhesion dynamics

Proefschrift

ter verkrijging van het doctoraat in de Medische Wetenschappen

aan de Rijksuniversiteit Groningen op gezag van de

Rector Magnificus, Dr. F. Zwarts, in het openbaar te verdedigen op

woensdag 14 januari 2009 om 16.15 uur

door

Niels Peter Boks geboren op 3 april 1979

te Apeldoorn

Promotores: Prof. dr. ir. H.J. Busscher Prof. dr. ir. W. Norde

Prof. dr. H.C. van der Mei Beoordelingscommissie: Prof. dr. Y. Ren Prof. dr. ir. M. Cohen-Stuart Prof. dr. ir. M. van Loosdrecht

Paranimfen: Anika Embrechts

Eefje Engels

Voor Froukje Visser - de Vries * 08-12-1906

† 24-03-1995

CONTENTS

Chapter 1 General introduction 1

Chapter 2 Forces involved in bacterial adhesion to hydrophilic

and hydrophobic surfaces

9

Chapter 3 Residence time dependent desorption of Staphylococcus epidermidis from hydrophilic and hydrophobic substrata

45

Chapter 4 Mobile and immobile adhesion of staphylococcal strains to hydrophilic and hydrophobic surfaces

57

Chapter 5 Bond-strengthening in staphylococcal adhesion to hydrophilic and hydrophobic surfaces using AFM

77

Chapter 6 Fibronectin interactions with Staphylococcus aureus with and without fibronectin-binding proteins and their role in adhesion and desorption

95

Chapter 7 General discussion

121

Summary

131

Samenvatting

137

Dankwoord

145

Curriculum vitae 150

CHAPTER 1

GENERAL INTRODUCTION

Chapter 1

2

Microbial adhesion

Adhesion of micro-organisms to surfaces and their subsequent growth into a

biofilm is a problem occurring in many fields of application. Bacterial biofilms

on pipes and heat exchangers in industry result in high costs when equipment

has to be cleaned or replaced [1,2]. In medicine, biofilms on biomedical

materials (like prosthetic implants or urinary catheters) lead to infections,

causing expensive treatments and enormous discomfort, and sometimes even

death of a patient [3-5]. In order to prevent these problems, it is important to

know more about microbial affinity for substratum surfaces, which governs the

first step in biofilm formation. Microbial affinity can be expressed in terms of

initial adhesion rate [6-8], deposition (or collision) efficiency [9] or in number

of adhering bacterial cells after a few hours [8]. However, none of these

parameters provide information on the adhesion strength between a micro-

organism and substratum surface.

The strength of microbial adhesion to substratum surfaces, is at least

equally important if not more so than data on numbers of adhering organisms. In

medicine bacterial desorption from one location may lead to an infection

elsewhere in the body. An example of the importance of bacterial adsorption

and desorption is found in the daily use of contact lenses. Wearing contact

lenses increases the risk of microbial keratitis (i.e. inflammation of the cornea)

[10]. Bacterial adhesion to contact lenses occurs during manual handling while

putting the lens onto the eye, but also during storage in a lens box [11]. Once the

contact lens is placed on the epithelium of the cornea, bacterial desorption from

the lens surfaces occurs and bacteria may adhere to the epithelium, with the

possibility to cause microbial keratitis.

General introduction

3

Adhesion forces

In literature, adhesion strengths between bacteria and surfaces are calculated

theoretically using the (extended-) DLVO theory (named after Derjaguin,

Landau, Verwey and Overbeek) [12-14] or measured e.g. by using centrifugal

force assays [15], laser tweezers [16-18] or total internal reflection microscopy

related techniques [19-22]. Most frequently used however, are atomic force

microscopy (AFM) [23-25] and fluid flow devices [26-29]. Because the flow

profile in these devices is well controlled [30], they are suitable to determine the

shear rate to prevent adhesion of bacteria or to detach adhering bacteria. These

shear rates provide an averaged adhesion force for a bacterial population.

Conversely, in AFM adhesion forces are probed directly between a substratum

surface and an individual bacterial cell.

As can be seen from Table 1, the magnitude of the force range estimated

for microbial interaction forces with substratum surfaces is greatly dependent on

the method used. For example, predicting interaction forces using the DLVO-

theory result in the weakest forces, while AFM yields forces that are up to 105

times stronger. It is unclear why these different techniques each yield their own

class of force values.

Table 1. Average force ranges for the interaction of micro-organisms with substratum

surfaces reported in the literature and obtained with different techniques.

Method of force measurement Force range (N) References

Fluid flow devices 10-13 – 10-11 [26,31]

Air bubble detachment 10-9 – 10-7 [32-34]

AFM 10-10 – 10-9 [35-38]

DLVO 10-14 – 10-10 [12,39,40]

Chapter 1

4

In conclusion, commonly accepted, well documented data on microbial

adhesion forces do not exist, because different methods yield widely varying

results and in many cases studies do not contain enough strains to warrant

generally valid conclusions [41].

Aim of this thesis

The main aim of this thesis is to develop an understanding of the reason(s) why

different techniques yield different ranges for microbial interaction forces with

substratum surfaces. To achieve this aim, adhesion forces, together with data on

adhesion dynamics, will be systematically obtained on hydrophobic and

hydrophilic surfaces for a wide variety of bacterial strains and using different

techniques.

References 1. Videla, H.A. (2002), Prevention and control of biocorrosion, Int Biodeter Biodegr 49,

259 - 270.

2. Visser, J. and Jeurnink, T.J.M. (1997), Fouling of heat exchangers in the dairy

industry, Exp Therm Fluid Sci 14, 407 - 424.

3. Dankert, J., Hogt, A.H. and Feijen, J. (1986), Biomedical polymers - Bacterial

adhesion, colonization, and infection, Crit Rev Biocompat 2, 219 - 301.

4. Harris, L.G. and Richards, R.G. (2006), Staphylococci and implant surfaces: A

review, Injury 37, 3 - 14.


5

5. Schierholz, J.M. and Beuth, J. (2001), Implant infections: A haven for opportunistic

bacteria, J Hosp Inf 49, 87 - 93.

6. Gallardo-Moreno, A.M., Gonzalez-Martin, M.L., Bruque, J.M. and Perez-Giraldo,

C. (2004), The adhesion strength of Candida parapsilosis to glass and silicone as a

function of hydrophobicity, roughness and cell morphology, Colloids and Surface A

249, 99 - 103.

7. Van Merode, A.E.J., Duval, J.F.L., Van der Mei, H.C., Busscher, H.J. and Krom,

B.P. (2008), Increased adhesion of Enterococcus faecalis strains with bimodal

electrophoretic mobility distributions, Colloids Surface B 64, 302 - 306.

8. Roosjen, A., Kaper, H.J., Van der Mei, H.C., Norde, W. and Busscher, H.J. (2003),

Inhibition of adhesion of yeasts and bacteria by poly(ethylene oxide)-brushes on glass

in a parallel plate flow chamber, Microbiol-Sgm 149, 3239 - 3246.

9. Cail, T.L. and Hochella, M.F. (2005), the effects of solution chemistry on the sticking

efficiencies of viable enterococcus faecalis: an atomic force microscopy and modeling

study, Geochim Cosmochim Ac 69, 2959 - 2969.

10. Bourcier, T., Thomas, F., Borderie, V., Chaumeil, C. and Laroche, L. (2003),

Bacterial keratitis: Predisposing factors, clinical and microbiological review of 300

cases, Brit J Ophthalmol 87, 834 - 838.

11. Vermeltfoort, P.B.J., van Kooten, T.G., Bruinsma, G.M., Hooymans, A.M.M., Van

der Mei, H.C. and Busscher, H.J. (2005), Bacterial transmission from contact lenses

to porcine corneas: An ex vivo study, Invest Ophth Vis Sci 46, 2042 - 2046.

12. Sharma, P.K. and Rao, K.H. (2003), Adhesion of Paenibacillus polymyxa on

chalcopyrite and pyrite: Surface thermodynamics and extended DLVO theory, Colloids

Surface B 29, 21 - 38.

13. Vijayalakshmi, S.P. and Raichur, A.M. (2003), The utility of Bacillus subtilis as a

bioflocculant for fine coal, Colloids Surface B 29, 265 - 275.

Chapter 1

6

14. Walker, S.L., Redman, J.A. and Elimelech, M. (2004), Role of cell surface

lipopolysaccharides in Escherichia coli K12 adhesion and transport, Langmuir 20,

7736 - 7746.

15. Prakobphol, A., Burdsal, C.A. and Fisher, S.J. (1995), Quantifying the strength of

bacterial adhesive interactions with salivary glycoproteins, J Dent Res 74, 1212 - 1218.

16. 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, Bios Bioelectron 19,

1429 - 1437.

17. Maier, B., Koomey, M. and Sheetz, M.P. (2004), A force-dependent switch reverses

type IV pilus retraction, P Natl Acad Sci USA 101, 10961 - 10966.

18. Liang, M.N., Smith, S.P., Metallo, S.J., Choi, I.S., Prentiss, M. and Whitesides,

G.M. (2000), Measuring the forces involved in polyvalent adhesion of uropathogenic

Escherichia coli to mannose-presenting surfaces, P Natl Acad Sci USA 97,

13092 - 13096.

19. Clapp, A.R., Ruta, A.G. and Dickinson, R.B. (1999), Three-dimensional optical

trapping and evanescent wave light scattering for direct measurement of long range

forces between a colloidal particle and a surface, Rev Sci Instrum 70, 2627 - 2636.

20. 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, Colloids Surface B 27, 355 - 364.

21. Geggier, P. and Fuhr, G. (1999), A time-resolved total internal reflection aqueous

fluorescence (TIRAF) microscope for the investigation of cell adhesion dynamics, Appl

Phys A-Mater 68, 505 - 513.

22. Prieve, D.C. (1999), Measurement of colloidal forces with TIRM, Adv Coll Int Sci 82,

93 - 125.


7

23. Bowen, W.R., Hilal, N., Lovitt, R.W. and Wright, C.J. (1999), Characterisation of

membrane surfaces: direct measurement of biological adhesion using an atomic force

microscope, J Membrane Sci 154, 205 - 212.

24. Van der Aa, B.C. and Dufrene, Y.F. (2002), In situ characterization of bacterial

extracellular polymeric substances by AFM, Colloids Surface B 23, 173 - 182.

25. Razatos, A., Ong, Y.L., Boulay, F., Elbert, D.L., Hubbell, J.A., Sharma, M.M. and

Georgiou, G. (2000), Force measurements between bacteria and poly(ethylene glycol)-

coated surfaces, Langmuir 16, 9155 - 9158.

26. Rutter, P.R. and Vincent, B. (1988), Attachment mechanisms in the surface growth of

microorganisms. In: Physiological models in microbiology. Bazin, M. J. and Prosser, J.

I. (Eds.), Boca Raton, Florida:CRC Press, Inc. pp 87 - 107.

27. 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.

28. Thomas, W.E., Nilsson, L.M., Forero, M., Sokurenko, E.V. and Vogel, V. (2004),

Shear-dependent 'stick-and-roll' adhesion of type 1 fimbriated Escherichia coli, Mol

Microbiol 53, 1545 - 1557.

29. Duddridge, J.E., Kent, C.A. and Laws, J.F. (1982), Effect of surface shear-stress on

the attachment of Pseudomonas fluorescens to stainless-steel under defined flow

conditions, Biotechnol Bioeng 24, 153 - 164.

30. Busscher, H.J. and Van der Mei, H.C. (2006), Microbial adhesion in flow

displacement systems, Clin Microbiol Rev 19, 127 - 141.

31. Shive, M.S., Hasan, S.M. and Anderson, J.M. (1999), Shear stress effects on bacterial

adhesion, leukocyte adhesion, and leukocyte oxidative capacity on a polyetherurethane,

J Biomed Mater Res 46, 511 - 519.

32. Gomez-Suarez, C., Busscher, H.J. and Van der Mei, H.C. (2001), Analysis of

bacterial detachment from substratum surfaces by the passage of air-liquid interfaces,

Appl Environ Microb 67, 2531 - 2537.

Chapter 1

8

33. Roosjen, A., Busscher, H.J., Norde, W. and Van der Mei, H.C. (2006), Bacterial

factors influencing adhesion of Pseudomonas aeruginosa strains to a poly(ethylene

oxide) brush, Microbiol-Sgm 152, 2673 - 2682.

34. Noordmans, J., Wit, P.J., Van der Mei, H.C. and Busscher, H.J. (1997), Detachment

of polystyrene particles from collector surfaces by surface tension forces induced by air-

bubble passage through a parallel plate flow chamber, J Adhes Sci Technol 11,

957 - 969.

35. Camesano, T.A. and Logan, B.E. (2000), Probing bacterial electrosteric interactions

using atomic force microscopy, Environ Sci Technol 34, 3354 - 3362.

36. Dufrene, Y.F., Boonaert, C.J.P., Gerin, P.A., Asther, M. and Rouxhet, P.G. (1999),

Direct probing of the surface ultrastructure and molecular interactions of dormant and

germinating spores of Phanerochaete chrysosporium, J Bacteriol 181, 5350 - 5354.

37. 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.

38. Emerson, R.J. and Camesano, T.A. (2004), Nanoscale investigation of pathogenic

microbial adhesion to a biomaterial, Appl Environ Microb 70, 6012 - 6022.

39. Busalmen, J.P. and de Sanchez, S.R. (2001), Adhesion of Pseudomonas fluorescens

(ATCC 17552) to nonpolarized and polarized thin films of gold, Appl Environ Microb

67, 3188 - 3194.

40. Jacobs, A., Lafolie, F., Herry, J.M. and Debroux, M. (2007), Kinetic adhesion of

bacterial cells to sand: Cell surface properties and adhesion rate, Colloids Surface B 59,

35 - 45.

41. Bakker, D.P., Postmus, B.R., Busscher, H.J. and Van der Mei, H.C. (2004),

Bacterial strains isolated from different niches can exhibit different patterns of adhesion

to substrata, Appl Environ Microb 70, 3758 - 3760.

CHAPTER 2

FORCES INVOLVED IN BACTERIAL ADHESION TO

HYDROPHILIC AND HYDROPHOBIC SURFACES

Parts of this chapter are reproduced with permission of the Society of General

Microbiology from : Boks, N.P., Norde, W., Van der Mei, H.C. and Busscher, H.J. (2008),

Microbiology 154, 3122-3133.

Chapter 2

10

Abstract

Using a parallel plate flow chamber, the hydrodynamic shear forces to prevent

bacterial adhesion (Fprev) and to detach adhering bacteria (Fdet) were evaluated

for hydrophilic glass, hydrophobic, dimethyldichlorosilane (DDS)-coated glass

and six different bacterial strains, in order to test the following three hypotheses:

1. A strong hydrodynamic shear force to prevent adhesion relates to a strong

hydrodynamic shear force to detach an adhering organism.

2. A weak hydrodynamic shear force to detach adhering bacteria implies that

more bacteria will be stimulated to detach by a passing air-liquid interface

through the flow chamber.

3. DLVO interactions determine the characteristic hydrodynamic shear

forces to prevent adhesion and to detach adhering micro-organisms as

well as the detachment induced by a passing air-liquid interface.

Fprev varied from 0.03 to 0.70 pN, while Fdet varied between 0.31 to over 19.64

pN, suggesting that after initial contact, strengthening of the bond occurs.

Generally, it was more difficult to detach bacteria from DDS-coated glass than

from hydrophilic glass, which was confirmed by air-bubble detachment studies.

Calculated attractive forces based on the DLVO theory (FDLVO) towards the

secondary interaction minimum were higher on glass than on DDS-coated glass.

In general, all three hypotheses had to be rejected, showing that it is of

importance to distinguish between forces acting parallel (hydrodynamic shear)

and perpendicular (DLVO, air-liquid interface passages) to the substratum

surface.

Forces involved in bacterial adhesion

11

Introduction

Microbial adhesion and subsequent biofilm formation occur in many fields of

industrial and medical applications, such as on ship hulls, heat exchanger plates,

food packaging materials and biomaterials implants, including urinary catheters,

contact lenses, and vascular grafts [1-3]. Common in most applications is the

deposition of micro-organisms to a surface from a flowing suspension. This

implies that a variety of forces act on depositing and already adhering

organisms. Deposition is mainly governed by Brownian motion, sedimentation

and hydrodynamic forces, while actual adhesion of micro-organisms to a

substratum surface is mediated by Lifshitz-Van der Waals, electrostatic, acid-

base and hydrophobic interaction forces [4].

Fluid flow is an important factor in microbial deposition [5]. An increase

in fluid flow velocity will in a first instance, yield increased microbial transport

towards a substratum surface (convective-diffusion), but at the same time causes

an increase in hydrodynamic detachment forces. Shear is the dominant effect of

fluid flow and can be well controlled in experimental systems, like on rotating

disks, at stagnation points and in parallel plate flow chambers. In principle, two

critical shear rates can be distinguished based on current literature (see Table 1):

a critical shear rate to prevent adhesion and a critical shear rate to stimulate

detachment of already adhering organisms. Both critical shear rates vary from

strain to strain and also depend on the substratum material involved. The shear

rates and, hence, the shear forces, required to stimulate detachment are generally

higher than the shear rates to prevent adhesion.

Detachment can also be invoked by allowing an air-bubble to pass over

adhering bacteria. The passage of an air-liquid interface is accompanied by a

perpendicularly oriented force of around 10-7 N, which is much higher than the

hydrodynamic shear forces acting parallel to a substratum surface. Yet, a

Cha

pter

2

4Tab

le 1

. Sum

mar

y of

inte

ract

ion

forc

es b

etw

een

bact

eria

and

subs

tratu

m su

rfac

es, t

oget

her w

ith th

e m

etho

d ap

plie

d.

Stra

in

Subs

trat

um

Forc

e (p

N)

Met

hod

Ref

eren

ce

Esch

eric

hia

coli

prot

ein

coat

ings

0.

2

[6]

Stap

hylo

cocc

us e

pide

rmid

is

seve

ral b

iom

ater

ials

1.

2 –

1.4

Hyd

rody

nam

ic fo

rce

[7-9

] St

aphy

loco

ccus

aur

eus

Col

lage

n 0.

4 to

pre

vent

adh

esio

n [1

0]

Pseu

dom

onas

fluo

resc

ens

stai

nles

s ste

el

9.2

– 12

.3

[1

1]

Stre

ptoc

occu

s san

guis

G

lass

22

.0

[1

2]

Baci

llus c

ereu

s gl

ass a

nd si

licon

ized

gla

ss

43.1

– 8

0.1

[1

2]

Esch

eric

hia

coli

hydr

opho

bic

subs

trate

s 3.

1 –

4.6

[1

3]

Stap

hylo

cocc

us e

pide

rmid

is

mod

ified

PV

C

0.1

– 1.

2 H

ydro

dyna

mic

forc

e [1

4]

Stap

hylo

cocc

us a

ureu

s C

olla

gen

>>3.

9 to

det

ach

adhe

ring

[1

5]

Pseu

dom

onas

fluo

resc

ens

stai

nles

s ste

el

18.5

ba

cter

ia

[11]

M

ix o

f Gra

m p

ositi

ve c

occi

gl

ass,

silic

oniz

ed g

lass

and

stee

l 20

.4 –

42.

4

[12]

Es

cher

ichi

a co

li Q

uartz

0.

3 –

2.4

[1

6]

Stap

hylo

cocc

us e

pide

rmid

is

PMM

A

11.1

[17]

Ps

eudo

mon

as fl

uore

scen

s G

old

32.1

– 5

7.9

[1

8]

Baci

llus c

ereu

s Sa

nd

0.03

D

LVO

cal

cula

tion

[19]

Ba

cillu

s sub

tilus

C

oal

0.09

[20]

Ba

cillu

s sub

tilus

Sa

nd

0.03

[19]

Pa

enib

acill

us p

olym

yxa

Pyrit

e –

chal

copy

rite

170

– 56

0

[21]

Sp

hing

omon

as p

auci

mob

ilis

Gla

ss

0.07

– 0

.7

[2

2]

Esch

eric

hia

coli

silic

on su

rfac

es

7400

– 2

2800

[23]

Es

cher

ichi

a co

li si

licon

nitr

ide

tip

400

– 21

00

Ato

mic

For

ce

[24]

St

aphy

loco

ccus

epi

derm

idis

si

licon

nitr

ide

tip

2000

M

icro

scop

e [2

5]

Spor

es o

f Bac

illus

myo

cide

s hy

drop

hobi

cally

coa

ted

glas

s 74

00 –

495

00

[2

6]

Esch

eric

hia

coli

gala

bios

e-fu

nctio

naliz

ed b

eads

50

– 1

00

[2

7]

Stap

hylo

cocc

us e

pide

rmid

is

fibro

nect

in c

oatin

gs

18

Opt

ical

Tw

eeze

rs

[28]

St

aphy

loco

ccus

aur

eus

fibro

nect

in c

oatin

gs

15 –

26

[2

9]

Hyd

rody

nam

ic fo

rces

are

cal

cula

ted

usin

g F

= η σ

Ap,

in w

hich

η th

e ab

solu

te v

isco

sity

of w

ater

and

Ap t

he a

rea

of th

e pa

rticl

e ex

pose

d to

shea

r. C

occi

wer

e as

sum

ed to

hav

e a

radi

us o

f 0.

5 μm

, whi

le ro

d-sh

aped

bac

teria

wer

e ap

prox

imat

ed b

y sp

here

s w

ith e

qual

vol

ume,

usi

ng 0

.7 μ

m a

s ra

dius

. DLV

O-f

orce

s are

take

n as

the

attra

ctiv

e fo

rce

tow

ards

the

pred

icte

d se

cond

ary

min

imum

in th

e to

tal i

nter

actio

n en

ergy

cur

ves.

Chapter 2

12


13

passing air-liquid interface does not cause complete bacterial detachment for all

combinations of strains and substratum surfaces.

Gomez-Suarez et al. [30] investigated detachment of several bacterial

strains from hydrophilic and hydrophobic surfaces by a passing air-bubble.

Depending on the strain involved, the presence of a conditioning film and the

velocity of the air-bubble, detachment ranged from 0 to 90%. Although air-

bubble induced detachment is relatively easy to measure, it only yields an

extremely rough estimate of a detachment force threshold and it cannot be used

to estimate the actual binding strength.

Perpendicularly oriented interaction forces can be measured more

directly, for instance using atomic force microscopy (AFM) or optical tweezers.

As can be seen in Table 1, forces obtained using these techniques, differ in

orders of magnitude. Forces measured with optical tweezers remain in the pN

range, while AFM yields stronger forces than any other method, which are

generally in the nN range.

Another, often used approach for assessing adhesion strength is the

(extended) DLVO theory (named after Derjaguin, Landau, Verwey and

Overbeek). In the DLVO theory the binding strength between colloidal particles,

such as micro-organisms, and substratum surfaces may be calculated on the

basis of Lifshitz-Van der Waals, (acid-base and) electrical double layer

interactions. Usually, also the theoretical values provide a distinct class of force

values, that cannot be easily matched with experimental values, as reported in

the literature.

From Table 1, it is obvious that throughout the literature different types of

forces may be distinguished for every strain-substratum combination.

Furthermore, conclusions on bacterial adhesion mechanisms are often based

on not more than two strains [31]. Comparing all reported data is further

complicated by the fact that different suspending media are used to determine

Chapter 2

14

adhesion parameters on different substrata. It is currently unclear why different

methods to evaluate bacterial binding forces yield distinct classes of force values

that often differ by orders of magnitude. The aim of our research is to gain more

insight in the relevance of the different bacterial interaction force indicators,

including theoretically predicted interaction forces from the DLVO-theory, and

their mutual relationships. To this end, the following hypotheses were tested:

1. A strong hydrodynamic shear force to prevent adhesion relates to a strong

hydrodynamic shear force to detach an adhering organism.

2. A weak hydrodynamic shear force to detach adhering bacteria implies that


through the flow chamber.

3. DLVO interactions determine the characteristic hydrodynamic shear

forces to prevent adhesion and to detach adhering micro-organisms as

well as the detachment induced by a passing air-liquid interface.

To test these hypotheses, the critical shear force to prevent bacterial adhesion

and to stimulate detachment of adhering bacteria are determined. Hydrophilic

glass and hydrophobic, dimethyldichlorosilane-coated, glass are employed as

substrata. To allow for more general conclusions to be drawn, six widely

different bacterial strains are included. In addition, theoretical DLVO interaction

forces, as calculated from measured zeta potentials and contact angles are

determined. Furthermore, the detachment force threshold is evaluated for

detachment caused by a passing air-liquid interface.


15

Materials and Methods

Bacterial strains and culture conditions. Staphylococcus epidermidis ATCC

35983, S. epidermidis HBH2 169, Pseudomonas aeruginosa D1, P. aeruginosa

KEI 1025 were cultured aerobically from blood agar plates in 10 ml Tryptone

Soya Broth (OXOID, Basingstoke, England) for 24 h at 37 ºC. Raoultella

terrigena ATCC 33527 was precultured aerobically from nutrient agar (Nutrient

Broth, OXOID) in 10 ml nutrient broth for 24 h at 37 ºC. Streptococcus

thermophilus ATCC 19258 was precultured from a frozen stock in 10 ml M17

broth for 24 h at 37 ºC. After 24 h, precultures were used to inoculate 200 ml

main cultures, which were grown for 16 h under similar conditions as the

corresponding precultures. S. epidermidis and P. aeruginosa strains were

harvested by centrifugation for 5 min at 6500 x g, while R. terrigena and S.

thermophilus were harvested at 10000 x g. All strains were washed twice with

10 mM potassium phosphate buffer at pH 7 and resuspended in the same buffer.

To break bacterial chains or clusters, sonication at 30 W (Vibra Cell model 375,

Sonics and Materials Inc., Danbury, CT, USA) was carried out for

staphylococcal (3 times 10 s) and streptococcal (2 times 10 s) suspensions, while

cooling in an ice/water bath. Subsequently, bacteria were resuspended to a

concentration of 3 x 108 cells ml-1. In the calculations discussed below, the cocci

were assumed to have a radius of 0.5 μm. Rod-shaped P. aeruginosa (2.5 μm x

0.9 μm) and R. terrigena (3.2 μm x 1.4 μm) were approximated as spheres with

equal volume, using a radius of 0.6 μm and 0.9 μm, respectively as they adhere

in different orientations, i.e. “end-on” and “side-on”.

Substratum surfaces. Glass slides were sonicated during 3 min in 2% RBS35

(Omnilabo International BV, The Netherlands) followed by thorough rinsing

with tap water, demineralised water, methanol, tap water and finally

Chapter 2

16

demineralised water again to obtain a hydrophilic surface. After washing, the

slides were either directly used or dried for 4 h at 80 ºC prior to applying a

hydrophobic coating. To obtain a hydrophobic surface, the dried glass slides

were submerged during 15 min in a solution of dimethyldichlorosilane (DDS,

Merck, Germany) in trichloroethylene (0.05 w/v %) and washed with

trichloroethylene, methanol and ultrapure water. Prepared slides were stored for

no longer than 3 days at room temperature and rinsed with 10 mM potassium

phosphate buffer before use.

Bacterial adhesion in the parallel plate flow chamber. The parallel plate flow

chamber (PPFC) and image analysis have been described previously [32]. The

flow chamber used in this study has a length of 175 mm, a depth of 0.75 mm

and a width of 17 mm. Prior to use, the flow chamber was washed with 2%

Extran (Merck, Germany) and rinsed thoroughly with tap water and

demineralised water before mounting a clean substratum surface in the PPFC.

Subsequently, the flow chamber was installed between two communicating

vessels and the system was filled with 10 mM potassium phosphate buffer while

care was taken to remove all air-bubbles. When the PPFC was positioned under

the microscope, the vessels containing bacterial suspension were positioned at

different heights to create a flow. The difference in fluid levels was maintained

by a roller-pump to ensure a circulating pulse free flow throughout the duration

of an entire experiment. Deposition of bacteria was monitored with a phase

contrast microscope (Olympus HB-2) equipped with a 40x ultra long working

distance objective (Olympus ULWD-CD Plan 40 PL) which was connected to a

CCD-MXRi camera (Basler A101F, Basler AG, Germany). Images were

obtained by summation of 15 consecutive images (time interval 0.25 s) in order

to enhance the signal to noise ratio and eliminate moving bacteria from analysis.

Analysis of the images was done using proprietary software based on the Matlab

Image processing Toolkit (The MathWorks, MA, USA)).


17

Shear rate dependent adhesion. The bacterial suspension was allowed to flow

through the flow chamber during 1 h at flow rates (Q) of 1, 5, 10, 19, 57, 77, 105

and 153 ml min-1 which corresponds to shear rates (σ) of 10, 50, 100, 200, 600,

800, 1100 and 1600 s-1. Under these conditions the flow is laminar and bacterial

transport occurs by convective-diffusion. Adhesion is monitored on both the top

(negative contribution of sedimentation) and bottom (positive contribution of

sedimentation) plate of the PPFC. For each shear rate, the number of bacteria

adhering per unit area was recorded as a function of time. Adhesion was then

expressed in initial deposition rates j0 (cm-2 s-1), while at the end of each

experiment an air-bubble was passed through the flow chamber to stimulate

detachment (only evaluated for the bottom plate).

Initial deposition rates for the top and bottom plate were averaged and expressed

as deposition efficiencies by normalization with respect to the Von

Smoluchowski-Levich (SL) theoretical upper limit for deposition in the parallel

plate flow chamber. The SL upper limit for bacterial deposition is an

approximate solution of the convective-diffusion equation and assumes perfect

sink conditions at the substratum surface (i.e. every particle that arrives at the

surface actually adheres) in the absence of sedimentation. The theoretical upper

limit for deposition is given by [33]:

31

*0 9

289.0 ⎥⎦

⎤⎢⎣⎡ ⋅= ∞

xbPe

rcDj (1)

in which D∞ is the diffusion coefficient of the particles (taken 3.1 x 10-13 m2 s-1

for micron-sized bacteria [34]), c the concentration of bacteria in suspension, r

the bacterial radius, x the longitudinal distance from the flow chamber entrance,

b the half-depth of the PPFC and Pe the dimensionless Péclet number. This

latter is defined as:

Chapter 2

18

∞

=Dwb

QrPe 3

3

43 (2)

in which Q is the applied flow rate and w the width of the flow chamber

Detachment induced by a passing air-liquid interface. Following the deposi-

tion measurement, an air-liquid interface was introduced by passing an air-

bubble through the flow chamber, which is accompanied by a perpendicularly

oriented detachment force equal to [35]:

sw,bw, ΘΘ

cos2

sin2 2max ⎟⎠

⎞⎜⎝

⎛⋅⋅= lvrF γπγ for Θw,s < 90 (3)

sw,bw, ΘΘ

cos2

sin2 2max ⎟⎠

⎞⎜⎝

⎛ +⋅⋅−=

πγπγ lvrF for Θw,s > 90 (4)

in which γlv represents the interfacial surface tension of the liquid and vapour,

Θw,b and Θw,s denote the bacterial- and substratum-water contact angles,

respectively.

Shear rate dependent detachment of adhering bacteria. The flow system was

filled and positioned as described before. Bacteria were resuspended in

potassium phosphate buffer to a high concentration of 7.5 x 108 cells ml-1 to

accelerate deposition and allowed to adhere to the collector surface at a shear

rate of 25 s-1. After 20 min, flow was switched to fresh buffer without bacteria at

25 s-1 to wash out the bacterial suspension for 30 min, after which the shear rate

was increased to either 250, 1000, 3000, 6650 or 7320 s-1 for 30 min. The

number of bacteria that remained adhering was enumerated after each step.


19

Surface characterization. To determine the zeta potentials of the substrata,

streaming potentials were measured in 10 mM phosphate buffer at pH 7.

Collector surfaces were mounted in a homemade parallel plate flow chamber,

separated by a 0.1 mm Teflon spacer. A platinum electrode was placed at either

end of the chamber. Streaming potentials were measured at 10 different

pressures ranging from 5 x 103 to 20 x 103 Pa. Each pressure was applied during

10 s in both directions. Zeta potentials were deduced by linear least squares

fitting from the pressure dependent streaming potentials [36].

For bacterial zeta potentials, bacteria were washed with demineralised water and

resuspended in 10 mM potassium phosphate buffer at pH 7 to a concentration of

1 x 108 cells ml-1. The electrophoretic mobilities of these suspensions were

measured at 150 V using a Lazer Zee Meter 501 (PenKem, USA). The

electrophoretic mobilities were converted to apparent zeta potentials assuming

the Helmholtz-Von Smoluchowski approximation holds, which is appropriate

considering the high value for κ r (i.e. ≈ 150) in the systems used (N.B. κ

denotes the reciprocal Debeye length which is directly related to the ionic

strength [37]).

To calculate surface free energies of the substratum and bacterial cell surfaces,

sessile drop contact angles were measured with water, formamide, α-

bromonaphthalene and methylene iodide. In order to measure contact angles

with liquids on bacteria, bacterial lawns were prepared by depositing bacteria

from suspension in demineralised water on cellulose acetate membrane filters

(Millipore, pore diameter 0.45 μm) under negative pressure until approximately

50 layers were stacked. Subsequently, filters were fixed on a sample holder and

left to dry until “plateau contact angles” could be measured, i.e. water contact

angles that remained stable over time for 30–60 min. All contact angles were

measured in triplicate, implying separate substrata and different bacterial

Chapter 2

20

cultures. Measured contact angles were converted into surface free energies

using:

lv

pluslv

plussv

lv

lvsv

lv

LWlv

LWsv

γγγ

γγγ

γγγ 222

1cos +++−=minusminus

Θ (5)

in which γLWsv is the Lifshitz-Van der Waals component of the surface free

energy of the surface of interest (i.e. substratum surface or bacterial lawn) and γlv

is the surface free energy of the liquid vapour interface. The acid-base

component of the surface free energies was separated into an electron donor

(γminussv) and electron acceptor (γplus

sv) parameter, according to: plussvsv

ABs γγγ minus2= (6)

Interaction forces using the extended DLVO theory. In the extended DLVO

theory, the interaction energy is divided in a Lifshitz-Van der Waals, acid-base

and electrostatic contribution, while accounting for their distance dependencies.

The Lifshitz-Van der Waals contribution can be derived by first calculating the

Lifshitz-Van der Waals component of the free energy of adhesion of a bacterium

to a substratum surface, which reads at contact [38]:

( )( )LWlv

LWsv

LWlv

LWbv

LWslbG γγγγ −−−=Δ 2 (7)

Equation 7 can be used to calculate the Hamaker constant A according to [39]: 2012 dGA LW

slb ⋅⋅Δ= π (8)

where d0 denotes the minimal separation distance (0.157 nm [40]) and ΔGLWslb is

obtained from thermodynamic analysis [38]. Lifshitz-Van der Waals attractive

interaction energies (ΔGLW) were subsequently calculated as a function of

distance assuming a sphere-plane geometry using [39]:

( )( ) ⎥

⎦

⎤⎢⎣

⎡⎟⎠⎞

⎜⎝⎛ +

−++

−=Δd

rdrddrdrAdG LW 2ln)2

26

)( (9)


21

in which d denotes the separation distance. The acid-base component, ΔGABslb,

can be calculated from [38]:

( ) ( ) ( )( ) ( ) ( )⎥⎥⎦

⎤

⎢⎢

⎣

⎡

−⋅−−−

⋅−−−⋅−=Δ

minusminusplusplusminusminus

pluspluminusminusplusplus

lvsvlvsvlvbv

lvs

bvsvbvsvbvABslbG

γγγγγγ

γγγγγγ2 (10)

in which the subscript “s” denotes the substratum surface and “b” the bacterial

cell surface.

Using Equation 10, the distance dependence of the acid-base interaction

energies (ΔGAB) can then be calculated according to [39]:

⎟⎠⎞

⎜⎝⎛ −

Δ⋅⋅=Δλ

λπdd

GrdG ABslb

AB 0exp2)( (11)

in which λ denotes the correlation length of molecules in the liquid medium

(estimated to be 0.6 nm [39]).

Distance dependent electrostatic interaction energies (ΔGEL) were calculated

using [41]:

( ) ( )( ) ( )[ ]

⎭⎬⎫

⎩⎨⎧

−−+⎥⎦

⎤⎢⎣

⎡−−−+

++=Δ d

ddrdG

sb

sbsb

EL κκκ

φφφφ

φφπεε 2exp1lnexp1exp1ln

2)( 22

220 (12)

in which εε0 denotes the dielectric permittivity of the medium (i.e. water), φb and

φs the surface (zeta) potentials of the bacterial cell surface and collector surface

and κ the reciprocal Debye length.

Summation and differentiation with respect to distance of these three

components lead to the total DLVO-interaction energy and interaction force,

respectively, as a function of separation distance. All DLVO interaction forces

reported in this chapter represent the maximal attractive force towards the

secondary interaction minimum, which was present in all bacterium-substratum

systems investigated.

Chapter 2

22

Results

Shear rates to prevent bacterial adhesion. Figure 1 presents an example of

bacterial deposition to the bottom and top plate of the parallel plate flow

chamber as a function of shear rate. Deposition is higher to the bottom plate than

to the top plate, especially at lower shear rates. Moreover, at low shear rates an

initial increase in deposition to the bottom plate can be seen with increasing

shear rate up to 200 s-1 due to increased mass transport, above which deposition

decreases with increasing shear due to detachment. A similar effect is observed

on the top plate.

Figure 1. Initial deposition rates (j0) for S. epidermidis ATCC 35983 on the bottom (●) and top plate (○) in a parallel plate flow chamber as a function of the shear rate (σ) applied on glass.


23

The influence of sedimentation on mass transport can be eliminated by

averaging bottom and top plate depositions. Figure 2 shows the deposition

efficiencies (α) in the absence of sedimentation, as calculated from averaged

initial deposition rates and the theoretical upper limit for deposition (Eq. 1) as a

function of shear rate. From Figure 2, critical shear rates to prevent adhesion

(σprev) were deduced using

⎪⎭

⎪⎬⎫

⎪⎩

⎪⎨⎧−⋅=

prevσσαα exp0 (13)

with α0 the extrapolated deposition efficiency in absence of shear. Subsequently,

values for σprev were expressed in shear forces using

ipi AF ση ⋅⋅= (14)

in which η is the absolute viscosity of the buffer (1 x 10-3 Pa s) and Ap is the area

of the adhering bacterium subject to shear flow. Furthermore, subscript “i”

denotes the type of hydrodynamic force calculated: prev for the hydrodynamic

force to prevent adhesion and det for the hydrodynamic force to detach adhering

micro-organisms. Hydrodynamic shear forces to prevent adhesion (Fprev) are

listed in Table 2. All values for Fprev remain in the low pN range and are

influenced by the substratum surface, although not consistently higher on any of

the two surfaces. Depending on the strain used, the difference between Fprev on

glass and DDS-coated glass can be as large as a factor 6.

Chapter 2

24

Figure 2. Bacterial deposition efficiency (α) in the absence of a mass transport contribution

from sedimentation as a function of the shear rate (σ) applied on glass (●) and DDS-coated

glass (○) for the six different bacterial strains included. Black and grey lines represent the

fits of equation 13 to the datapoints on glass and DDS-coated glass, respectively.


25

Table 2. Critical shear forces to prevent (Fprev) bacterial adhesion and to detach (Fdet) adhering bacteria from a hydrophilic (glass) and hydrophobic (DDS-coated) substratum, as derived for different bacterial strains, together with the theoretically calculated DLVO interaction forces. Reported uncertainties are based on the standard error of the predicted fitting curve. Fprev (pN) Fdet (pN) FDLVO (pN)

Bacterial

strain

Glass DDS Glass DDS Glass DDS

S. epidermidis HBH2 169

0.13 ± 0.06 0.40 ± 0.12 0.31 ± 0.03 5.52 ± 0.09 0.08 0.05

S. epidermidis ATCC 35983

0.57 ± 0.22 0.10 ± 0.03 5.39 ± 0.19 >5.75* 0.05 0.03

R. terrigena ATCC 33527

0.10 ± 0.00 0.11 ± 0.01 > 19.64* 14.30 ± 2.45 0.10 0.06

S. thermophilus ATCC 19258

0.12 ± 0.34 0.03 ± 0.01 0.55 ± 0.05 0.68 ± 0.04 0.00 0.00

P. aeruginosa D1

0.06 ± 0.02 0.04 ± 0.01 0 3.41 ± 1.00 0.05 0.03

P. aeruginosa KEI 1025

0.24 ± 0.02 0.70 ± 0.28 4.53 ± 0.82 9.93 ± 0.03 0.08 0.05

* No detachment could be stimulated within the shear rates applied and the value indicated denotes the highest shear applied.

Shear rates to remove adhering bacteria. Figure 3 presents the detachment of

bacteria from glass and DDS as a function of the shear rate applied, expressed as

the fraction (f) of bacteria removed from the substratum surface. For a given

shear rate, f is defined as the number of removed bacteria after 30 min exposure

to that shear divided by the number of adhering bacteria before application of

the shear. From the plots in Figure 3 critical shear rates to detach adhering

bacteria (σdet) were derived, defined as the shear rate at which 63% of the

adhering bacteria had detached. Subsequently, these shear rates were expressed

in detachment forces (Fdet) using Equation 14, and their values are listed in

Chapter 2

26

Table 2. In most cases, bacteria are more readily detached from glass than from

DDS-coated glass. All forces remain in the pN range, but are an order of

magnitude larger than Fprev. Note that the critical detachment level could not be

reached within the range of shear rates possible in our experimental set-up for S.

epidermidis ATCC 35983 on DDS-coated glass and for R. terrigena ATCC

33527 on glass.

Air-bubble induced bacterial detachment. Table 3 summarizes the effect of

an air-bubble passing over the adhering bacteria. At first sight, binding affinity

on DDS-coated glass seems to be less than on hydrophilic glass, as judged from

air-bubble-induced detachment. However, on DDS-coated glass, the force

exerted by an air-liquid interface on adhering bacteria is calculated to be up to

five times larger than on glass. For the two Staphylococcus epidermidis strains

and R. terrigena, this results in higher detachment percentages from DDS-coated

glass. In contrast, the percentages detached from glass and DDS-coated glass,

respectively, are for the pseudomonas strains and S. thermophilus not

significantly different. It should be noted that detachment by a passing air-

bubble does not give any indication of the magnitude of the interaction forces.

For example, for the staphylococcal strains and R. terrigena on glass, it cannot

be established at which force detachment would be stimulated to a larger extent.

Air-bubble detachment studies are indecisive here with respect to binding

strength information. However, for the pseudomonas strains and S. thermophilus

it is clear that, even though the exerted force on DDS-coated glass is stronger,

detachment percentages are not higher. Results for these strains suggest stronger

interaction forces with the hydrophobic DDS-coated glass.


27

Figure 3. Shear-induced detachment, expressed as the fraction (f) of bacteria that are removed, as a function of the shear rate (σ) applied for glass (●) and DDS-coated glass (○) after 30 min of flow.

Surface characterization and calculation of theoretical interaction forces.

Measured contact angles, together with the surface free energy components of

the wetting liquids used, are listed in Table 4. All bacteria have a surface

hydrophilicity comparable to the one of glass, as judged from the water contact

angles. DDS-coated glass is significantly more hydrophobic.

Chapter 2

28

Table 3. Number of adhering bacteria on the bottom plate of the parallel plate flow chamber after 1 h of flow (N1h, averaged over adhesion experiments at σ = 10, 50, 100 and 200 s-1; n=1 for each shear rate), detachment percentages from glass and a DDS-coating and the corresponding maximal detachment force (Fγ

max) a liquid/air interface exerts. Glass DDS

Bacterial strain N1h

(x 106 cm-2)

Detach-

ment

(%)

Fγmax

(nN)

N1h

(x 106 cm-2)

Detach-

ment

(%)

Fγmax

(nN)

S. epidermidis HBH2 169

4.9 ± 0.5 9 ± 10 14 3.8 ± 0.5 92 ± 9 40

S. epidermidis ATCC 35983

4.0 ± 0.8 4 ± 5 20 3.5 ± 0.8 62 ± 47 39

R. terrigena ATCC 33527

0.8 ± 0.8 27 ± 6 16 1.4 ± 1.0 87 ± 14 72

S .thermophilus ATCC 19258

0.4 ± 0.3 56 ± 16 17 0.5 ± 0.5 47 ± 21 39

P. aeruginosa D1

0.3 ± 0.4 71 ± 40 37 0.4 ± 0.2 40 ± 14 48

P. aeruginosa KEI 1025

1.3 ± 2.1 53 ± 10 12 2.9 ± 0.7 51 ± 13 54

Bacterial cell surfaces and the glass substratum surface are predominantly

electron-donating, as evidenced by their larger γminus surface free energy

parameter as compared with γplus. Hydrophobic DDS-coated glass is neither a

good electron donor nor acceptor. All surfaces are negatively charged and

whereas bacterial zeta potentials vary between -22 and -50 mV, the zeta

potentials of glass and DDS-coated glass are similarly negative (-33 to -35 mV).

The bacterial cell and substratum surface properties listed in Table 4 have

been used in the extended DLVO theory, yielding interaction free energy- and

force-distance profiles for all combinations of bacteria and substratum surfaces,

as illustrated in Figure 4 for P. aeruginosa KEI 1025. Note the reversed force-


29

axis (right hand side) in Figure 4 indicating that negative values correspond to

attractive interaction forces according to the definition of force:

( ) ( )dEd

dFδδ

−= (15)

Residing in the secondary minimum of the interaction energy corresponds to

zero interaction force, resulting from compensating attractive (Van der Waals)

and repulsive (electrostatic) forces. However, the approach toward the

secondary minimum yields a maximum net attraction force (Figure 4) at a

distance of about 40 nm from the surface. On glass, these interaction forces are

generally higher than on DDS-coated glass (see also Table 2), due to larger

Hamaker constants for glass as a substratum. Additionally, on DDS-coated glass

a primary minimum (closer to the surface) is predicted due to acid-base

interaction. The height of the energy barrier between the secondary and primary

minimum varies from 229 kT for S. thermophilus to 1030 kT for R. terrigena and

therefore it is very unlikely that a depositing micro-organism will cross the

barrier to adhere in the primary minimum. On glass, a primary interaction

minimum is absent.

Discussion

The forces that govern microbial deposition, adhesion and detachment are still

not fully understood, and difficult to relate with each other. In a previous study

we successfully investigated the characteristic shear force to prevent adhesion of

microbial strains [42]. In the current research we used a more systematic

approach by including not only the shear forces to prevent adhesion, but also

those that stimulate detachment of adhering bacteria, as well as theoretical

adhesion forces calculated using the extended DLVO theory. In addition, the

effect of a passing air-liquid interface, which invokes a high, perpendicularly

Cha

pter

2

30

Tab

le 4

. Phy

sico

-che

mic

al c

hara

cter

istic

s of

the

bact

eria

l stra

ins

and

colle

ctor

sur

face

s us

ed. B

acte

rial c

hara

cter

izat

ions

wer

e ba

sed

on th

ree

sepa

rate

ly g

row

n cu

lture

s. Pe

r cul

ture

, con

tact

ang

les

of w

ater

( Θw),

form

amid

e (Θ

form

), α

-bro

mon

apht

hale

ne (Θ

br) a

nd m

ethy

lene

iodi

de (Θ

met)

wer

e m

easu

red

on fo

ur b

acte

rial l

awns

usi

ng o

ne d

ropl

et p

er li

quid

per

bac

teria

l law

n. Z

eta

pote

ntia

ls (ζ

) wer

e de

term

ined

in tr

iplic

ate.

Con

tact

an

gle

and

stre

amin

g po

tent

ial

mea

sure

men

ts o

n su

bstra

tum

sur

face

s w

ere

perf

orm

ed i

n qu

adru

plic

ate.

Fre

e su

rfac

e en

ergy

com

pone

nts

are

deriv

ed fr

om c

onta

ct a

ngle

mea

sure

men

ts g

ivin

g an

ele

ctro

n-do

natin

g ( γ

min

us) a

nd -a

ccep

ting

(γpl

us) p

aram

eter

for t

he a

cid-

base

com

pone

nt (γ

AB),

the

Lifs

hitz

-Van

der

Waa

ls c

ompo

nent

(γLW

) and

the

tota

l sur

face

free

ene

rgy

(γTo

t ). B

acte

rial

stra

in

Θw

(º)

Θfo

rm

(º)

Θbr

(º

) Θ

met

(º)

γmin

us

(mJ

m-2

) γpl

us

(mJ

m-2

) γA

B

(mJ

m-2

) γLW

(m

J m

-2)

γTot

(mJ

m-2

) ζ

(m

V)

S. e

pide

rmid

is

HB

H2 1

69

31 ±

4

31 ±

4

34 ±

5

50 ±

3

47.8

0.

4 9

40

49

-50

± 6

S. e

pide

rmid

is

ATC

C 3

5983

38

± 5

40

± 5

36

± 1

54

± 4

45

.8

0.5

10

34

44

-51

± 2

R. te

rrig

ena

ATC

C 3

3527

24

± 3

24

± 3

40

± 4

51

± 4

49

.9

1.8

19

34

53

-49

± 5

S. th

erm

ophi

lus

ATC

C 1

9258

35

± 2

31

± 4

58

± 2

77

± 2

41

.2

5.3

30

22

52

-22

± 5

P. a

erug

inos

a

D1

44 ±

6

42 ±

4

48 ±

8

58 ±

6

38.8

1.

2 14

30

44

-3

0 ±

3

P. a

erug

inos

a

KEI

102

5 25

± 2

31

± 2

40

± 2

49

± 4

54

.8

0.8

14

35

49

-39

± 5

Subs

trat

um su

rfac

e

Gla

ss

28

± 8

25

± 3

51

± 2

64

± 1

45

.8

3.7

26

28

54

-35

± 5

DD

S-co

atin

g

101

± 2

85 ±

3

59 ±

4

65 ±

4

2.2

0.0

0 26

26

-3

3 ±

2

Chapter 2

30


31

Figure 4. Example of the extended DLVO interaction energy (⎯) and –force (⋅⋅⋅⋅) as a function of distance for P. aeruginosa KEI 1025 on glass and DDS-coated glass. Arrows indicate the correct axis for both plots. Note the reversed force-axis.

Chapter 2

32

oriented detachment force on adhering bacteria, was determined. Furthermore,

all experiments were carried out with six different bacterial strains in order to

allow general conclusions to be drawn. As a first step in the experimental

analysis, the gravitational force and its impact on bacterial deposition [43,44]

and adhesion was eliminated by averaging the deposition rates on bottom-and

top plate. At low shear rates, deposition efficiencies (α) exceed unity especially

for the S. epidermidis strains, indicating that deposition is more favourable than

theoretically predicted. Often such deviations are ascribed to the presence of

surface structures [45]. With respect to possible relations between the different

forces distinguished, we test the following hypotheses:

1) A strong hydrodynamic shear force to prevent adhesion relates to a strong

hydrodynamic shear force to detach an adhering organism. This hypothesis

implies a positive correlation between attachment and detachment. Comparison

between Fprev and Fdet (Table 2) show that regardless of the substratum involved,

Fdet is always larger than Fprev. In the experimental set-up used, bacteria are

adhering to the substratum surface for at least half an hour before being subject

to high shear. Therewith, over time the bond between a bacterium and the

substratum surface may become stronger. Supporting evidence for this is

provided by others who have used AFM and found that the adhesion force

increases with prolonged contact time [46,47]. Thus, even though initial

adhesion forces are rather weak, they may be indicative for forces after a

prolonged time, i.e. a relatively strong Fprev might be expected to correspond

with a relatively strong Fdet. However, from Figure 5 it is clear that no

correlation exists between Fprev and Fdet. It implies that attachment and

detachment should be regarded as independent processes and the hypothesis of

an unambiguous relation between attachment and detachment forces should be

rejected.


33

Figure 5. Graphical presentation of possible relations between Fprev and Fdet (A), FDLVO and Fprev (B), FDLVO and Fdet (C) and detachment percentage and Fmax (D).

2) A weak hydrodynamic shear force to detach adhering bacteria implies that


through the flow chamber. Table 2 clearly indicates that Fdet for hydrophobic

DDS-coated glass is larger than for hydrophilic glass, indicating stronger

interaction forces on the hydrophobic substratum. Table 3 summarizes

parameters involved in air-bubble-induced detachment. An air-liquid interface

exerts forces 104 times larger than Fdet, yet it does not result in complete

detachment. Combining the data in Tables 2 and 3, reveals the absence of a clear

relation between shear-induced detachment and detachment by passing an air-

bubble. Thus a weaker Fdet does not result in higher air-bubble-stimulated

detachment and this hypothesis has to be rejected too. In this respect it must be

realized that different mechanisms of detachment are involved in both processes.

Chapter 2

34

Hydrodynamic detachment forces are measured while the system is completely

submerged in liquid whereas an extra phase is introduced in air-bubble-induced

detachment. Furthermore, Fdet is a force acting parallel to the substratum surface,

whereas the air-liquid interface acts perpendicularly to the substratum surface.

3) DLVO interactions determine the characteristic hydrodynamic shear forces

to prevent adhesion and to detach adhering micro-organisms as well as the

detachment induced by a passing air-liquid interface. Further analysis revealed

the absence of quantitative relations between FDLVO and Fprev, as well as between

FDLVO and Fdet (Figure 5). DLVO-predictions have often been demonstrated to

deviate from experimental observations of bacterial interaction phenomena,

which is usually ascribed to the presence of surface appendages [48,49] or

chemical surface heterogeneities. However, the direction of action of the

DLVO-forces should be taken into account as well. DLVO-forces act

perpendicularly to the substratum surface, whereas both Fprev and Fdet are

directed parallel to the substratum surface.

When the fluid flow is increased to high enough values, the bacterium

most likely detaches in a rolling fashion [50]. It can be argued that in this mode

of detachment, forces normal to the surface (i.e. DLVO- and lift forces) are

related to forces directed parallel to the surface. However, in similar detachment

studies it was found that lift forces are negligible and surface roughness may

play a decisive role in determining the hydrodynamic force to remove adhering

particles from the surface [51,52]. This feature is not accounted for in the

DLVO-theory. Table 2 shows only slight differences between the theoretical

FDLVO-values for the various microbial strains, but substantial differences

between the experimentally obtained forces Fprev and Fdet. If a correlation

between DLVO forces and shear forces would exist, an increase of these parallel

directed forces implies an increase in normally directed forces. However, this is


35

not observed in FDLVO. Hence, the parallel detachment forces do not correlate to

the perpendicularly directed DLVO-forces.

The DLVO theory predicts a secondary minimum of interaction at a

distance of about 30 to 40 nm away from the surface (see Figure 4). On

hydrophilic glass, closer approach is impossible due to strong repulsion and

adhesion can only occur in the secondary minimum. On DDS-coated glass, also

primary minimum interactions are predicted. However, due to the prohibitive

high barrier of the free energy (ranging from 229 kT to 1030 kT depending on

the strain used), it is very unlikely that adhesion in the primary minimum can

occur. Therefore, also on the hydrophobic DDS-coated glass, only adhesion in

the secondary minimum is expected to occur. As can be seen in Table 2, Fdet-

values are much higher than FDLVO. Often, a transition of adhesion from the

secondary interaction minimum towards the primary minimum is used as

explanation [53]. However, in this study this is considered to be impossible as

on glass a primary minimum is absent and on DDS-coated glass it is considered

to be unreachable due to the high energy barrier. It is therefore more likely that

the higher Fdet values are the result of attachment of surface appendages, or

“extracellular polymeric substances” produced, capable to reach the surface.

These structures are known to extend as much as hundreds of nm away from the

bacterial cell wall [38], which is more than enough to bridge the distance

between secondary minimum and the substratum surface. Unfortunately,

although it is known for instance that some streptococci may possess surface

fibrils, structural information about the cell surface of far most all strains studied

in the literature are lacking, let alone detailed knowledge about the length,

diameter and micro(nano-)scopic physico-chemical properties of these

structures. The use of the DLVO-theory as currently done in the literature as

well as in this chapter, can therefore only pertain to long-distance approach,

where fine surface structures do not play a role. Up to what distance of approach

and up to what extent this statement is valid, is hard to say. However, while the

Chapter 2

36

DLVO-theory predicts interactions for the entire micro-organism, it is likely that

the experimentally obtained detachment forces are related to a number of

distinct (hydrogen) bonds. When these linkages break, due to parallel directed

forces, the bacterium can be transported away from the surface due to lift forces

which are induced by the tangential flow [54]. In this respect, parallel directed

hydrodynamic forces (i.e. Fprev and Fdet) can serve as useful parameters to

indicate adhesion strength.

When combining the detachment parameters (i.e. Fdet and the air-bubble

detachment percentage), our results suggest that bridging between the bacterium

and the substrate surface is more favourable for DDS-coated glass. Fdet on

hydrophobic DDS-coated glass is always higher than on glass and even though

one has to be cautious in interpreting air-bubble detachment percentages, the

higher detachment force exerted by the air-bubble on DDS-coated glass does not

necessarily lead to more detachment. The hydrophobicity of the surface likely

enhances the possibility of bridging as removal of water from in between the

interacting surfaces is more favourable. This matter is further complicated by the

influence of the type of medium in which adhesion occurs. The DLVO-theory is

based on averaged properties of the surfaces of the bacterial cell and substratum.

However, it was found that ions in the suspending medium, especially divalent

ions, can greatly influence the adhesion of bacteria to a surface, probably due to

surface charge heterogeneities resulting from complexation of different ions

with the (bacterial cell) surface(s) [55]. Since our experiments were performed

in potassium phosphate buffer, we cannot rule out similar effects caused by the

divalent phosphate anion.

Even though no quantitative correlation between the DLVO theory and

detachment behaviour could be established, and the above forwarded hypothesis

should therefore be rejected, this theory does help to provide a better insight in

the mechanism of bacterial adhesion to a substratum surface.


37

Conclusions

The hydrodynamic force to prevent adhesion (Fprev) is lower than the

hydrodynamic force to stimulate detachment (Fdet) showing that the bond

between a substratum surface and a bacterium becomes stronger after initial

adhesion. Consequently, Fprev and Fdet should be considered as independent

parameters.

There is no unambiguous relation between the hydrodynamic forces (Fprev

and Fdet) directed parallel to the substratum surface and perpendicularly oriented

parameters (FDLVO, air-liquid interface detachment), because these forces act in

different directions. DLVO forces maybe wrongfully estimated because of local

charge heterogeneities and bridging between cell appendages and/or exudates on

the one hand and substrate surface on the other. Furthermore, air-liquid interface

induced detachment relies on a three-phase system, whereas the other forces are

obtained for a two-phase environment, complicating establishment of a possible

correlation.

Chapter 2

38

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Chapter 2

44

CHAPTER 3

RESIDENCE TIME DEPENDENT DESORPTION OF

STAPHYLOCOCCUS EPIDERMIDIS FROM HYDROPHILIC AND

HYDROPHOBIC SUBSTRATA

Reproduced with permission of Elsevier b.v. from: Boks, N.P., Kaper, H.J., Norde, W.,

Busscher, H.J. and Van der Mei, H.C. (2008), Colloids and Surfaces B: Biointerfaces 67,

276-278.

Chapter 3

46

Abstract

Adhesion and desorption are simultaneous events during bacterial adhesion to

surfaces, although desorption is far less studied than adhesion. Here, desorption

of Staphylococcus epidermidis from substratum surfaces is demonstrated to be

residence time dependent. Initial desorption rate coefficients were similar for

hydrophilic and hydrophobic dimethyldichlorosilane (DDS) -coated glass, likely

because initial desorption is controlled by attractive Lifshitz-Van der Waals

interactions, which are comparable on both substratum surfaces. However,

significantly slower decay times of the desorption rate coefficients are found for

hydrophilic glass than for hydrophobic DDS-coated glass. This difference is

suggested to be due to the acid-base interactions between staphylococci and

these surfaces, which are repulsive on hydrophilic glass and attractive on

hydrophobic DDS-coated glass. Final desorption rate coefficients are higher on

hydrophilic glass than on hydrophobic DDS-coated glass, due to the so called

hydrophobic effect, facilitating a closer contact on hydrophobic DDS-coated

glass.

Residence time dependent desorption

47

Introduction

Microbial adhesion to substratum surfaces is generally considered to consist of

two steps [1,2]. In the first step, adhesion is reversible and detachment may

occur spontaneously. Gradually, as a second step, adhesive forces increase to

cause more irreversible adhesion [3-6] with a lower desorption probability [7].

Although residence time dependent desorption has been investigated previously,

these studies were subject to technical limitations [8]. Analyses were based on a

series of images taken with 12 s time interval between consecutive pictures.

Nowadays, consecutive images can be taken with a time interval of 1 s, or even

shorter if desired, enabling more accurate registration and analysis of adhesion

and desorption events in microbial adhesion.

Desorption of bacteria from surfaces can be described by a so called

residence time dependent desorption rate coefficient (β(t-τ)). After adhesion,

immediate spontaneous detachment may occur, which is reflected in an initial

desorption rate coefficient (β0). However, due to bond strengthening effects, the

desorption probability will decrease with prolonged contact time and, as a result,

β0 will decay with a characteristic decay time τc to a final desorption rate

constant, β∞. The bond strength between bacteria and surfaces might be

influenced by several factors, like for example hydrodynamic shear [9,10],

substratum surface hydrophobicty or biosurfactant release. Initial desorption rate

coefficients of Streptococcus thermophilus B from hydrophilic glass and

hydrophobic fluoroethylenepropylene, for instance, were 7.4 x 10-3 s-1 and 7.7 x

10-3 s-1, respectively and decreased with residence time on both surfaces. Final

desorption rate coefficients were achieved within 60 s and were slightly larger

on the hydrophilic than on the hydrophobic surface (1.0 x 10-5 s-1 and 0.7 x 10-5

s-1, respectively), indicating that desorption was easiest from the hydrophilic

surface [8].

Chapter 3

48

Staphylococcus epidermidis is normally a non-pathogenic skin organism,

but it is involved in many biomaterial-related infections. S. epidermidis can

adhere to a variety of different hydrophobic and hydrophilic biomaterials, but

little is known about its ability to desorb from substratum surfaces. The aim of

this study is to investigate the residence time dependent desorption of 4 S.

epidermidis strains from a hydrophilic and a hydrophobic substratum surface.


Staphylococcal strains and culture conditions. S. epidermidis strains 3399,

ATCC 35983, HBH2 3 and HBH2 169 were cultured aerobically in 10 ml

Tryptone Soy Broth (OXOID) for 24 h at 37 ºC. After 24 h, cultures were used

to inoculate 200 ml main cultures, which were grown for 16 h under similar

conditions as the precultures. Bacteria were harvested by centrifugation for 5

min at 5000 x g, washed twice with 10 mM potassium phosphate buffer at pH 7

and suspended in the same buffer. To break bacterial aggregates, the bacterial

suspension was sonicated 3 times for 10 s at 30 W (Vibra Cell model 375,

Sonics and Materials Inc., Danbury, CT, USA), while cooling the suspension in

a water/ice bath. Staphylococci were suspended to a concentration of 3 x 108 per

ml in 10 mM potassium phosphate buffer at pH 7.

Desorption studies. Experiments were conducted in a parallel plate flow

chamber [11]. The top and bottom plates of the flow chamber were comprised of

two microscope glass slides. As a hydrophilic substratum surface, glass (water

contact angle 28 ± 8 degrees) was used, while a hydrophobic surface was

obtained by silanization of glass slides in 0.05% (w/v) dimethyldichlorosilane

(DDS, water contact angle 101 ± 2 degrees) in trichloroethylene [12]. After

filling the system with a bacterial suspension, flow was maintained at a wall


49

shear rate of 15 s-1 and adhesion and desorption was monitored microscopically

on the bottom plate of the flow chamber. Consecutive images were taken at 1 s

time intervals for a period of 25 min with a CCD camera (Basler A102F, Basler

AG, Germany). All experiments were conducted in six fold with three separately

grown cultures.

Analysis. Image analysis consisted of registering the time of arrival and the

location of adhering staphylococci on the substratum surface and comparison of

their positions in following images using proprietary software based on the

Matlab Image processing Toolkit (The MathWorks, MA, USA)). This analysis

allows us to follow individual bacteria in time and distinguishes between

moving and adhering bacteria. Subsequently, when the time of adsorption (τ)

and the time of desorption (t) were known, the residence time dependent

desorption rate (β(t-τ)) can be calculated according to [12]:

( ) ( )( )( )∑ ∑

−

= += −− −ΔΔ

−−=−

1

1 1 111N

j

N

ji iijiads

ides

ttntn

jNt

ττβ (1)

In this summation, which runs over the number of images taken, Δndes(ti) is the

number of bacteria desorbing between time ti-1 and ti and adsorbing between

time τi-j-1 and τi-j, and Δnads(ti-j) is the total number of adsorbed bacteria between

time τi-j-1 and τi-j.

The measured β(t-τ) was fitted according to [13]:

( ) ( )⎟⎟⎠

⎞⎜⎜⎝

⎛ −−−−=− ∞∞

c

ttττβββτβ exp)( 0 (2)

yielding the initial desorption rate coefficient (β0), which decays to a final

desorption rate coefficient (β∞) with a relaxation time (τc).

Chapter 3

50

Statistical analysis (non-parametric Mann-Whitney U test) was performed using

SPSS 14.0 to identify significant differences in staphylococcal desorption from

the two surfaces, taking p < 0.05 as a level of significance.

Results

Figure 1 gives an example of the residence time dependent desorption of S.

epidermidis HBH2 3 from hydrophilic glass and hydrophobic DDS-coated glass.

Similar curves were obtained for the other three staphylococcal strains. An

exponential decay pattern justifies the use of equation 2 to determine initial and

final desorption rate coefficients, as well as their relaxation times, as

summarized in Table 1. On average, the desorption rate coefficients decay from

0.5 s-1 to 2.0 x 10-3 s-1 in less than 1 s, indicating a rapid bond strengthening.

Table 1. Initial and final desorption rate coefficients (β0 and β∞, respectively) and their characteristic decay time (τc) of S. epidermidis strains from hydrophilic glass and hydrophobic DDS-coated, glass. Values represent averages of six measurements with three separately grown cultures. Average standard deviations amount ± 0.2 s-1 and ± 0.6 x 10-3 s-1 over β0 and β∞, respectively, and ± 0.2 s over τc. Glass DDS-coated glass

Strain β0 (s-1) β∞ (10-3 s-1) τc (s) β0 (s-1) β∞ (10-3 s-1) τc (s)

3399 0.4 2.8 1.1 0.5 1.9 0.7

ATCC 35983 0.4 2.4 1.1 0.4 1.2 0.7

HBH2 3 0.5 2.2 0.9 0.5 1.3 0.7

HBH2 169 0.8 2.5 1.1 0.5 1.4 0.8

Initial desorption rate coefficients β0 are not statistically different on hydrophilic

glass and hydrophobic DDS-coated glass. However, final desorption rate

coefficients β∞ and relaxation times τc differ significantly between both


51

substratum surfaces. On DDS-coated glass, bond strengthening is faster and

yields lower final desorption rate coefficients than on hydrophilic glass.

Figure 1. Examples of the residence time dependent desorption rate coefficient (β(t-τ)) as a function of the residence time (t-τ) for S. epidermidis HBH2 3 from hydrophilic glass (●) and hydrophobic DDS-coated glass (○).

Discussion

Adhesion and desorption are outer cell surface events, and attractive Lifshitz-

Van der Waals forces are among the forces that become effective at some

separation distance between a bacterium approaching a surface. This is different

from interactions such as hydrogen bonding, which require close contact. The

Lifshitz-Van der Waals free energies of interaction are attractive and of

comparable magnitude for all staphylococci on both hydrophilic and DDS-

Chapter 3

52

coated hydrophobic glass, as can be seen from Table 2, constructed from

previously published contact angles on staphylococcal lawns and the solid

substrata involved [15]. Since the initial desorption rates on hydrophilic and

hydrophobic DDS-coated glass are comparable as well for all staphylococcal

strains, it is suggested here that initial desorption of adhering bacteria is

controlled by attractive Lifshitz-Van der Waals forces.

Hydrogen bonding between a bacterium (b) and a substratum surface (s)

requires direct contact between the two components. It is a competitive process

involving hydrogen bonding between b and water (l) and between s and l on the

one hand, and between s and b and l and l, on the other. The values for ΔGABslb,

reported in Table 2, indicate that this competition for hydrogen bonding results

in attraction of the staphylococci to the hydrophobic DDS-coated glass and in

repulsion from the hydrophilic glass surface.

Table 2. Lifshitz-Van der Waals and Acid-Base components of interaction free energy (ΔGLW

slb and ΔGABslb, respectively) between staphylococci and hydrophilic glass or

hydrophobic DDS-coated glass, calculated from published contact angles [14]. Glass DDS-coated glass

Strain ΔGLWslb

(mJ m-2)

ΔGABslb

(mJ m-2)

ΔGLWslb

(mJ m-2)

ΔGABslb

(mJ m-2)

3399 -1.7 +34.6 -1.2 -7.4

ATCC 35983 -1.4 +25.7 -1.0 -14.7

HBH2 3 -1.7 +22.1 -1.2 -13.3

HBH2 169 -2.1 +26.8 -1.4 -13.7

Since ΔGAB is evaluated from interfacial tensions [16] it is a macroscopic

property. However, the substratum surface and, even more so, the bacterial

surface are highly heterogeneous. Hence, even though for the whole bacterial


53

cell ΔGABslb is repulsive on glass, favourable bonds between the cell and this

surface may be formed locally. Indeed, such favourable bonds must have been

formed, because otherwise no significant adhesion of the staphylococci on glass

would have been observed. It is understood that, unlike in adhesion to DDS-

coated glass, bacteria non-specifically adsorb to glass in the first, fast step of

adhesion by sampling multiple interaction sites until favourable conditions

enable progression to stronger adhesion. This process requires rearrangements of

bacterial surface structures and it explains why the strength of the adhesive bond

increases at a lower rate at the glass surface. This is reflected in the larger decay

times for the desorption rate coefficient, τc, on glass.

Although desorption rate coefficients do not provide information on the

magnitude of adhesion force or bond strength [17], the differences in final

desorption rate coefficients β∞ show that the rate of desorption at equilibrium is

higher for the staphylococci interacting with hydrophilic glass than those

interacting with hydrophobic DDS-coated glass. Apparently, the contribution of

the hydrophobic effect, i.e. the entropy-driven dehydration of a hydrophobic

surface [18], is responsible for the relatively low desorption probability. Also

other surface characteristics might aid bacterial adhesion. For example, Lichter

et al. recently showed that an increasing elastic modulus of the substratum

surface has a positive effect on the adhesion of viable S. epidermidis cells [19].

Such an effect cannot be ruled out in our case as we use glass and DDS-coated

glass.

At this point, it should be noted that previously reported values for the

desorption rate constants β0 and β∞ were 100 to 1000 times smaller and

relaxation time constants τc were about 50 times slower than reported in our

study [8]. This is a clear result of faster computing capabilities, allowing shorter

time intervals between recorded images. Interestingly, although the time interval

Chapter 3

54

between consecutive images is shorter, in our current study initial desorption

rate coefficients also decay by a factor 100 to 1000.

Conclusions

Staphylococcal desorption from hydrophobic DDS-coated glass and hydrophilic

glass is residence time dependent. Under the experimental conditions employed,

including hydrodynamic shear, temperature and the buffer, initial desorption rate

coefficients for a collection of staphylococcal strains were similar for all strains

and substrata and are suggested to be controlled by attractive Lifshitz-Van der

Waals interactions, acting immediately upon approach of a bacterium toward a

surface. Stable desorption rate coefficients were achieved faster for all four

staphylococcal strains on hydrophobic DDS-coated glass than on hydrophilic

glass, due to favourable acid-base interactions between the staphylococci and

DDS-coated glass. We propose that final desorption rate coefficients are

controlled by the hydrophobic effects, facilitating removal of interfacial water,

enhancing contact with DDS-coated glass and resulting in lower final desorption

rate coefficients for the hydrophobic surface.


55

References 1. Hermansson, M. (1999), The DLVO theory in microbial adhesion, Colloids Surface B

14, 105 - 119.

2. Palmer, J., Flint, S. and Brooks, J. (2007), Bacterial cell attachment, the beginning of

a biofilm, J Ind Microbiol Biot 34, 577 - 588.

3. Castelain, M., Pignon, F., Piau, J.M., Magnin, A., Mercier-Bonin, M. and Schmitz,

P. (2007), Removal forces and adhesion properties of Saccharomyces cerevisiae on

glass substrates probed by optical tweezer, J Chem Phys 127, 135104-1 - 135104-14.

4. Mercier-Bonin, M., Ouazzani, K., Schmitz, P. and Lorthois, S. (2004), Study of

bioadhesion on a flat plate with a yeast/glass model system, J Colloid Interf Sci 271,

342 - 350.

5. Simpson, K.H., Bowden, M.G., Hook, M. and Anvari, B. (2002), Measurement of

adhesive forces between S. epidermidis and fibronectin-coated surfaces using optical

tweezers, Laser Surg Med 31, 45 - 52.

6. Xu, L.C. and Siedlecki, C.A. (2007), Effects of surface wettability and contact time on

protein adhesion to biomaterial surfaces, Biomaterials 28, 3273 - 3283.

7. Cowan, M.M., Taylor, K.G. and Doyle, R.J. (1986), Kinetic-analysis of Streptococcus

sanguis adhesion to artificial pellicle, J Dent Res 65, 1278 - 1283.



9. Thomas, W., Forero, M., Yakovenko, O., Nilsson, L., Vicini, P., Sokurenko, E. and

Vogel, V. (2006), Catch-bond model derived from allostery explains force-activated

bacterial adhesion, Biophys J 90, 753 - 764.

10. Meinders, J.M., Noordmans, J. and Busscher, H.J. (1992), Simultaneous monitoring

of the adsorption and desorption of colloidal particles during deposition in a parallel

plate flow chamber, J Colloid Interf Sci 152, 265 - 280.

Chapter 3

56



12. Ruardy, T.G., Schakenraad, J.M., Van der Mei, H.C. and Busscher, H.J. (1995),

Adhesion and spreading of human skin fibroblasts on physicochemically characterized

gradient surfaces, J Biomed Mater Res 29, 1415 - 1423.

13. Meinders, J.M., Van der Mei, H.C. and Busscher, H.J. (1994), Physicochemical

aspects of deposition of streptococcus-thermophilus-b to hydrophobic and hydrophilic

substrata in a parallel-plate flow chamber, J Colloid Interf Sci 164, 355 - 363.

14. Dabros, T. and Van de Ven, T.G.M. (1982), Kinetics of coating by colloidal particles,

J Colloid Interf Sci 89, 232 - 244.

15. Boks, N.P., Norde, W., Van der Mei, H.C. and Busscher, H.J. (2008), Forces

involved in bacterial adhesion to hydrophilic and hydrophobic surfaces, Microbiology

154, 3122 - 3133.

16. Van Oss, C.J. (1994), Polar or Lewis acid-base interactions. In: Interfacial forces in

aqueous media. Van Oss, C.J. (Eds.), New York:Marcel Dekker. pp 18 - 45.

17. Walton, E.B., Lee, S. and Van Vliet, K.J. (2008), Extending bell's model: How force

transducer stiffness alters measured unbinding forces and kinetics of molecular

complexes, Biophys J 94, 2621 - 2630.

18. Norde, W. (2003), Water. In: Colloids and interfaces in life sciences. Norde, W. (Eds.),

New York:Marcel Dekker Inc. pp 47 - 61.

19. Lichter, J.A., Thompson, M.T., Delgadillo, M., Nishikawa, T., Rubner, M.F. and

Van Vliet, K.J. (2008), Substrata mechanical stiffness can regulate adhesion of viable

bacteria, Biomacromolecules 9, 1571 - 1578.

CHAPTER 4

MOBILE AND IMMOBILE ADHESION OF STAPHYLOCOCCAL

STRAINS TO HYDROPHILIC AND HYDROPHOBIC SURFACES

Boks, N.P., Kaper, H.J., Norde, W., Van der Mei, H.C. and Busscher, H.J. (2008), Journal

of Colloid and Interface Science (in press).

Chapter 4

58

Abstract

Staphylococcus epidermidis adheres to hydrophilic glass and hydrophobic

dimethyldichlorosilane (DDS)-coated glass in similarly high numbers, but in

different modes. Real-time observation of staphylococcal adhesion under a shear

rate of 15 s-1 in a parallel plate flow chamber revealed different adhesion

dynamics on both substratum surfaces. The total number of adsorption and

desorption events to achieve a similar total number of adhering bacteria was

twice as high on hydrophilic glass than on hydrophobic DDS-coated glass.

Moreover, 22% of all staphylococci on hydrophilic glass slid over the surface

prior to adhering on a fixed site (“mobile adhesion mode”), but mobile adhesion

was virtually absent (1%) on hydrophobic DDS-coated glass. Similarly, sliding

preceded desorption on hydrophilic glass in about 20 % of all desorption events,

while on hydrophobic DDS-coated glass 2% of all staphylococci desorbed

straight from the site where they had adhered. Since acid-base interactions

between the staphylococci and a hydrophobic DDS-coating are attractive, it is

suggested that these interactions facilitate a closer approach of the bacteria to the

substratum and therewith enhance immobile adhesion at local, high affinity

surface heterogeneities. Alternatively, if the local site is low affinity, surface

heterogeneities may lead to desorption from the surface. In the absence of

attractive acid-base interactions, as the case on hydrophilic glass, bacteria can be

captured in the minimum of the DLVO-interaction energy curve, but this does

not prevent them from sliding under the influence of flow at a fixed distance

from a substratum surface until immobilization or desorption at or from a local

high or low affinity site, respectively.

Mobile and immobile adhesion of staphylococcal strains

59

Introduction

Microbial adhesion to surfaces is a phenomenon occurring in many fields of

application [1,2], as e.g. dairy processing, food and paper industry,

bioremediation of contaminated soils and in the restoration of human function

using biomaterials. Microbial adhesion can be detrimental or beneficial. For

example, the colonization of biomedical implants by Staphylococcus

epidermidis strains may lead to biomaterial related infections and usually

necessitates removal of the implant [3]. Alternatively, effective bioremediation

of soil or wastewater requires bacteria to adhere [4,5].

Adhesion starts with the transport of micro-organisms towards a

substratum surface by means of gravity, convection and diffusion [6,7]. Upon

close approach, adhesion is mediated by so-called non-specific DLVO

interactions, including Lifshitz-Van der Waals-, electrostatic- and Lewis acid-

base interactions [8] and ultimately via specific ligand-receptor interactions [9].

Microbial adhesion can be a dynamic process consisting of an ongoing series of

adsorption and desorption events, depending on the degree of reversibility of the

interaction. Micro-organisms may adhere reversibly or irreversibly and the

desorption probability was found to depend on the contact time between bacteria

and a substratum surface [10]. Generally, the desorption probability decreases

with increasing contact time, suggesting an increase in bond strength in time, as

evidenced for the time-dependent interaction between Streptococcus

thermophilus and a silicon nitride AFM tip [11].

Fluid flow may have a profound influence on microbial adhesion. Fluid

flow is not only responsible for convective mass transport toward a substratum

surface, but can also stimulate detachment once exceeding a critical limit [12] or

alternatively, facilitate stronger binding [13]. A basic feature of microbial

adhesion is, that the micro-organisms reside at a fixed distance from a

Chapter 4

60

substratum surface, or at least in the close vicinity of that surface for an

extended period of time. Therewith, adhesion does not rule out that organisms

can slide over a substratum surface due to fluid flow and it seems appropriate to

introduce the terms “mobile” and “immobile” adhesion. Sliding may occur

directly after a bacterium has arrived at a substratum surface until it has reached

its final site, or as the onset of a desorption event. We want to stress that in this

respect, our notion of mobile is not related to bacterial motility, like in

flagellated bacteria [14] or bacterial gliding, as expressed by some non-

flagellated bacteria [15,16].

In this chapter a method is proposed to distinguish between mobile and

immobile adhesion and the modes of adhesion of S. epidermidis strains on

hydrophilic glass and hydrophobic DDS-coated glass under flow are compared.


Bacterial strains and culture conditions. S. epidermidis strains 3399, ATCC

35983, HBH2 3 and HBH2 169 were cultured aerobically from blood agar plates

in 10 ml Tryptone Soya Broth (OXOID, Basingstoke, England) for 24 h at 37

ºC. After 24 h, precultures were used to inoculate 200 ml main cultures, which

were grown for 16 h. Bacteria were harvested by centrifugation for 5 min at

6500 x g, washed twice with 10 mM potassium phosphate buffer at pH 7 and

resuspended in the same buffer. To break bacterial aggregates, 3 times 10 s

sonication at 30 W (Vibra Cell model 375, Sonics and Materials Inc., Danbury,

CT, USA) was carried out while cooling the suspension in a water/ice bath.


(Omnilabo International BV, Breda, The Netherlands) followed by thorough

rinsing with tap water, demineralised water, methanol, tap water and finally


61

demineralised water again to obtain a hydrophilic surface (water contact angle

28 ± 8 degrees). After washing, the slides were either directly used or dried for 4

h at 80 ºC for coating. A hydrophobic coating (water contact angle 101 ± 2

degrees) was achieved by submerging the dried glass slides during 15 min in a

solution of dimethyldichlorosilane (DDS, Merck, Darmstadt, Germany) in

trichloroethylene (0.05 w/v%) and subsequent washing with trichloroethylene,

methanol and ultrapure water. Coated slides were never stored longer than 3

days at room temperature prior to experiments and rinsed with 10 mM

potassium phosphate buffer before use.

Mobile and immobile bacterial adhesion in a parallel plate flow chamber.

The parallel plate flow chamber (PPFC) and image analysis have been described

previously [10]. The PPFC used in this study has a length of 175 mm, a depth

of 0.75 mm and a width of 17 mm. Prior to use, the flow chamber was washed

with 2% Extran (Merck, Darmstadt, Germany) and rinsed thoroughly with tap

water and demineralised water. After cleansing, the PPFC was equipped with a

glass top plate and a glass or DDS-coated bottom plate, to which staphylococcal

adhesion, including all individual adsorption and desorption events, was

observed. The PPFC was installed on a microscope stage between two

communicating vessels and the system was filled with 10 mM potassium

phosphate buffer while care was taken to remove all air bubbles. The vessels

containing bacterial suspension were positioned at different heights to create a

flow and the difference in fluid levels was maintained by a roller-pump to

ensure a circulating pulse free flow throughout the duration of an experiment.

Phosphate buffer was allowed to flow through the system at a flow rate of 1.5 ml

min-1 for half an hour. Subsequently, buffer was switched to bacterial

suspension, which was allowed to flow through the PPFC during 25 min at a

rate of 1.5 ml min-1. Under these conditions, flow is laminar and bacterial

Chapter 4

62

A

C

B

D

transport occurs by convective-diffusion. Deposition of bacteria was monitored

with a phase contrast microscope (Olympus BH-2) equipped with a 40x ultra

long working distance objective (Olympus ULWD-CD Plan 40 PL) which was

connected to a CCD-MXRi camera (Basler A102F, Basler AG, Hamburg,

Germany). Image frames (1392 x 1040 pixels) were grabbed at a rate of 15 s-1

and averaged to remove in focus flowing bacteria from the analysis, yielding

images with a time interval of 1 s between each image. Further analysis

consisted of registering the location of the bacteria on the substratum surface

and comparison of their positions in subsequent images in order to determine the

mode of adhesion of individual bacteria. Mobile and immobile adhesion were

distinguished both with respect to adsorption and desorption events (see Fig. 1).

Figure 1. Schematic presentation of the different modes of adhesion distinguished in this study: Immobile adsorption (A): A bacterium arrives at the surface and adheres without sliding along the surface until possible detachment. Mobile adsorption (B): A bacterium arrives at the surface and adheres, but slides along the surface in the direction of the flow under the perpendicularly oriented attractive forces originating from the substratum, until it has found its final adhesion site or possible detachment. Immobile desorption (C): A bacterium detaches from a substratum surface directly from its adhesion site, without sliding prior to desorption. Mobile desorption (D): A bacterium starts to slide away from its adhesion site, but remains adhering under the perpendicularly oriented attractive forces originating from the substratum, until it finally desorbs.


63

The distinction between adsorption and desorption can simply be made on the

basis of appearance or disappearance of a bacterium. The distinction between

mobile and immobile is a more difficult one to make. A bacterium was

identified as adhering in an immobile mode, when it showed a total

displacement immediately after adsorption or just prior to desorption of less than

1 µm around its adhesion site. Sometimes bacteria were observed to “wobble”

around their adhesion site, but since this was always within the displacement

limit of 1 µm, these bacteria were classified as immobile ones. Sliding once

adhering at a fixed distance above a substratum surface is flow-induced and

establishing mobile adhesion requires a distinction between sliding bacteria that

are under the influence of the perpendicularly oriented attractive forces

originating from the substratum and bacteria that are moving in the fluid flow

without an interaction with the substratum. In order to make this distinction (see

also Fig. 2), it was first calculated that under the experimental conditions, the

fluid velocity at a distance of 0.5 µm above the bottom plate of the flow

chamber (i.e. the radius of a single bacterium) was 5 µm s-1 [6]. Allowing a

minor displacement of 0.5 µm perpendicular to the flow direction, all bacteria

travelling a distance of less than 5 µm in two consecutive images in the direction

of flow were considered to have an interaction with the substratum surface and

were classified as adhering in a mobile mode.

Statistical analysis. Data were analyzed with the Statistical Package for the

Social Sciences (version 14.0, SPSS, Chicago Illinois, USA). Differences

between the two modes of adhesion on both substratum surfaces were analyzed

using the Mann-Whitney U test. The level of significance was set at p < 0.05.

Chapter 4

64

ΔX = 1-5 μm

2 μm

Δt = 1 s

Y-c

oord

inat

e

X-coordinate

Direction of flow

Figure 2. Graphical presentation of the criteria used to identify a mobile mode of adhesion. The black dot represents the X,Y location in the field of 1392 x 1040 pixels of a bacterium in the first image. If the X-displacement in the direction of the flow between two subsequent images is between 1 µm and 5 µm and the Y-displacement is less than 0.5 µm (i.e. the radius of the bacterium), the bacterium is considered to adhere in a mobile mode.

Results

Table 1 summarizes the total number of adhering bacteria to hydrophilic glass

and hydrophobic DDS-coated glass after 25 min of flow for all four

staphylococcal strains. It is of interest to note that three out of the four strains

adhere in similar numbers to the hydrophilic and hydrophobic substratum, while

only strain HBH2 169 adheres in higher numbers to hydrophobic DDS-coated

glass. Table 1 also comprises the number of adsorption and desorption events

leading to the total number of adhering bacteria given. Clearly, staphylococcal

adhesion is a dynamic process on both substrata.

Mob

ile a

nd im

mob

ile a

dhes

ion

of st

aphy

loco

ccal

stra

ins

65

Tab

le 1

. Tot

al n

umbe

rs o

f bac

teria

that

hav

e ad

sorb

ed (N

ads)

and

deso

rbed

(Nde

s) du

ring

25 m

in o

f flo

w. N

end r

epre

sent

s th

e nu

mbe

r of b

acte

ria

pres

ent

by t

he e

nd o

f th

e ex

perim

ent

(i.e.

pre

sent

in

the

final

im

age

afte

r 25

min

). V

alue

s pr

esen

t th

e av

erag

e ±

stan

dard

dev

iatio

n of

six

in

depe

nden

t exp

erim

ents

with

thre

e se

para

tely

gro

wn

cultu

res o

f S. e

pide

rmid

is st

rain

s.

Gla

ss

DD

S-co

ated

gla

ss

Stra

in

Nad

s

(x 1

06 cm

-2)

Nde

s

(x 1

06 cm

-2)

Nen

d

(x 1

06 cm

-2)

Nad

s

(x 1

06 cm

-2)

Nde

s

(x 1

06 cm

-2)

Nen

d

(x 1

06 cm

-2)

3399

18

± 1

2 16

± 1

1 2.

5 ±

1.0

7 ±

4 5 ±

1 2.

4 ±

0.9

ATC

C 3

5983

9 ±

3 7 ±

3 1.

7 ±

0.5

4 ±

1 2 ±

1 1.

9 ±

0.3

HB

H2 3

8 ±

2 6 ±

2 2.

1 ±

0.2

5 ±

1 3 ±

1 2.

0 ±

0.1

HB

H2 1

69

20 ±

7

19 ±

7

1.0 ±

0.4

9 ±

7 6 ±

7 3.

1 ±

0.4


65

Chapter 4

66

However, on the hydrophilic glass surface, the number of adsorption and

desorption events observed are two- to threefold higher than on hydrophobic

DDS-coated glass and staphylococcal adhesion to the hydrophilic surface is thus

more dynamic than on the hydrophobic surface.

Figure 3 presents an example of staphylococcal sliding for S. epidermidis 3399

on hydrophilic glass and hydrophobic DDS-coated glass prior to desorption.

Tracks indicative of mobile desorption are clearly present on hydrophilic glass

(Fig. 3A) but not on hydrophobic DDS-coated glass (Fig. 3B). The modes of

adsorption and desorption for all four staphylococcal strains and both substrata

are summarized in Table 2. Interestingly, the percentages of staphylococci

showing immobile or mobile adsorption are similar to the percentages of

immobile and mobile desorption, respectively, with no considerable differences

between strains: 19-24% of the staphylococci adhering to hydrophilic glass do

so in a mobile mode, whereas on hydrophobic DDS-coated glass virtually no

(0-2%) staphylococci adhere in this mode.

Discussion

Over the past decades, many researchers have attempted to relate numbers of

bacteria adhering to substratum surfaces with surface free energies. Indeed for

specific collections of strains, or sometimes even one strain, the substratum

surface free energies, its Lifshitz-Van der Waals components and/or electron-

donating and electron-accepting parameters related with numbers of adhering

bacteria [17]. However, in other studies such a relation is rather weak [18], or

even absent [19,20]. Also in our current study, changing the substratum surface

hydrophobicity, and therewith the surface free energy, does not clearly influence

the number of adhering staphylococci.

Mob

ile a

nd im

mob

ile a

dhes

ion

of st

aphy

loco

ccal

stra

ins

67

Tab

le 2

. Fra

ctio

ns o

f im

mob

ile a

nd m

obile

ads

orpt

ion

and

deso

rptio

n ev

ents

in s

taph

yloc

occa

l adh

esio

n to

hyd

roph

ilic

glas

s an

d hy

drop

hobi

c D

DS-

coat

ed g

lass

. V

alue

s gi

ve t

he a

vera

ge o

f si

x in

depe

nden

t ex

perim

ents

with

thr

ee s

epar

atel

y gr

own

cultu

res

of S

. ep

ider

mid

is s

train

s. St

anda

rd d

evia

tions

are

, on

aver

age,

± 9

% o

n gl

ass a

nd ±

2%

on

DD

S-co

ated

gla

ss.

G

lass

D

DS-

coat

ed g

lass

A

dsor

ptio

n ev

ents

D

esor

ptio

n ev

ents

A

dsor

ptio

n ev

ents

D

esor

ptio

n ev

ents

Stra

in

Imm

obile

(%)

Mob

ile (%

)Im

mob

ile (%

) M

obile

(%)

Imm

obile

(%)

Mob

ile (%

) Im

mob

ile (%

) M

obile

(%)

3399

78

22

86

14

99

1

99

1

ATC

C 3

5983

81

19

82

18

99

1

99

1

HB

H2 3

76

24

82

18

10

0

0 99

1

HB

H2 1

69

77

23

71

29

98

2

94

6

67


Chapter 4

68

Figure 3. Example of the sliding for S. epidermidis 3399 on hydrophilic glass (A) and hydrophobic DDS-coated glass (B), prior to desorption. Single dots represent immobile desorption events, while lines indicate the tracks along which mobile desorption events occur.

However, it is still generally believed that surface free energies play a definitive

role in certain aspects of bacterial adhesion to surfaces.

In this chapter, we compared the mode of adhesion of four staphylococcal

strains under flow on hydrophilic glass and hydrophobic DDS-coated glass. The

total number of adsorption and desorption events appeared two times higher on

hydrophilic glass as compared to hydrophobic DDS-coated glass, despite the

fact that the total numbers of adhering bacteria were roughly similar on both


69

substratum surfaces. It was further shown that on hydrophilic glass, on average,

21% of the staphylococci adhered in a mobile mode and slid over the surface

prior to adhering on a fixed site or desorbing. Mobile adhesion occurred tenfold

less frequently on hydrophobic DDS-coated glass than on hydrophilic glass.

These results demonstrate that substratum hydrophobicity is much more

influential on the dynamics of bacterial adhesion to biologically inactive

surfaces, i.e. adhesion in the absence of specific ligand-receptor bonding, than it

is on the number of actually adhering bacteria. Interestingly, using a similar

experimental set-up and analysis, De Kerchove at al. [21] showed that sliding

of carboxylated latex particles was virtually absent on hydrophilic quartz but

present when an alginate conditioning film was used. It was stated that the

properties of the alginate (i.e. surface roughness and local charge/chemical

heterogeneities) were likely to facilitate the irreversible adhesion.

All four staphylococcal strains behaved similarly with respect to their

dynamic adhesion to glass and DDS-coated glass, respectively. This is in line

with their cell surface properties, including cell surface energetics and

electrostatic potentials (i.e. zeta-potentials), being similar [22]. Both substratum

surfaces were also negatively charged, and the major difference between glass

and DDS-coated glass is their hydrophobicity. Hydrophobicity is determined by

the Lifshitz-Van der Waals and acid-base (AB) free energy components of a

surface [23], which are both incorporated in the extended DLVO-theory (named

after Derjaguin, Landau, Verwey and Overbeek). Previously reported values of

surface free energies, including AB-interactions, for the staphylococci used in

this study [22], are employed here to calculate the distance-dependent total free

energy of interaction. By way of example, Fig. 4 presents the distance-

dependent free energy of interaction between S. epidermidis ATCC 35983 and

hydrophilic glass and hydrophobic DDS-coated glass, respectively. The distance

dependence of the interaction is very similar for both surfaces, except for short

separation distances where acid-base interactions become influential and

Chapter 4

70

dominate. On hydrophilic glass, the total free energy of interaction at close

approach is positive due to repulsive AB-interaction. Conversely, the AB-

interaction is attractive on DDS-coated glass, resulting in a negative total free

energy of adhesion at close approach. Here we suggest, that adhesion on

hydrophilic glass is more dynamic than on hydrophobic DDS-coated glass

because of attractive AB- interactions between the staphylococci and DDS-

coated glass. It has been reported, already more than twenty years ago, that for a

collection of oral streptococci adhesion is more reversible when the total free

energy of adhesion at contact is positive, i.e. repulsive [24]. However, at that

time the contribution of AB-interactions to surface energetics was not well

defined yet. Based on the current study we now conclude that highly reversible

bacterial adhesion can be ascribed to repulsive AB-interactions.

In the curves in Fig. 4, the depths of the minima in the DLVO-interaction

free energy, occurring at around 40 nm away from the surface, are about -0.5

kT. This is in the same range as the energy of thermal motion of a bacterial cell

(in one direction). However, when the surface is chemically non-homogeneous,

the depth of these minima varies over the substratum surface. Indeed, Wit and

Busscher [25] identified scattered positively charged spots on an overall

negatively charged glass surface by noticing that negatively charged polystyrene

particles repeatedly adhered first to the same positions after removal from the

glass surface. The DDS-coated glass surfaces are unlikely to be devoid of

heterogeneities as well. These heterogeneities may include local charge

variations, similar to glass but may also be physical in nature as the thickness of

the DDS-coating might vary. Furthermore, the bacterial cell surfaces, often

consisting of polymeric substances, may exhibit chemical and physical

heterogeneities. Thus, chemical and physical variations of the surface

characteristics of the substrata and bacteria used in our study may influence the

depth of the minima in the interaction free energy considerably. Consequently, a

bacterium sliding over the surface may arrive and become captured at a site


71

Figure 4. Example of the distance-dependent free energy of interaction between S. epidermidis ATCC 35983 and hydrophilic glass and hydrophobic DDS-coated glass, using the extended DLVO-theory and surface characteristics as reported previously [21].

Chapter 4

72

Figure 5. Schematic presentation of bacterial sliding on a heterogeneous surface. Bacteria adhering in the interaction minimum near the surface can slide until becoming immobilized in a local, high affinity site (top panel), or until desorption from a local, low affinity site (bottom panel). The grey area represents the hydrophilic glass; arrows denote the flow-induced movement of a bacterium.


73

where it is more strongly attracted or arrive at a site of weaker attraction from

where it desorbs. Both events are schematically displayed in Fig. 5.

Since adhesion was found to be more mobile on hydrophilic glass than on

hydrophobic DDS-coatings, the question has to be addressed why bacteria spot

high affinity sites more readily on the hydrophobic than on the hydrophilic

surface. Extracellular polymeric substances and/or appendages at the surface of

S. epidermidis may well be able to bridge across the DLVO-free energy barrier

near the DDS-coated glass surface (Fig. 4, lower panel) [26], thereby displacing

water molecules from that surface. With the AB-interaction between the

bacterium and the hydrophobic substratum being attractive, removal of

interfacial water allows the bacteria to approach the substratum surface more

closely. In contrast to hydrophobic surfaces, water molecules are attracted to the

hydrophilic glass surface by hydrogen bonding. Hence, adsorption of bacterial

cell surface components at the expense of displacement of interfacial water is far

less favourable, i.e. AB interaction between the bacterium and the substratum is

repulsive. We suggest that removal of the interfacial water from the hydrophobic

surface makes spotting of high affinity adsorption sites more easy. Hydrophobic

surfaces are thus expected to facilitate the immobile mode of adhesion.

Conclusions

Bacterial adhesion to hydrophilic glass and hydrophobic DDS-coated glass was

found to be comparable in terms of numbers of adhering bacteria for four

different staphylococcal strains. The main difference in staphylococcal adhesion

to both substrata was in the mode of adhesion and adhesion dynamics.

Significantly more adsorption and desorption events were found on hydrophilic

glass as compared to hydrophobic DDS coated glass. This difference is

attributed to repulsive acid-base interactions between the staphylococci and the

Chapter 4

74

hydrophilic glass surface, as opposed to the hydrophobic DDS-coating exerting

attractive acid-base interactions. Sliding over the surface, prior to fixed adhesion

or desorption, providing the basis for our notion of ”mobile” versus “immobile”

adhesion, constituted another important difference between staphylococcal

adhesion to both substrata. Immobile adhesion occurred tenfold more frequently

on hydrophobic DDS-coated glass than on hydrophilic glass. Sliding was

associated with surface chemical heterogeneity, inherent to virtually all surfaces,

and the capacity of adhering bacteria to become locally fixed at high affinity

sites which is facilitated by removal of interfacial water. The latter is clearly

easier at a hydrophobic than at a hydrophilic substratum.

References



2. Von Eiff, C., Jansen, B., Kohnen, W. and Becker, K. (2005), Infections associated

with medical devices - Pathogenesis, management and prophylaxis, Drugs 65, 179 -

214.

3. Vuong, C. and Otto, M. (2002), Staphylococcus epidermidis infections, Microbes

Infect 4, 481 - 489.

4. Nicolella, C., van Loosdrecht, M.C.M. and Heijnen, J.J. (2000), Wastewater

treatment with particulate biofilm reactors, J Biotechnol 80, 1 - 33.

5. Satoh, H., Ono, H., Rulin, B., Kamo, J., Okabe, S. and Fukushi, K.I. (2004),

Macroscale and microscale analyses of nitrification and denitrification in biofilms

attached on membrane aerated biofilm reactors, Water Research 38, 1633 - 1641.


suspensions - Mathematical formulation, numerical solution, and simulations, Separ

Technol 4, 186 - 212.


75

7. Korber, D.R., Lawrence, J.R., Zhang, L. and Caldwell, D.E. (1990), Effect of

gravity on bacterial deposition and orientation in laminar flow environments, Biofouling

2, 335 - 350.

8. Hermansson, M. (1999), The DLVO theory in microbial adhesion, Colloids Surface B

14, 105 - 119.











342 - 350.

13. Anderson, B.N., Ding, A.M., Nilsson, L.M., Kusuma, K., Tchesnokova, V., Vogel,

V., Sokurenko, E.V. and Thomas, W.E. (2007), Weak rolling adhesion enhances

bacterial surface colonization, J. Bacteriol. 189, 1794 - 1802.

14. Vigeant, M.A.S., Ford, R.M., Wagner, M. and Tamm, L.K. (2002), Reversible and

irreversible adhesion of motile Escherichia coli cells analyzed by total internal

reflection aqueous fluorescence microscopy, Appl Environ Microb 68, 2794 - 2801.

15. Kolari, M., Schmidt, U., Kuismanen, E. and Salkinoja-Salonen, M.S. (2002), Firm

but slippery attachment of Deinococcus geothermalis, J Bacteriol 184, 2473 - 2480.

16. Hoiczyk, E. (2000), Gliding motility in cyanobacteria: Observations and possible

explanations, Arch Microbiol 174, 11 - 17.

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17. Absolom, D.R., Lamberti, F.V., Policova, Z., Zingg, W., Van Oss, C.J. and

Neumann, A.W. (1983), Surface thermodynamics of bacterial adhesion, Appl Environ

Microb 46, 90 - 97.

18. Van Pelt, A.W.J., Weerkamp, A.H., Uyen, M.H.W.J., Busscher, H.J., De Jong, H.P.

and Arends, J. (1985), adhesion of Streptococcus sanguis CH3 to polymers with

different surface free energies, Appl Environ Microb 49, 1270 - 1275.

19. Bayoudh, S., Othmane, A., Bettaieb, F., Bakhrouf, A., Ben Ouada, H. and

Ponsonnet, L. (2006), Quantification of the adhesion free energy between bacteria and

hydrophobic and hydrophilic substrata, Mat Sci Eng C-Bio S 26, 300 - 305.

20. Pringle, J.H. and Fletcher, M. (1983), Influence of substratum wettability on

attachment of freshwater bacteria to solid surfaces, Appl Environ Microb 45, 811 - 817.

21. De Kerchove, A.J., Weronski, P. and Elimelech, M. (2007), Adhesion of nonmotile

Pseudomonsa aeruginosa on "soft" polyelectrolyte layer in a radial stagnation point

flow system: Measurements and model prediction, Langmuir 23, 12301 - 12308.

22. Boks, N.P., Norde, W., Van der Mei, H.C. and Busscher, H.J. (2008), Forces

involved in bacterial adhesion to hydrophilic and hydrophobic surfaces, Microbiology

154, 3122 - 3133.

23. Van Oss, C.J. (1995), Hydrophobicity of biosurfaces - Origin, quantitative

determination and interaction energies, Colloids Surface B 5, 91 - 110.

24. Busscher, H.J., Uyen, M.H.M.J., Weerkamp, A.H., Postma, W.J. and Arends, J.

(1986), Reversibility of adhesion of oral streptococci to solids, FEMS Microbiol Lett 35,

303 - 306.

25. Wit, P.J. and Busscher, H.J. (1998), Site selectivity in the deposition and redeposition

of polystyrene particles to glass, J Colloid Interf Sci 208, 351 - 352.

26. Palmer, J., Flint, S. and Brooks, J. (2007), Bacterial cell attachment, the beginning of

a biofilm, J Ind Microbiol Biot 34, 577 - 588.

CHAPTER 5

BOND-STRENGTHENING IN STAPHYLOCOCCAL ADHESION TO

HYDROPHILIC AND HYDROPHOBIC SURFACES USING AFM

Reproduced with permission of the American Chemical Society from: Boks, N.P.,

Busscher, H.J., Van der Mei, H.C. and Norde W. (2008), Langmuir 24, 12990-12994.

Chapter 5

78

Abstract

Time-dependent bacterial adhesion forces of four strains of Staphylococcus

epidermidis to hydrophobic and hydrophilic surfaces were investigated. Initial

adhesion forces differed significantly between the two surfaces and hovered

around -0.4 nN. No unambiguous effect of substratum surface hydrophobicity

on initial adhesion forces for the four different S. epidermidis strains was

observed. Over time, strengthening of the adhesion forces was virtually absent

on hydrophobic dimethyldichlorosilane (DDS)-coated glass, although in a few

cases multiple adhesion peaks developed in the retract curves. Bond-

strengthening on hydrophilic glass occurred within 5 to 35 s to maximum

adhesion forces of -1.9 ± 0.7 nN, and was concurrent with the development of

multiple adhesion peaks upon retract. Poisson analysis of the multiple adhesion

peaks allowed to separate contributions of hydrogen bonding from other non-

specific interaction forces and revealed a force contribution of -0.8 nN for

hydrogen bonding and +0.3 nN for other non-specific interaction forces. Time-

dependent bacterial adhesion forces were comparable for all four staphylococcal

strains. It is concluded that on DDS-coated glass, the hydrophobic effect causes

instantaneous adhesion, while strengthening of the bonds on hydrophilic glass is

dominated by non-instantaneous hydrogen bond formation.

Bond strengthening in staphylococcal adhesion

79

Introduction

Bacterial adhesion to surfaces is a crucial step in biofilm formation and

associated problems, such as in biomaterials implant surgery. Staphylococcus

epidermidis is one of the most often isolated bacterial pathogens in biomaterials-

implant related infections [1,2]. One of the first steps in biofilm formation is

transport of the bacterial cells towards the surface, which may be governed by

sedimentation, convection and diffusion [3,4]. Once brought within the range of

the interaction forces, bacteria can come in close contact with a substratum

surface and adhere. Initially, adhesion is reversible but over time adhesion

becomes irreversible [5,6], although the exact mechanism of this transition

remains poorly understood.

Flow displacements systems have been extensively used to determine the

close-range affinity of bacteria for a substratum surface [7]. One of the

possibilities in such systems is to determine the hydrodynamic forces to prevent

adhesion or to detach adhering bacteria. Usually, the forces to prevent adhesion

are smaller than the forces needed to establish detachment, suggesting that the

adhesion bond strengthens over time [8-10]. As a disadvantage, however, flow

displacement systems only provide an indirect measure of the actual adhesion

force, without a clear view on time dependence of strengthening of the bond

between individual bacteria and the substratum surface. Atomic force

microscopy (AFM) is a promising technique to directly measure the interaction

forces between bacteria and substratum surfaces.

For example, Cao et al. [11] found adhesion forces upon retract between 5

and 24 nN for Escherichia coli and various hydrophilic and hydrophobic

surfaces, while Sheng et al. [12] found adhesion forces upon retract of 0.5 to 5.6

nN for Pseudomonas aeruginosa on various metal surfaces. Others showed that

Chapter 5

80

the presence of surface structures influence bacterial adhesion properties

substantially, sometimes already in the approach curve [5,6,13].

Measuring bond-strengthening with AFM requires accurate control of the

z-displacement during contact of the probe with a substratum surface.

Previously, AFM systems were not fully capable of achieving this. Nowadays,

precise feedback in z-displacement, ensures that the probe can remain at the

same distance from a surface within a force-curve bandwidth of 0.1 nm root

mean square. Bond-strengthening effects have been reported for eukaryotic cells

with mica and silica [14,15], or polymer coated model colloids interacting with

bovine serum albumin, lysozyme and dextran [16]. Furthermore, Vadillo-

Rodriguez et al. [17] probed whole bacterial cells with silicon nitride tips at two

different pH values and found bond-strengthening to occur within 60 s,

independent of pH.

However, no study has been undertaken hitherto to demonstrate a

potential role of substratum hydrophobicity on bond-strengthening between

bacteria and substratum surfaces. Therefore, the aim of this chapter is to

compare bond-strengthening of four S. epidermidis strains on a hydrophobic

(dimethyldichlorosilane-coated glass) and a hydrophilic (glass) substratum.


Staphylococcal strains and culture conditions. S. epidermidis strains ATCC

35983, 3399, HBH2 3 and HBH2 169 were cultured aerobically from blood agar

plates in 10 ml Tryptone Soy Broth (OXOID, Basingstoke, England) for 24 h at

37ºC (Note that the latter three strains are clinical isolates). After 24 h,

precultures were used to inoculate 200 ml main cultures, which were grown for

16 h under similar conditions as the precultures. Bacteria were harvested by

centrifugation for 5 min at 5000 x g, washed twice with 10 mM potassium


81

phosphate buffer at pH 7 and resuspended in the same buffer. To break bacterial

aggregates, 3 times 10 s sonication at 30 W (Vibra Cell model 375, Sonics and

Materials Inc., Danbury, CT, USA) was carried out while cooling the suspension

in a water/ice bath.


(Omnilabo International BV, The Netherlands) followed by thorough rinsing

with tap water, demineralised water, methanol, tap water and finally

demineralized water again to obtain a hydrophilic surface (water contact angle

28 ± 8 degrees). After washing, the slides were either directly used or dried for 4

h at 80ºC prior to applying of a hydrophobic coating.

To obtain a hydrophobic surface (water contact angle 101 ± 2 degrees),

the dried glass slides were submerged during 15 min in a solution of

dimethyldichlorosilane (DDS, Merck, Germany) in trichloroethylene (0.05

w/v%) and washed with trichloroethylene, methanol and ultrapure water.

Prepared slides were stored for no longer than 3 days at room temperature and

rinsed with 10 mM potassium phosphate buffer before use.

Bacterial probe preparation. Staphylococci were immobilized to tipless “V”-

shaped cantilevers (VEECO, DNP-0) by means of electrostatic attraction with

positively charged poly-L-lysine. To this end, cantilevers were mounted in a

micromanipulator under microscopic observation to allow only the tip of the

cantilever to be coated. A droplet of poly-L-lysine solution was placed on a

glass slide and the tip of the cantilever was dipped in the droplet for 1 min. After

air drying the cantilever for 2 min, it was dipped in bacterial suspension for 1

min. Bacterial probes were freshly prepared for each experiment and checked

regularly during an experiment for staphylococcal presence.

Chapter 5

82

Atomic Force Microscopy. AFM experiments were carried out at room

temperature in 10 mM potassium phosphate buffer (pH 7) using an optical lever

microscope (Nanoscope IV Digital instruments). For each probe, force curves

were measured for different surface delay times on the same, randomly chosen,

spot on a hydrophobic or hydrophilic substratum surface. Interaction forces were

measured after 0, 10, 30, 45, 60, 90 and 120 s of contact time (Δt) between the

bacterial probe and the substratum surface with z-scan rates of less than 1 Hz.

To ensure that no staphylococci detached from the cantilever during the

experiment, 5 control force-distance curves were made with 0 s contact time

after each measurement with a certain surface delay, as schematically outlined in

Figure 1. Whenever the “0 s contact time” forces measured were out of range, a

bacterial probe was replaced. For each combination of a bacterial strain and

substratum surface, six probes were employed on average and the number of

staphylococcal probes used depended on the outcome of the control

measurements. Calibration of bacterial probes was done using the thermal

tuning method (Nanoscope V6.13r1), yielding spring constants of 0.044 ± 0.008

Nm-1 .

Subsequently, for each staphylococcal probe the maximum adhesion

forces were plotted as a function of the surface delay time and fitted to:

( ) ⎟⎠

⎞⎜⎝

⎛⎭⎬⎫

⎩⎨⎧ Δ−−−+=Δ ∞ τ

tFFFtF ss exp1)( 00 (1)

with F0s the maximum adhesion force at 0 s contact time, F∞ the maximum

adhesion force after bond-strengthening and τ the characteristic time needed for

the adhesion force to strengthen.


83

Prepare a new probe

5curves at Δt = 0 s

5 curves at Δt = 10 s

5 control curves at Δt = 0 s

5 curves at Δt = i s

Are control curves and curves at Δt = 0 s similar?

Yes No

Figure 1. Schematics of the experimental time line, including go/no points after control measurements with a staphylococcal probe to determine the need to prepare a new bacterial probe. In the absence of the need for new probe preparation, the same probe was used for a new contact time i (30, 45, 60, 90 and 120 s).

Statistical Analysis. Data was analyzed with the Statistical Package for the

Social Sciences (version 14.0, SPSS, Chicago Illinois, USA). A Wilcoxon

signed rank test was used to analyze adhesion forces measured within a probe.

Differences between the above bond-strengthening parameters on the two

substratum surfaces were analyzed using the Mann-Whitney U test; this test was

also used to analyze the total ageing of the adhesion force. The level of

significance was set at p < 0.05.

Results

As an example, Figure 2 presents force-distance curves obtained for S.

epidermidis ATCC 35983 on hydrophobic DDS-coated and on hydrophilic

glass. A downward peak in the retract curves indicates attractive forces between

the bacterium and surface and the maximum adhesion force corresponds to the

Chapter 5

84

largest peak. From Figure 2A it is clear that there is hardly any strengthening of

the maximum adhesion force on hydrophobic DDS-coated glass, although upon

longer contact times (i.e. 120 s) a few multiple adhesion peaks may be observed.

On hydrophilic glass (Figure 2B), the maximum adhesion force increases

strongly with increasing surface delay time and multiple adhesion peaks, that

may already be observed at 0 s contact time, strongly develop during prolonged

contact between bacterial cell and substratum surface. Note that the long-range

detachment events at several hundred nanometers in the retract curves represent

stretching of staphylococcal cell surface structures.

Figures 3 and 4 show the maximum adhesion forces as a function of

contact time for the four staphylococcal strains involved in this study for both

DDS-coated glass (Figure 3) and bare glass (Figure 4). In both figures grey

regions represent the force window of the control curves at 0 s contact time. All

control curves had similar appearances and within each set of measurements no

significant changes in their adhesion force was observed. On a hydrophobic

substratum (Figure 3), adhesion forces hardly increase upon increasing the

contact time between the staphylococcal probe and the substratum surface. On

hydrophilic glass, however, adhesion forces increase significantly with longer

contact times (Figure 4). Adhesion forces already significantly strengthen within

10 s of contact for all strains and reach stable values within approximately 60 s.

Table 1 summarizes the bond-strengthening parameters, as can be determined by

using Eq. (1) for all four S. epidermidis strains investigated. There is no

unambiguous influence of the substratum on the initial maximal adhesion forces

(F0s) as can be seen from Table 1. However, in line with the qualitative features

of Figures 3 and 4, the increases in maximum adhesion force on hydrophobic

DDS-coated glass are limited to 0.3 nN and only significant for strains 3399 and

HBH2 3. Conversely, on the hydrophilic glass the maximum adhesion forces

increase significantly for all strains with increments ranging between 0.7 nN and


85

Figure 2. Examples of force-distance curves for S. epidermidis ATCC 35983 on hydrophobic DDS-coated glass (A) and hydrophilic glass (B) with retract curves after 0, 10, 60 and 120 s. Maximum adhesion forces (Fmax) are defined as the force associated with the largest adhesion peak.

Chapter 5

86

1.5 nN. The characteristic time τ needed for the adhesion force to strengthen is

maximally 32 s (see Table 1). Table 1. Bond-strengthening parameters for four S. epidermidis strains on DDS-coated glass (hydrophobic) and glass (hydrophilic). Values represent the averages and standard deviations of 6 staphylococcal probes, each used to measure 5 force distance curves.

DDS-coated glass Glass

Strain F0s (nN) F∞ (nN) τ (s) F0s (nN) F∞ (nN) τ (s)

3399 -0.4 ± 0.3 -0.6 ± 0.4 17 ± 21 -0.2 ± 0.2 -0.9 ± 0.2 17 ± 13

ATCC 35983 -0.4 ± 0.3 -0.6 ± 0.5 16 ± 14 -0.5 ± 0.2 -1.9 ± 0.7 17 ± 15

HBH2 3 -0.3 ± 0.1 -0.6 ± 0.3 8 ± 6 -0.3 ± 0.1 -1.8 ± 0.9 32 ± 27

HBH2 169 -0.1 ± 0.1 -0.1 ± 0.1 -* -0.6 ± 0.3 -1.8 ± 0.4 3 ± 1

* No bond-strengthening found

Discussion

In this chapter, we investigated the time-dependent adhesion forces between

four S. epidermidis strains and hydrophilic and hydrophobic substratum

surfaces. Hydrophobicity was created by DDS-coating of glass surfaces, and

thus had little effect on the roughness of the substratum surface. Both glass as

well as DDS-coated glass are negatively charged with comparable zeta

potentials [18]. Therefore, hydrophobicity is considered to be the main

difference between the two surfaces. Strengthening of the bond was virtually

absent on hydrophobic DDS-coated glass, but on hydrophilic glass strengthening

of the bond by factors up to 6 occurred within a few tens of seconds, concurrent

with the consistent development of multiple adhesion peaks in the retract force-

distance curves. All four staphylococcal strains roughly exhibited a similar

behaviour with respect to bond-strengthening. In order to rule out artefacts due


87

to bacterial detachment from the probe and/or due to bacterial footprints on the

substratum surface [19], force-distance curves with 0 s contact time were

recorded after each measurement with a given surface delay time. Control

experiments indicated that staphylococci did not detach from the probe and that

there were no bacterial footprints left on the substratum surface after

measurement of a force-distance curve, with a measurable influence on the 0 s

force-distance curves.

Figure 3. Adhesion forces of four S. epidermidis strains on DDS-coated glass as a function of the surface delay time. Grey regions denote the force window of the control curves at 0 s contact time, based on the average and standard deviation of 180 measurements. Each point represents the average and standard deviation of 30 measurements divided over 6 bacterial probes.

Bond-strengthening was significantly different on hydrophobic DDS-coated

glass than on hydrophilic glass. The hydrophobicity of DDS-coated glass is

Chapter 5

88

caused by the presence of apolar CH3-groups. Water molecules adjacent to the

substratum surface are not able to form hydrogen bonds with the apolar surface,

and therefore they will do so as much as possible with other water molecules at

the solution side of the surface [20]. This phenomenon is known as the hydro

phobic effect. As a result the water molecules near the surface are restricted in

their rotational freedom. Consequently, bacterial adhesion to a DDS-coated

substratum is driven by an entropically favourable release of DDS-associated

Figure 4. Adhesion forces of four S. epidermidis strains on glass as a function of the surface delay time. Grey regions denote the force window of the control curves at 0 second contact time, based on the average and standard deviation of 180 measurements. Each point represents the average and standard deviation of 30 measurements divided over 6 bacterial probes.

water molecules, giving relatively weak adhesion forces between bacteria and

substratum surface. This release of water molecules from the surface region is a


89

fast process, and from the current data can be expected to be completed within

the first 10 s of contact.

Alternatively, hydrophilic glass offers numerous sites for hydrogen

bonding and the hydrophobic effect is not likely to play any significant role in

bond-strengthening between staphylococci and glass. Similar to the glass

surface, also the staphylococcal strains involved are negatively charged,

hydrophilic and able to form hydrogen bonds [21]. Upon approach of the

bacteria towards the hydrophilic surface, the outer cell surface first forms

hydrogen bonds with the substratum, therewith expelling surface-associated

water molecules. This implies a gain in entropy and a relatively fast rise in

adhesion force. However, upon prolonged contact times, more extensive

rearrangements of bacterial surface structures may occur to create additional

bonds and cause the adhesion force to strengthen further. This is reflected in

Figure 2, by the development of multiple adhesion peaks upon prolonged

contact times. Interestingly, Abu-Lail and Camesano [22] recently performed a

Poisson analysis of these multiple adhesion peaks in the interaction of E. coli

with silicon nitride AFM tips and associated these peaks with multiple hydrogen

bonds with an individual force value of -0.13 nN.

In the Poisson analysis of multiple adhesion forces, it is assumed that the

average force of all adhesion peaks (μF) is related to the variance (σF2) of the

adhesion force according to:

specificNonbondHbondHFF FFF −−− −= μσ 2 (2)

in which FH-bond and FNon-specific represent the contributions of hydrogen bonding

and other non-specific interaction forces (i.e. contributions of Lifshitz-Van der

Waals-, electrostatic- and steric interactions) to the adhesion force, respectively.

Thus, in a plot of σF2 versus μF , the slope of a linear fit will yield FH-bond, while

FNon-specific can be calculated from the intercept.

Chapter 5

90

Figure 5. Example of a Poisson analysis of the multiple adhesion peaks appearing after 120 s contact between S. epidermidis ATCC 35983 and hydrophilic glass. The linear dependency of the average adhesion force of all peaks (μF) versus the variance (σF

2) of the adhesion force is denoted by the solid line (r2 = 0.86). The slope of the regression yields FH-bond, while from the intercept FNon-specific is calculated.

Poisson analysis of our retract curves of staphylococci from glass obtained after

120 s contact time were completely in line with the observations by Abu-Lail

and Camesano [22] as can be seen in Figure 5 for a selected example. From the

straight line dependencies as in Figure 5, contributions of H-bonding and non-

specific interaction forces to the total adhesion forces between staphylococci and

glass after bond-strengthening could be calculated, as summarized in Table 2.


91

Table 2. Poisson analysis on the multiple adhesion peaks as observed for four S. epidermidis strains on glass after a contact time of 120 s. Adhesion forces were separated in a hydrogen bonding component (FH-bond) and a component for non-specific interactions (FNon-specific).

Strain

FH-bond (nN)

FNon-specific (nN)

Net Force (nN)

(FH-bond + FNon-specific)

3399 -0.24 +0.07 -0.17

ATCC 35983 -0.79 +0.33 -0.46

HBH2 3 -1.02 +0.58 -0.45

HBH2 169 -0.75 +0.41 -0.34

All FNon-specific values are positive, indicating that non-specific interactions have a

net repulsive contribution due to the forced nature of the contact in AFM.

Whereas under non-forced conditions, it is difficult to establish contact closer

than in the secondary minimum of the DLVO interaction curve [23]. It is clear

from the current results that in AFM close contact beyond the secondary

minimum is imposed, causing the non-specific forces, attractive at long range, to

become repulsive at short range. FH-bond is negative for all four staphylococcal

strains involved and amounts to -0.70 nN on average. Abu-Lail et al. [22]

measured a force contribution FH-bond of -0.13 nN, but their measurements

involved a smaller contact area, i.e. a native AFM tip versus a bacterial cell

surface, whereas we used a bacterial probe versus a macroscopic glass surface.

Note that the net interaction force (i.e. FH-bond + FNon-specific) is attractive in all

cases, but less than the maximum adhesion forces after bond-strengthening

measured, because the Poisson analysis is based on average forces and not on

the maximum forces observed.

Chapter 5

92

Conclusions

Staphylococcal bond-strengthening between hydrophobic DDS-coated and

hydrophilic glass proceeds according to different mechanisms, as revealed here

by using atomic force microscopy. On hydrophobic DDS-coated glass bond-

strengthening was fast (less than 10 s), limited to a minor increase in adhesion

force and likely governed by hydrophobic interaction. On hydrophilic glass,

bond strength increased during contact time and is ascribed to progressive

formation of hydrogen bonds made possible by ongoing rearrangements of outer

cell surface structures over a time scale of, typically, a few tens of seconds. As a

consequence, adhesion forces strengthened considerably more on hydrophilic

glass than on hydrophobic DDS-coated glass, as confirmed by Poisson analysis

of the multiple adhesion peaks upon retract of the staphylococci from

hydrophilic glass surfaces.

References 1. Presterl, E., Lassnigg, A., Parschalk, B., Yassin, F., Adametz, H. and Graninger, I.

(2005), Clinical behavior of implant infections due to staphylococcus epidermidis, Int J

Artif Org 28, 1110 - 1118.

2. Vuong, C. and Otto, M. (2002), Staphylococcus epidermidis infections, Microbes

Infect 4, 481 - 489.


suspensions - Mathematical formulation, numerical solution, and simulations, Separ

Technol 4, 186 - 212.

4. Korber, D.R., Lawrence, J.R., Zhang, L. and Caldwell, D.E. (1990), Effect of

gravity on bacterial deposition and orientation in laminar flow environments, Biofouling

2, 335 - 350.


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5. Bell, C.H., Arora, B.S. and Camesano, T.A. (2005), Adhesion of Pseudomonas putida

KT2442 is mediated by surface polymers at the nano- and microscale, Environ Eng Sci

22, 629 - 641.

6. Salerno, M.B., Li, X. and Logan, B.E. (2007), Adhesion characteristics of two

Burkholderia cepacia strains examined using colloid probe microscopy and gradient

force analysis, Colloid Surface B 59, 46 - 51.



8. Duddridge, J.E., Kent, C.A. and Laws, J.F. (1982), Effect of surface shear stress on

the attachment of Pseudomonas fluorescens to stainless steel under defined flow







342 - 350.

11. Cao, T., Tang, H.Y., Liang, X.M., Wang, A.F., Auner, G.W., Salley, S.O. and Ng,

K.Y.S. (2006), Nanoscale investigation on adhesion of E. coli surface modified silicone

using atomic force microscopy, Biotechnol Bioeng 94, 167 - 176.

12. Sheng, X.X., Ting, Y.P. and Pehkonen, S.O. (2007), Force measurements of bacterial

adhesion on metals using a cell probe atomic force microscope, J Colloid Interf Sci 310,

661 - 669.

13. Ong, Y.L., Razatos, A., Georgiou, G. and Sharma, M.M. (1999), Adhesion forces

between E. coli bacteria and biomaterial surfaces, Langmuir 15, 2719 - 2725.

14. Bowen, W.R., Lovitt, R.W. and Wright, C.J. (2001), Atomic force microscopy study

of the adhesion of Saccharomyces cerevisiae, J Colloid Interf Sci 237, 54 - 61.

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15. McNamee, C.E., Pyo, N., Tanaka, S., Vakarelski, I.U., Kanda, Y. and Higashitani,

K. (2006), Parameters affecting the adhesion strength between a living cell and a colloid

probe when measured by the atomic force microscope, Colloid Surface B 48, 176 - 182.

16. Xu, L.C., Vadillo-Rodriguez, V. and Logan, B.E. (2005), Residence time, loading

force, pH, and ionic strength affect adhesion forces between colloids and biopolymer-





18. Gomez-Suarez, C., Pasma, J., Van der Borden, A.J., Wingender, J., Flemming,

H.C., Busscher, H.J. and Van der Mei, H.C. (2002), Influence of extracellular

polymeric substances on deposition and redeposition of Pseudomonas aeruginosa to

surfaces, Microbiol-Sgm 148, 1161 - 1169.

19. Paul, J.H. and Jeffrey, W.H. (1985), Evidence for separate adhesion mechanisms for

hydrophilic and hydrophobic surfaces in Vibrio proteolytica, Appl Environ Microb 50,

431 - 437.

20. Norde, W. (2003), Water. In: Colloids and interfaces in life sciences. Norde, W. (Eds.),

New York:Marcel Dekker Inc. pp 47 - 61.

21. Kiers, P.J.M., Bos, R., Van der Mei, H.C. and Busscher, H.J. (2001), The

electrophoretic softness of the surface of Staphylococcus epidermidis cells grown in a

liquid medium and on a solid agar, Microbiology 147, 757 - 762.

22. Abu-Lail, N.I. and Camesano, T.A. (2006), Specific and nonspecific interaction forces

between Escherichia coli and silicon nitride, determined by poisson statistical analysis,

Langmuir 22, 7296 - 7301.

23. Hermansson, M. (1999), The DLVO theory in microbial adhesion, Colloid Surface B

14, 105 - 119.

CHAPTER 6

FIBRONECTIN INTERACTIONS WITH STAPHYLOCOCCUS

AUREUS WITH AND WITHOUT FIBRONECTIN-BINDING

PROTEINS AND THEIR ROLE IN ADHESION AND DESORPTION

Xu, C.-P., Boks, N.P., De Vries, J., Kaper, H.J., Norde, W., Busscher, H.J. and Van der

Mei, H.C. (2008), Applied and Environmental Microbiology (in press).

Chapter 6

96

Abstract

Adhesion and residence-time dependent desorption of two Staphylococcus

aureus strains with and without fibronectin binding proteins (FnBPs) on Fn-

coated glass were compared in a parallel plate flow chamber. To gain a better

understanding of the role of Fn-FnBP binding, adsorption enthalpies of Fn to

staphylococcal cell surfaces were determined using isothermal titration

calorimetry (ITC). Interaction forces between staphylococci and Fn-coatings

were measured using atomic force microscopy (AFM). The strain with FnBPs

adhered faster and initially stronger to a Fn-coating than the strain without

FnBPs and its Fn-adsorption enthalpies were higher. Initial desorption was high

for both strains, but decreased strongly within 2 s. These time scales of

staphylococcal bond ageing were confirmed by AFM adhesion force

measurements. After exposure of either Fn-coating or staphylococcal cell

surfaces to bovine serum albumin (BSA), adhesion of both strains to Fn-coatings

was reduced, suggesting that BSA not only suppresses non-specific, but also

specific Fn-FnBPs interactions. Adhesion forces and adsorption enthalpies were

only slightly affected by BSA adsorption. This implies that under the mild

contact conditions of convective-diffusion in the flow chamber, adsorbed BSA

prevents specific interactions, but does allow forced Fn-FnBPs binding during

AFM or stirring in ITC. Bond strength energies calculated from retract force-

distance curves in AFM were orders of magnitude larger than from desorption

data, confirming that the penetrating Fn-coated AFM tip probes multiple

adhesins in the outermost cell surface that remain hidden during mild landing of

an organism on a Fn-coated substratum as during convective-diffusional flow.

Fibronectin interactions

97

Introduction

Staphylococcus aureus is an extremely versatile pathogen, which can adhere to

epithelial cells, endothelial cells, fibroblasts as well as to plasma exposed

biomaterials implant surfaces in the human body [1], causing potentially

persistent infections. The best described mechanism of S. aureus adhesion to

eukaryotic cells and other fibronectin-coated surfaces involves the fibronectin

(Fn) binding proteins FnBP A and FnBP B on the surface of S. aureus [2,3].

Peacock et al. [4] demonstrated the significant role played by the FnBPs by

comparing adhesion of different isogenic S. aureus strains to human endothelial

cells. Moreover, in vitro adhesion of S. aureus strain Wood 46 to Fn-coated

surfaces was demonstrated to be inhibited in a dose-dependent manner by anti-

Fn antibodies [5,6].

At constant temperature and pressure, which is usually the case in

biological systems, all physico-chemical interactions, including adsorption, (co-)

adhesion and (co-)aggregation, are determined by changes in the Gibbs energy

(G) of a system. These interactions can either be evaluated at a macroscopic

level, in terms of Lifshitz-Van der Waals, electrostatic and hydrophobic forces

originating from overall characteristics of bacteria and substrata, or at a more

microscopic or even nanoscopic level, where highly specific interactions

between stereo-chemical surface components, such as fibronectin and FnBPs are

considered. For a spontaneous process, the change in Gibbs energy (ΔG) is

negative. ΔG is composed of a change in enthalpy (ΔH) and in entropy (ΔS),

according to

ΔG = ΔH – T ΔS (1)

where T is the temperature in Kelvin. The enthalpy tends to reach a minimum

value, whereas the entropy strives for a maximum. The enthalpy of a system is

Chapter 6

98

directly related to its heat content. At constant pressure, and if no work other

than that related to volume change is involved, the enthalpy change can be

determined as the heat exchange between a system and its environment. Direct

determination of the entropy, however, is practically impossible as it would

require counting all conformational and configurational possibilities before and

after a process. Many biological processes are characterized by strong enthalpy-

entropy compensation [7], that is, they occur spontaneously by virtue of an

entropy increase that compensates for an unfavourable enthalpy effect, or vice

versa. The enthalpy of interaction between bacterial cell surfaces and proteins

can be assessed using isothermal titration calorimetry (ITC). ITC measures the

enthalpy change of formation of a complex at constant temperature. Xu et al. [8]

determined the adsorption enthalpies of salivary proteins to Streptococcus

mutans and found that S. mutans LT11 with antigen I/II, a cell surface binding

protein involved in bacterial adhesion to extracellular matrix proteins, yielded a

much higher, exothermic adsorption enthalpy when mixed with saliva at pH 6.8

than did S. mutans IB03987, lacking surface antigen I/II. It was thus inferred

that antigen I/II at the surface of S. mutans LT11 specifically binds different

proteins with different affinities from the large pool of proteins present in whole

saliva. Furthermore, Busscher et al. [9] used ITC to evaluate the adsorption of a

single protein, laminin, to these streptococcal cell surfaces and found that

enthalpy is released upon adsorption of laminin to the surface of the parent

strain LT11, but not upon adsorption to IB03987. Whereas ITC operates at a

macroscopic level, atomic force microscopy (AFM) senses at the nanometer

level and allows to determine the force between a sharp probe attached to a

flexible cantilever and a cell surface and can thus distinguish between different

functional surface proteins [10]. Using AFM, differences in interaction forces

between protein-coated AFM probes and streptococcal strains with and without

antigen I/II have been measured. Generally, upon retraction of streptococci from


99

saliva- or laminin-coated probes, stronger forces were observed when the

streptococcal strain possessed antigen I/II than when it did not.

Initial microbial adhesion is reversible, but over time the bond strength

may increase and adhesion becomes less reversible. The kinetics of microbial

adhesion and desorption can be investigated simultaneously in a parallel plate

flow chamber with in situ observation and real-time image analysis. Moreover,

by registering the time of arrival and detachment of an adhering microorganism,

desorption can be measured as a function of the residence time of an adhering

organism [11-13]. Dabros and Van de Ven [11] proposed that the desorption rate

coefficient of a particle adsorbed at time τ and desorbing at time t, i.e. after

residing on the surface for a time (t−τ), changes exponentially from an initial

value β0 to a final value β∞ during ageing of the bond with a relaxation time 1/δ

according to:

β(t−τ) = β∞−(β∞−β0)e−δ(t−τ) (2)

Meinders et al. [14] applied this equation to analyze the residence time-

dependent desorption of Streptococcus thermophilus B during non-specific

adhesion on glass, and found that the desorption rate coefficient decreased

according to Eq. (2) from an initially high value β0 (2.5 × 10-3 s-1) to an almost

negligibly low value β∞ (0.01 × 10-3 s-1) over a time scale of approximately 50 s.

Many years later, atomic force microscopy (AFM) was applied to directly

measure the strengthening of the adhesion force between S. thermophilus B and

a silicon nitride (Si3Ni4) AFM tip and bond strengthening by a factor of 2 to 3

was found to occur over a similar time scale as the residence-time dependent

desorption [15]. However, S. thermophilus B adheres to glass utilizing non-

specific adhesion mechanisms, which are very different from the specific

mechanisms applied by S. aureus strains in their adhesion to Fn-films.

Therefore, the aim of this study is to analyze the role of FnBPs on S.

aureus cell surfaces in their interaction with (adsorbed) Fn using ITC and AFM,

Chapter 6

100

in particular in relation to adhesion and residence-time dependent desorption on

Fn-coated surfaces under flow. To this end, we first determined adhesion and

desorption of a S. aureus wild type strain 8325-4 and of an isogenic mutant

DU5883 lacking FnBPs, deposited by convective-diffusion on Fn-coated glass

slides in a parallel plate flow chamber. Subsequently, interaction forces between

Fn-coated AFM tips and the cell surfaces were compared, while furthermore the

enthalpies of adsorption of Fn to the surfaces of the S. aureus strains were

measured. In order to determine to what extent Fn-binding to S. aureus cell

surfaces is dominated by specific interactions, additional experiments were

performed after coating either the substrata or the staphylococcal cells with a

layer of bovine serum albumin (BSA).


Bacterial strains and culture conditions. S. aureus strain 8325-4 and its

isogenic mutant lacking FnBPs, DU5883 (kindly provided by Dr. T.J. Foster,

Moyne Institute of Preventive Medicine, Dublin, Ireland), were used in this

study. The bacterial cells were maintained at -80oC in tryptone soya broth (TSB;

OXOID, Basingstoke, UK) containing 7% dimethylsulfoxide (DMSO; MERCK,

Germany). For culturing, both strains were plated onto TSB agar plates

overnight at 37oC. Subsequently, bacterial colonies were precultured in 10 ml

TSB batch culture overnight under constant rotation. This preculture was used to

inoculate a main culture of 190 ml TSB. After approximately 2 h of growth to

early stationary phase, corresponding with peak expression of FnBPs in S.

aureus 8325-4 [16], bacteria were harvested by centrifugation at 6500g for 5

min at 10oC and washed twice with demineralised water. Bacterial chains and

aggregates were broken by mild sonication on ice for 3 × 10 s at 30 W (Vibra

Cell model 375, Sonics and Materials Inc., Danbury, Connecticut, USA). Then


101

bacteria were resuspended in phosphate-buffered saline (PBS; 10 mM potassium

phosphate and 0.15 M NaCl, pH 7), to a concentration of 3 × 108 or 5 × 109 per

ml for adhesion experiments or ITC, respectively, as determined in a Bürker-

Türk counting chamber. In order to block FnBPs on the staphylococcal cell

surfaces, staphylococci were also incubated for 60 min at 37oC in PBS

supplemented with 1% BSA.

Bacterial deposition to a Fn-film in a parallel plate flow chamber. The

deposition experiments were carried out in a parallel plate flow chamber

(internal dimensions: length × width × height, 175 × 17 × 0.75 mm) equipped

with image analysis options [17]. The bottom glass plate (76 × 26 mm) of the

flow chamber was first cleaned by sonication for 3 min in a surfactant solution

(2 % RBS 35 detergent in water; Omniclean), rinsed thoroughly with tap water,

and then washed with methanol, thoroughly rinsed with tap water and finally

with demineralised water. Subsequently, the centre of the glass plate was drop-

coated with 0.05 ml Fn (25 µg ml-1 human Fn, Sigma-Aldrich BV, Zwijndrecht,

The Netherlands) for 2 h at room temperature to create a circular Fn-coated

region with a diameter of approximately 1 cm on which staphylococcal adhesion

was monitored. In addition, glass plates were prepared on which non-specific

adhesion sites were blocked by immersing the entire glass plate, including the

Fn-coated region for 1 min in PBS containing 1% BSA. Glass plates were rinsed

after protein coating with demineralised water. Bacterial adhesion was

monitored with a phase-contrast microscope (Olympus BH-2) equipped with a

×40 ultra-long-working-distance lens (Olympus ULWD-CD plan 40 PL) and

coupled to a Firewire CCD camera (Basler AG, Germany).

The flow rate during the experiments was adjusted to 1.4 ml min-1 under

the influence of a hydrostatic pressure yielding a shear rate of 15 s-1. During

flow experiments, 15 images (1392 × 1040 pixels) were grabbed every second.

These 15 frames were averaged on a pixel by pixel basis in order to distinguish

Chapter 6

102

between adhered and in focus moving bacteria. The averaged frame was

computer-stored for subsequent offline analysis using proprietary software based

on the Matlab Image Processing Toolkit (The Mathworks, MA, USA). Further

analysis consisted of locating the staphylococci on the substratum surface and

comparison of their positions in a current image with their positions in previous

images to determine the total number of adhering bacteria n(t) as a function of

time during 4 h as well as their residence times. The affinity of an organism for

the Fn-coated glass surface was expressed as the initial deposition rate j0,

representing the initial increase of n(t) with time. Note that since the initial

deposition rate is derived only from the first adhering bacteria, it represents the

affinity of the organisms for the adsorbed Fn-coatings without intervening

influences of interactions between adhering bacteria, as occurs due to crowding

at the surface, such as after 4 h [17]. Finally, the staphylococcal desorption rate

coefficient β(t−τ) as a function of residence-time (t−τ) was calculated according

to [14]:

∑∑+= −−

−

= −ΔΔ

−−=−

N

ji iijiads

idesN

j ttntn

jNt

1 1

1

1 ))(()(

11)(

ττβ (3)

where the summation runs over the number of images taken, Δndes(ti) is the

number of bacterial desorbing between time ti-1 and ti and adsorbing between

time τi-j-1 and τi-j, and Δnads(ti-j) is the total number of adsorbed bacteria between

time τi-j-1 and τi-j. The residence–time dependent desorption rates calculated were

fitted to Eq. (2) to yield the initial and final desorption rate coefficients (β0 and

β∞, respectively) and their relaxation time 1/δ.

All adhesion and desorption experiments in the parallel plate flow

chamber were done in four-fold with separate bacterial cultures.

Atomic force microscopy. For AFM, the negatively charged bacteria were

attached through electrostatic interactions to a glass slide, made positively


103

charged through pre-adsorption of poly-L-lysine, as described before [18]. AFM

tips (DNP from Veeco, Woodbury, USA) were coated with a Fn-film by

immersion for 30 min in a Fn-solution (25 µg ml-1 in PBS, pH 7) with the aid of

a micromanipulator. All glass slides with immobilized bacteria and Fn-coated

AFM tips were immediately used after preparation. To block non-specific

binding sites on the bacterial cell surfaces, the glass slides with attached bacteria

were also immersed for 1 min in PBS containing 1% BSA and rinsed with

demineralised water.

AFM measurements were done at room temperature in PBS using a

Dimension 3100 system (Nanoscope IV Digital Instrument, Woodbury, USA).

Nanoscope imaging software (version 6.13r1, Veeco) was used to analyze the

resulting images. All AFM cantilevers were calibrated using resonant frequency

measurements [19] and the slopes of the retract force curves, in the region where

probe and sample are in contact, were used to translate the voltage into

cantilever deflection. Force-distance curves were generated and approach curves

analyzed for the repulsive force at contact. Retraction of the tip from the

bacterial surface was carried out after 0 and 2 s contact time between the AFM

tip and staphylococcal cell surface to demonstrate strengthening of the adhesion

force. Retract curves were integrated to yield the bond strength energy for the

two surface delay times evaluated.

Three different bacterial cells were examined at ten locations for each

particular case, yielding 30 force-distance curves. This resulted in a non-

parametric distribution, from which median, mode and range values were

derived.

Isothermal titration calorimetry. The adsorption enthalpy of Fn to the

bacterial cell surfaces was measured in a twin-type, isothermal microcalorimeter

TAM 2277 (Thermometric, Sweden). The calorimeter was positioned in a

Chapter 6

104

temperature-controlled environment (20 ± 0.1oC), allowing a baseline stability

of ± 0.1 µW over 24 h [20]. The instrument had an electrical calibration with a

precision better than 1% and proper calibration was regularly checked by

measuring the dilution enthalpy of concentrated sucrose solutions [21].

Experiments were performed isothermally at 25oC in stainless steel ampoules of

4 ml. Four ampoules, connected with separate titration systems, were used inside

the microcalorimeter. The use of a twin-type microcalorimeter allows the

measurement of the heat (Q) flowing from the reaction ampoule as compared

with a reference ampoule. The output signal was collected as power, P, versus

time, t, and was integrated to evaluate the isobaric heat exchange (the enthalpy

change) during adsorption, using the dedicated Digitam 4.1 software

(Thermometric, Sweden). Notably, the measured heat effect should be corrected

for the heat of dilution of the proteins to obtain the net adsorption enthalpy [22].

Typically, all four reaction ampoules including the reference ampoule,

were filled with 1.5 ml of bacterial suspension (5 ×109 cells per ml) in PBS

under constant stirring (90 rpm) with a specially designed two-blades stirrer.

The ampoules were lowered gradually into the microcalorimeter and left in the

measuring position to reach thermal equilibrium before data collection started.

After equilibration, a stable baseline was obtained and Fn was titrated into the

reaction ampoules. Titration was done at a controlled rate of 2 µl s-1 via a

stainless steel cannula connected to a syringe. In order to study possible

saturation of adsorption sites, Fn solution (25 µg ml-1) was added in four

consecutive injections of 60 µl into the ampoule with intervals of 40 min. All

calorimetric experiments were done in fourfold.

Statistical analysis. Data were analyzed with the statistical package for the

social sciences (Version 11.0, SPSS, Chicago, Illinois, USA). Median values of

the repulsive force at contact (F0) upon approach, the adhesion force (Fadh) upon


105

retract, as well as of the bond strength energy were analyzed using the Wilcoxon

signed rank test for the median. A Student's t-test was used to determine

significant differences in initial deposition rates, adhesion numbers after 4 h,

initial and final desorption rate coefficients, and their relaxation time as well as

in interaction enthalpies. The level of significance was set at p < 0.05.

Time (s)

0 4000 8000 12000 16000

Bac

teria

(106

cm-2

)

0

2

4

6

8

Figure 1. Representative examples of the adhesion kinetics of S. aureus 8325-4 (●) and DU5883 (○) to Fn-films in PBS.

Results

Adhesion and residence time dependent desorption of S. aureus from Fn-

films. Figure 1 shows representative examples of the adhesion kinetics of S.

aureus 8325-4 and DU5883 to Fn-coatings in a parallel plate flow chamber in

PBS at pH 7. The adhesion kinetics of both S. aureus strains are linear during

approximately 4000-5000 s prior to levelling off toward stationary numbers. The

linear trajectories of the curves are taken to calculate the initial deposition rates,

as summarized in Table 1. The initial deposition rate of S. aureus 8325-4 is

about twice as high as the one of FnBPs deficient DU5883, which indicates the

Cha

pter

6

106

Tab

le 1

. M

ean

valu

es f

or th

e in

itial

dep

ositi

on r

ate

(j 0)

and

num

bers

adh

erin

g af

ter

4 h

(n4h

), in

itial

(β 0

) an

d fin

al d

esor

ptio

n ra

te c

oeff

icie

nts

(β∞) t

oget

her w

ith th

e re

laxa

tion

time

for b

ond

agei

ng (1

/δ) f

or S

. aur

eus

8325

-4 w

ith fi

bron

ectin

bin

ding

pro

tein

s (F

nBPs

) and

isog

enic

mut

ant

DU

5883

with

out F

nBPs

from

Fn-

coat

ings

. Exp

erim

ents

wer

e pe

rfor

med

prio

r to

and

afte

r exp

osur

e of

the

subs

trata

or t

he s

taph

yloc

occi

to a

1%

B

SA s

olut

ion.

Ave

rage

sta

ndar

d de

viat

ions

ove

r fou

r sep

arat

e ex

perim

ents

am

ount

± 1

10 c

m-2

s-1

and

± 0

.5 ×

106 c

m-2

ove

r the

initi

al d

epos

ition

ra

tes

and

num

bers

of

bact

eria

adh

erin

g af

ter

4 h,

res

pect

ivel

y; ±

124

x 1

0-3 s

-1 a

nd ±

0.3

x 1

0-3 s

-1 o

ver

the

initi

al a

nd f

inal

des

orpt

ion

rate

co

effic

ient

s, re

spec

tivel

y, a

nd ±

0.3

s in

the

rela

xatio

n tim

e fo

r bon

d ag

eing

.

j 0 (c

m-2

s-1)

n 4h

(106 c

m-2

) β

0 (1

0-3 s-1

) β ∞

(10-3

s-1)

1/δ

(s)

Subs

trat

um

8325

-4

DU

5883

83

25-4

D

U58

83

8325

-4

DU

5883

83

25-4

D

U58

83

8325

-4

DU

5883

Fn-c

oate

d gl

ass

2438

12

90

7.0

5.2

307

463

1.0

1.2

0.9

0.9

Fn a

nd B

SA-

coat

ed g

lass

81

5 67

8 5.

2 4.

4 20

0 17

0 0.

6 0

.4

1.0

1.2

Fn-c

oate

d gl

ass*

70

4 52

7 3.

9 3.

5 33

4

504

0.6

1.8

0.9

0.9

* Th

ese

expe

rimen

ts w

ere

carr

ied

out w

ith st

aphy

loco

cci e

xpos

ed to

1%

BSA

prio

r to

the

expe

rimen

ts

Chapter 6

106


107

Figure 2. Representative examples of force-distance curve between an Fn-coated AFM tip and staphylococcal cell surfaces: S. aureus 8325-4 after 0 s surface delay (A), S. aureus 8325-4 after 2 s surface delay (B), S. aureus DU5883 after 0 s surface delay (C), S. aureus DU5883 after 2 s surface delay (D), S. aureus 8325-4 coated with BSA after 0 s surface delay (E), S. aureus 8325-4 coated with BSA after 2 s surface delay (F), S. aureus DU5883 coated with BSA after 0 s surface delay (G) and S. aureus DU5883 coated with BSA after 2 s surface delay (H).

Separation distance (nm)

Chapter 6

108

relatively high affinity of strain 8325-4 for Fn-coatings. Also after 4 h of

deposition, strain 8325-4 adheres in higher numbers than FnBPs deficient

DU5883, but the difference is not two-fold anymore as in initial deposition rates.

After exposure of either the Fn-coating or the staphylococci to BSA, initial

deposition rates and the numbers of bacteria adhering after 4 h decreased

significantly for both strains.

Table 1 also summarizes desorption characteristics of the two

staphylococcal strains. Exposure of either the Fn-coated surface or the bacterial

cells to BSA has only a minor effect, if any, on the initial desorption rate

coefficients (β0), which suggests that the desorbing bacteria mainly leave non-

specific binding sites. Desorption rates decrease with increasing residence-times

for both strains, regardless of the absence or presence of a BSA-coating on the

surfaces with relaxation times for bond ageing less than 2 s. Final desorption

rate coefficients (β∞) are similar for both strains without significant influences of

bacterial exposure to BSA and with a slight reduction in final desorption rates

after exposure of the Fn-coated surface to BSA.

Bond strengthening between Fn-coatings and S. aureus cell surfaces.

Median values of the interaction forces measured using AFM are summarized in

Table 2. The repulsive force at contact F0, is significantly (p < 0.05) stronger for

S. aureus 8325-4 with FnBPs than for S. aureus DU5883. Blocking of non-

specific binding sites on the staphylococcal cell surfaces has little (S. aureus

DU5883) or no (S. aureus 8325-4) influence on the repulsive force upon

approach. However, upon retract, median adhesion forces were significantly

stronger after a 2 s surface delay than when measured immediately, i.e. with a 0

s surface delay. There is no significant difference in adhesion forces between the

two strains. Interestingly, the distance over which the adhesion forces are

operative varies considerably between the different conditions applied, (see Fig.

2) which translates in significant differences in bond strength energies. Initial

Fib

rone

ctin

inte

ract

ions

109

Tab

le 2

. M

edia

n va

lues

1) f

or th

e re

puls

ive

forc

es a

t con

tact

F0

upon

app

roac

h2), a

dhes

ion

forc

e F a

dh u

pon

retra

ct a

nd a

ssoc

iate

d bo

nd s

treng

th

ener

gies

for t

he in

tera

ctio

n be

twee

n Fn

-coa

ted

AFM

tips

and

S. a

ureu

s 832

5-4

and

an is

ogen

ic m

utan

t with

out F

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109

Chapter 6

110

bond strength energies of S. aureus 8325-4 with FnBPs are significantly (p <

0.05) higher than for S. aureus DU5883 without FnBPs, regardless of exposure

of the staphylococci to a 1% BSA solution. Both strains show a significant

increase in bond strength energy by a factor 2 to 3 when the surface delay time

is increased from 0 to 2 s for S. aureus 8325-4 and even more (factor 4 to 5) for

S. aureus DU5883 (Table 2). Furthermore, after a surface delay, effects of BSA

exposure of the staphylococci on bond strength energies disappear.

Figure 3. Adsorption enthalpies (µJ), after correction for dilution effects, of Fn to S. aureus cell surfaces upon consecutive injections of 60 µl Fn solution (25 µg/ml) into 1.5 ml bacterial suspension of S. aureus 8325-4 (●) and S. aureus DU5883 (○) in PBS. Right panel data refer to staphylococci first exposed to 1% BSA. Error bars indicate standard deviation based on four independent measurements.

Enthalpies of adsorption of Fn to S. aureus cell surface. The measurement of

adsorption enthalpies of Fn to the S. aureus cell surfaces requires correction for

the heat of diluting of the proteins in PBS. For four consecutive injections of 60

μl of a 25 μg ml-1 Fn-solution into 1.5 ml of PBS yielded heat effects of,

respectively, -55, -56, -37 and -37 × 10-9 μJ. Figure 3 summarizes the adsorption

enthalpies upon consecutive injections of Fn to the staphylococcal suspensions,

after correction for protein dilution. For the parent strain 8325-4 with FnBPs

adsorption enthalpies decrease with the number of injections done, but no

saturation of adsorption sites seems to be reached within four injections. For


111

strain DU5883 the enthalpy effects are essentially invariant with the number of

injections. The cumulative adsorption enthalpies after the four injections are

shown in Table 3, as expressed per bacterium and per m2 bacterial cell surface.

Fn adsorption to the bacterial cell surfaces is an exothermic process in all cases,

i.e. enthalpy is released upon adsorption. Adsorption of Fn to S. aureus 8325-4

with FnBPs is enthalpically significantly more favourable than to S. aureus

DU5883. After exposure to BSA of S. aureus 8325-4, the adsorption enthalpy

decreases significantly, but remains larger than those for S. aureus DU5883. No

significant effect is seen for exposure to BSA on the adsorption enthalpy of S.

aureus DU5883, lacking FnBPs.

Table 3. Cumulative adsorption enthalpies per bacterium (10-9µJ) and per m2 bacterial cell surface (mJ m-2) after four consecutive injections of 60 µl Fn solution (25 µg ml-1) into 1.5 ml bacterial suspensions in PBS. Average standard deviations over 4 separate experiments amount ± 25 × 10-9 µJ per bacterium and ± 8 mJ m-2, respectively.

Cumulative adsorption

enthalpies per bacterium

(10-9µJ)

Cumulative adsorption

enthalpies per m2

(mJ)

Experiments

8325-4 DU5883 8325-4 DU5883

No BSA -140 -43 -44 -14

1% BSA -102 -54 -32 -17

* for calculation of the adsorption enthalpies per m2, it was assumed that the bacterial cell radius was 0.5 µm.

Discussion

In this chapter, we compare the interactions mediating adhesion to Fn-coated

surfaces as well as desorption of two S. aureus strains, one containing FnBPs

and the other one being FnBP-deficient, using three entirely different

techniques. Adhesion and residence-time dependent desorption of the two

Chapter 6

112

strains is determined in a parallel plate flow chamber under convective-

diffusion. In addition, the adhesion forces to Fn-coated AFM tips were measured

as well as the adsorption enthalpies of Fn to the staphylococcal cell surfaces. In

general, adhesion of the strain with FnBPs to Fn-coated substrata occurs faster

and in higher numbers than that of the strain deficient of FnBPs and, in line,

adhesive bonds are stronger and adsorption enthalpies higher. Surprisingly,

adsorption of BSA to either the Fn-coated substrata or the staphylococcal cell

surfaces not only blocks non-specific adhesion/adsorption sites, but also

obstructs the accessibility of the FnBPs on strain 8325-4 during convective-

diffusional mass transport, as, in this case, adhesion approaches that of the

FnBP-deficient strain DU5883. Influences of a BSA-coating on interaction

forces and adsorption enthalpies are far less significant, however. Strengthening

of the bond is evident from the resident-time dependent desorption of both

strains as measured in a parallel plate flow chamber as well as from a

comparison of the adhesion forces and adhesive bond strength energies

measured after 0 and 2 s surface delays in AFM.

Adsorption enthalpies. The enthalpy changes associated with the interaction

between Fn and the S. aureus cells are all exothermic, but differ markedly

between the two strains, as shown in Fig. 3 and Table 3. For the FnBP-deficient

DU5883 strain the enthalpy change is essentially the same for each injection

step, as is to be expected for non-specific adsorption in the sub-saturation range.

Assuming that the staphylococcal cell diameter equals 1 μm, it can be calculated

that in the ITC ampoule there is 23.6 × 10-3 m2 of bacterial surface area available

for adsorption. Since each fibronectin injection adds 1.5 ×10-3 mg Fn, the

maximal cell surface coverage by fibronectin after 4 consecutive injections

amounts 0.25 mg Fn per m2 bacterial cell surface, which is far below the

saturation limit for non-specifically adsorbed Fn, which would amount to at least


113

a few mg m-2 [23]. Assuming that all Fn added is adsorbed, the cumulative

enthalpy effect measured of -14 mJ m-2 corresponds to -13.8 × 103 kJ per mol Fn

(which corresponds to about 5600 kT per molecule Fn at 25°C), as calculated

using a molar mass of 250 kDa. Taking into account the large molar mass of Fn,

this value is quite reasonable when compared with enthalpy effects reported for

non-specific adsorption of various proteins to different surfaces [24]. The

enthalpy effects measured for the FnBPs containing strain 8325-4 are more

exothermic than for the FnBP-deficient strain DU5883. This indicates

involvement of enthalpically favourable specific Fn-binding sites. The

downward trend of the interaction enthalpy with consecutive injection steps,

displayed in Fig. 3 (left panel), suggests that not all specific binding sites are

equally favourable, or, alternatively, that they become gradually saturated, so

that for each subsequent addition a smaller fraction of Fn binds to FnBPs on the

cell surface. Assuming that all Fn added during the first injection binds to

FnBPs, the measured -250 μJ corresponds to -41.7 × 103 kJ per mol Fn. This is

about 300× higher than the enthalpy of the biotin-streptavidin interaction [25].

However, it should be realized that the much larger Fn molecule may interact

through more binding sites than the number of sites involved in e.g. a single

biotin-streptavidin interaction.

Exposure of the bacteria to a BSA solution hardly influences the enthalpy

of interaction between Fn and the FnBP-deficient strain DU5883. In contrast,

BSA exposure of the FnBP-containing strain 8325-4 significantly suppresses the

enthalpy of interaction with Fn, but not even nearly to the level of a non-specific

interaction. However, the downward trend in enthalpy for the BSA-coated strain

8325-4 (Fig. 3, right panel) seems to indicate that for the later Fn injections,

smaller fractions of Fn added finds FnBPs, as compared to the non-BSA-coated

cells. This is completely in line with the lack of effects of BSA coating on

adhesion forces observed by AFM and attests to the forceful contact established

Chapter 6

114

during AFM or stirring in the microcalorimeter as compared with the

spontaneous and relatively mild nature of cell-surface interaction during

convective-diffusion in the parallel plate flow chamber.

Interaction forces. AFM adhesion forces to Fn-coated surfaces are similar for

both S. aureus strains. This is unexpected considering their different abilities to

adsorb fibronectin [16]. Adsorption of fibronectin is a process occurring at the

outermost cell surface. However, the Fn-coated AFM tip penetrates the cell

surface therewith probing underneath the outermost cell surface. It is clear that,

upon penetration, for both S. aureus strains FnBPs or other adhesins are

encountered in the cell wall, even for strain DU5883 generally considered to be

devoid of FnBPs [16]. However, the spatial distribution of adhesins in strain

8325-4 must be completely different than in strain DU5883, as its adhesion

forces reach out much further and consequently strain 8325-4 has a higher Fn-

bond strength energy than strain DU5883 (see Table 2). Under the conditions of

convective-diffusion prevailing in the parallel plate flow chamber, it can be

envisaged that bacteria land mildly at the substratum surface, therewith invoking

interaction with only the outermost region of the cell wall. Contrary, the

penetrating AFM tip senses similar bond strength energies with no influence of

an adsorbed BSA-film over the cell surface. Mendez-Vilas et al. [26,27] has

suggested that a penetrating AFM tip may cause irreversible damage to the inner

cell surface, as concluded from saw-tooth patterns in the force-distance curves at

close approach. As we observed no such patterns in our force-distance curves

(see also Fig. 2), it is considered unlikely that the AFM tip has caused such cell

surface damage. Moreover, we regularly checked whether interaction of our Fn-

coated tips with clean glass yielded the same force values, and this was always

the case within one series of experiments.


115

Residence-time dependent desorption. Desorption rate coefficients from a Fn-

coated surface decrease for both S. aureus strains within 2 s by a factor of about

300-400. It is remarkable that the desorption rates and their residence time

dependence are insensitive to whether or not specific Fn-FnBP interactions were

involved in the adhesion. Apparently, the cells of strain 8325-4 that desorb

belong to the fraction of the population that have not been able to adhere

through strong specific bonds. Accordingly, exposure of either the bacterial cells

or the Fn-coated surface to BSA, therewith blocking specific Fn-FnBP

interactions has no or little effect on desorption kinetics. The decreasing

desorption rate coefficients could be confirmed by independent AFM

measurements.

Assuming that for a given condition, all bacteria adhere with the same

bond strength, a staphylococcal bond strength energy can also be calculated

from the desorption rate coefficients measured in the parallel plate flow

chamber, by applying

βesc= kTmehc

j /0 ϕ

Δ (4)

where βesc is the desorption rate coefficient, j0 the initial deposition rate, c the

bacterial cell concentration at the entrance of the flow chamber, Δh the width of

the energy minimum, φm depth of the energy minimum and kT the energy of

thermal motion [14,28]. The initial bond strength energies of our staphylococcal

strains to a Fn-film in the absence of a BSA-coating can be calculated from the

initial desorption rate coefficients and ranges between 2.2 to 3.3 kT, which

seems quite reasonable for non-specific binding. After bond ageing, the use of

the final desorption rate coefficients yields much higher bond strength energies

between 8.2 and 9.0 kT. Yet, these bond strength energies are orders of

magnitude smaller than derived from AFM, and conversion of the bond strength

energies from Table 2 to a thermal energy scale yields values of around 106 kT.

Chapter 6

116

This huge number attests to the fact that the penetrating, Fn-coated AFM tip

must have encountered numerous adhesins in the cell surface.

Conclusions

The combined use of a parallel plate flow chamber, AFM and ITC has yielded

new insights in the mechanisms of interaction between adsorbed Fn-films and S.

aureus strains, one of which (strain 8325-4) has FnBPs on its cell surface

whereas the other one (strain DU5883) is generally considered to be devoid of

FnBPs. First of all, the differences between the two strains with respect to (a)

initial deposition rate at a Fn-coated surface, (b) strength of binding to a Fn-

coated AFM tip and (c) enthalpy of interaction with Fn indicate that for 8325-4

additional attractive forces are involved which are ascribed to specific FnBP-Fn

interaction. Most interestingly, exposure of either Fn-coatings or staphylococcal

cell surfaces to BSA, strongly reduces staphylococcal adhesion under

convective-diffusion, but their enthalpy of Fn adsorption and their adhesion

force upon retracting a Fn-coated tip from the staphylococcal cell surface are

much less, if at all, influenced by a BSA-coating. It suggests that AFM and

calorimetry not only probe interactions at the outermost surfaces of the

interacting species but also those occurring underneath the BSA coating.

Residence-time dependent desorption data and AFM measurements reveal

considerable bond strengthening within a few seconds of contact for both S.

aureus strains. Bond strength energies calculated from the retract force-distance

curves in AFM were orders of magnitude larger than calculated from desorption

rate coefficients. This is another indication that the penetrating Fn-coated AFM

tip probes multiple receptor sites in the cell surface, for S. aureus 8325-4 as well

as DU5883. Apparently, mild landing of an organism on a Fn-coated substratum


117

as during convective-diffusion in the parallel plate flow chamber clearly does

not invoke specific interactions with deeper located adhesins.

Acknowledgements We like to thank ZON-MW for grant 91105005 enabling the purchase of the

Nanoscope IV Digital Instrument.

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Scheie, A.A. and Van der Mei, H.C. (2007), Intermolecular forces and enthalpies in

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119

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Staphylococcus aureus fibronectin binding protein genes is negatively regulated by agr

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Dynamics of fibronectin adsorption on TiO2 surfaces, Langmuir 23, 7046 - 7054.

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26. Mendez-Vilas, A., Gallardo-Moreno, A.M. and Gonzalez-Martin, M.L. (2006),

Nano-mechanical exploration of the surface and sub-surface of hydrated cells of

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Martin, M.L. (2008), AFM probing in aqueous environment of Staphylococcus

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Biophys J 66, 1222 - 1230.

CHAPTER 7

GENERAL DISCUSSION

Chapter 7

122

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

General discussion

123

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

Chapter 7

124

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 AB-

interaction on hydrophilic glass is repulsive (Figure 1). Conversely, on

General discussion

125

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

Chapter 7

126

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, DDS-

coated 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

General discussion

127

stronger attachment to a hydrophobic substratum surface, probably caused by

bridging of extracellular polymeric substances. On hydrophilic glass, AB-

interactions 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

Chapter 7

128

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.



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.

General discussion

129

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., Calzado-

Montero, R., Nuevo, M.J., Bruque, J.M. and Perez-Giraldo, C. (2004), Surface

characterisation of two strains of Staphylococcus epidermidis with different slime-

production by AFM, Appl Surf Sci 238, 18 - 23.


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.



system design and application for the study of bacterial adhesion, Biosens Bioelectron

19, 1429 - 1437.

Chapter 7

130

SUMMARY

Summary

132

Microbial adhesion to surfaces is a problem occurring in many fields of

application, including biomaterials associated repair of human function. Several

experimental techniques as well as predictive theoretical models are available to

gain a better understanding of the strength by which bacteria adhere to a

substratum.

Chapter 1 gives an introduction on the importance of understanding the

adhesion strength between micro-organisms, like bacteria, and substratum

surfaces. Adhesion strengths can be determined experimentally, but also

predicted theoretically. Experimental forces not only differ orders of magnitude,

but also theoretical force calculations present their own class of force values.

Furthermore, little research exists, in which adhesion parameters are

investigated under identical experimental conditions (i.e. substratum surface,

suspending media, growth conditions and bacterial strains).

Therefore, the main aim of this thesis is to develop an understanding of

the reason(s) why different techniques yield different ranges for microbial

interaction forces with substratum surfaces. The possible relations between

hydrodynamic shear forces obtained in the PPFC, predicted DLVO-forces and

air bubble detachment percentages for six different bacterial strains are

investigated in Chapter 2 by testing three hypotheses:

1. A strong hydrodynamic shear force to prevent adhesion (Fprev) relates to a

strong hydrodynamic shear force to detach (Fdet) an adhering organism.

2. A weak Fdet implies that more bacteria will be stimulated to detach by a

passing air-liquid interface through the flow chamber.

3. DLVO interactions determine Fprev and Fdet as well as the detachment

induced by a passing air-liquid interface.

However, every hypothesis had to be rejected showing the importance to

distinguish between forces acting parallel (hydrodynamic shear) and

perpendicular (DLVO, air-liquid interface passages) to the substratum surface.

Summary

133

Substratum surface hydrophobicity did not have an unambiguous

influence on Fprev. However, on the hydrophobic, dimethyldichlorosilane (DDS)-

coated glass it was more difficult to detach adhering bacteria, which was

confirmed by air-liquid interface induced detachment. These results showed that

the hydrophobic surface is more favourable for bacterial adhesion.

Another indication for interaction strength is the residence time dependent

desorption rate coefficient, which is studied in Chapter 3. Initial desorption rate

coefficients of four strains of Staphylococcus epidermidis were similar for

hydrophilic and hydrophobic DDS-coated glass, likely because initial desorption

is controlled by attractive Lifshitz-Van der Waals interactions, which are

comparable on both substratum surfaces. However, contact time allows a

significant effect of substratum surface hydrophobicity. On DDS-coated glass,

decay times are slower and final desorption rate coefficients smaller, suggesting

adhesion is more favourable on the hydrophobic surface. It is concluded that the

hydrophobic effect is the probable cause for these observations, because of the

more close contact between bacterium and substratum surface on hydrophobic

DDS-coated glass.

In Chapter 4 the adhesion dynamics, a more qualitative way of

describing bacterial affinity, were investigated for the same strains of S.

epidermidis as those in Chapter 3. Two modes of adhesion were distinguished:

immobile and mobile adhesion in which, despite an interaction with the

substratum surface, sliding along a substratum surface is possible. On

hydrophilic glass significantly more bacteria were found to adhere mobile, while

this feature was virtually absent on hydrophobic DDS-coated glass. It was

concluded that the presence of fully mobile adhesion depends on the Lewis acid-

base component of the free energy of interaction between the bacterium and

substratum. On hydrophilic glass this component is repulsive, and bacteria

cannot bind locally with a strong enough force for immobile adhesion. On DDS-

Summary

134

coated glass, the Lewis acid-base component is attractive, creating stronger local

bonds and preventing fully mobile adhesion.

Residence time dependent adhesion forces of these staphylococcal strains

with hydrophilic glass and DDS-coated glass can be investigated directly by

using atomic force microscopy (AFM), as described in Chapter 5. No

unambiguous effect of substratum surface hydrophobicity on initial adhesion

forces was observed. However, over time, strengthening of the adhesion forces

was virtually absent on DDS-coated glass, although in a few cases multiple

adhesion peaks developed in the retract curves. Significant bond-strengthening

on hydrophilic glass was observed for all four staphylococcal strains and was

concurrent with the development of multiple adhesion peaks upon retract. It is

concluded that on DDS-coated glass, the hydrophobic effect causes

instantaneous adhesion, while strengthening of the bonds on hydrophilic glass is

dominated by non-instantaneous hydrogen bond formation as determined by

using Poisson analysis.

So far adhesion parameters were studied for systems where only non-

specific interactions were possible. Therefore, the adhesion of two

Staphylococcus aureus strains, one with fibronectin binding proteins (FnBP’s)

and the other without these proteins, was studied in Chapter 6. The strain with

FnBP’s adhered significantly faster and stronger to a fibronectin (Fn) coated

surface. In line with this, this strain also had higher adsorption enthalpies

(measured with isothermal titration calorimetry, ITC) of Fn as compared to the

strain without FnBP’s. Initial desorption rate coefficients were high for both

strains, but decreased orders of magnitude within 2 s. Bond ageing was

confirmed by AFM adhesion force measurements. After exposure of either Fn-

coatings or staphylococcal cell surfaces to bovine serum albumin (BSA), the

adhesion of both strains to the substratum surface was strongly reduced,

suggesting that BSA not only suppresses non-specific cell-surface interactions

but also specific Fn-FnBP interactions. However, adhesion forces and

Summary

135

adsorption enthalpies were only slightly affected by BSA adsorption.

Furthermore, desorption rate coefficients were insensitive to whether or not

specific Fn-FnBP interactions were involved. From this study it was concluded

that the forced contact during AFM or stirring in ITC allows Fn-FnBP binding

with deeper located FnBP’s, despite an adsorbed BSA film and that mild landing

of an organism on a Fn-coated substratum as during convective-diffusion in the

parallel plate flow chamber does not invoke deeper located receptors.

In the general discussion, Chapter 7, it was noted that direct correlation

of adhesion parameters measured with the PPFC and AFM, is obscured by the

nature of both experimental techniques. Parameters measured in a flow chamber,

resemble the natural process best and give the most accurate approximation of

bioadhesion in vivo. In AFM experiments, interaction forces are measured that

are, due to the forced contact in AFM, very often not reached in naturally

occurring adhesion. It is concluded 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 differ in hydrophobicity.

However, in this thesis both experimental techniques have indicated the

importance of hydrogen bonding in bacterial adhesion to non-specifically

binding (inert) surfaces, especially with respect to bond strength and associated

adhesion dynamics.

Summary

136

SAMENVATTING

Samenvatting

138

Aanhechting van micro-organismen, zoals bacteriën, aan oppervlakken komt in

veel situaties voor. Een heel bekend voorbeeld van dit proces vindt plaats in de

mond. In de mond zijn veel verschillende soorten oppervlakken aanwezig

waaraan bacteriën zich kunnen hechten. Als dit gebeurt op tanden en kiezen, dan

kunnen zij zich na verloop van tijd vermenigvuldigen en een leefgemeenschap

vormen die, toepasselijk, “tandplaque” wordt genoemd. Wanneer plaque niet

wordt verwijderd, door bijvoorbeeld tandenpoetsen, zijn tandvleesontstekingen

of cariës (gaatjes in tanden en kiezen) het gevolg. De algemene term voor een

dergelijke microbiële leefgemeenschap is een “biofilm”. Biofilms komen in veel

praktische situaties voor en kunnen op metaaloppervlakken roestvorming

veroorzaken (bijvoorbeeld in pijpleidingen en warmtewisselaars in de industrie),

maar kunnen ook bij diverse medische toepassingen, zoals kunstgewrichten of

katheters, voor infecties zorgen. Voordat een biofilm kan ontstaan, moeten de

bacteriën sterk genoeg aan een oppervlak gaan hechten zodat ze niet wegspoelen

door een vloeistofstroom, zoals bijvoorbeeld speeksel in de mondholte. Om een

beter begrip te krijgen van de kracht waarmee de aanhechting plaatsvindt, is een

aantal experimentele technieken en theoretische beschrijvingen beschikbaar.

In Hoofdstuk 1 wordt het belang van kennis over de sterkte waarmee

bacteriën aan oppervlakken hechten kort weergegeven. Deze hechtingskrachten

kunnen experimenteel bepaald worden met, bijvoorbeeld, de Parallel Plate Flow

Chamber (afgekort tot PPFC) en de Atomic Force Microscope (afgekort tot

AFM). Ook kunnen ze berekend worden met behulp van een theorie die

ontwikkeld is door Derjaguin, Landau, Verwey en Overbeek (en dus de DLVO-

theorie genoemd wordt). De krachten die bepaald zijn met de verschillende

benaderingen hebben allemaal hun eigen orde van grootte. Een verdere

complicatie is dat er in de literatuur weinig experimenten worden beschreven die

bij dezelfde omstandigheden zijn uitgevoerd (bijvoorbeeld de te gebruiken

testoppervlakken, bacteriële stammen en de vloeistof waarin de hechting plaats

Samenvatting

139

vindt). Dit is echter wel een vereiste om de uitkomsten van de verschillende

technieken goed met elkaar te kunnen vergelijken.

Het doel van het onderzoek, beschreven in dit proefschrift, is om meer

inzicht te krijgen in de mechanismen die verantwoordelijk zijn voor de variatie

in grootte-orde van hechtingskrachten gemeten met verschillende technieken.

Om dit doel te bereiken werden experimenten in de PPFC en AFM uitgevoerd

bij dezelfde omstandigheden (vloeistof, temperatuur) en werden twee

testoppervlakken met verschillende affiniteiten voor water gebruikt: een

waterminnend (hydrofiel) en een waterafstotend (hydrofoob) oppervlak.

In Hoofdstuk 2 worden voor zes verschillende bacteriële stammen

hydrodynamische schuifkrachten, zoals bepaald in een PPFC, de berekende

DLVO-krachten en het percentage bacteriën dat van het oppervlak verwijderd

wordt als gevolg van het passeren van een luchtbel, getoetst aan een drie-tal

hypothesen:

1. Als een sterke hydrodynamische schuifkracht nodig is om aanhechting te

voorkomen (Fprev) zal ook een sterke hydrodynamische schuifkracht om

hechtende bacteriën te verwijderen (Fdet) nodig zijn.

2. Een zwakke Fdet impliceert dat meer bacteriën verwijderd zullen worden

als een luchtbel door de PPFC geleid wordt.

3. DLVO interacties bepalen Fprev, Fdet en het percentage bacteriën dat door

een passerende luchtbel verwijderd wordt.

Het bleek dat alle hypothesen verworpen moesten worden, waarmee duidelijk

werd dat er een onderscheid gemaakt moet worden tussen krachten die parallel

aan het oppervlak werken (de schuifkrachten) en de krachten die loodrecht op

het oppervlak werken (DLVO-krachten en de door de luchtbel uitgeoefende

krachten om hechtende bacteriën te verwijderen).

Uit het onderzoek bleek verder dat de affiniteit voor water van het

testoppervlak geen eenduidige invloed had op Fprev. Wel was het veel moeilijker

om bacteriën met schuifkrachten van het hydrofobe oppervlak te verwijderen,

Samenvatting

140

wat bevestigd werd door de verwijderingexperimenten met luchtbellen. Deze

resultaten laten zien dat aanhechting van bacteriën gunstiger is, als het oppervlak

waterafstotend is.

Een andere parameter voor hechtingssterkte is de zogenoemde

desorptiesnelheidscoëfficiënt die afhankelijk is van de contacttijd tussen bacterie

en testoppervlak. Voor vier verschillende Staphylococcus epidermidis stammen

werd deze coëfficiënt onderzocht en in Hoofdstuk 3 beschreven. De initiële

waarde van deze parameter werd niet beïnvloed door de water-affiniteit van het

testoppervlak. Waarschijnlijk komt dit doordat de krachten die voor aantrekking

zorgen (de zogenoemde Lifshitz-Van der Waals interacties) voor beide

oppervlakken gelijk zijn. Dat betekent dat onafhankelijk van het oppervlak

initieel evenveel hechtingsenergie overwonnen moet worden, om aan de

aantrekking van het oppervlak te kunnen ontsnappen. Met het toenemen van de

contacttijd tussen bacterie en oppervlak, wordt de desorptiesnelheidscoëfficiënt

kleiner. Met andere woorden, hechtende bacteriën gaan moeilijker van het

oppervlak af. In dit proces speelt de affiniteit voor water van het testoppervlak

wel een rol. Op het hydrofobe oppervlak gaat dit proces namelijk significant

sneller en zijn de eindwaarden van de coëfficiënt ook significant kleiner. Dit

suggereert dat hechten aan het water afstotende oppervlak gunstiger is. Aan het

einde van het hoofdstuk wordt geconcludeerd dat het “hydrofobe effect” hier de

oorzaak van is. Anders gezegd, doordat watermoleculen niet graag tegen een

waterafstotend oppervlak gedrukt zitten, is het gunstiger als hun plek wordt

ingenomen door de hechtende bacterie.

In Hoofdstuk 4 wordt beschreven hoe de PPFC gebruikt kan worden voor

een meer kwalitatieve benadering van adhesie, namelijk het dynamische

karakter waarmee bacteriën kunnen hechten. In dit hoofdstuk worden, voor

aanhechting van dezelfde vier S. epidermidis stammen als in Hoofdstuk 3, twee

verschillende hechtingsmechanismen beschreven: immobiel (bacteriën hechten

op een locatie op het oppervlak en blijven op die plek) en mobiel (bacteriën

Samenvatting

141

worden wel door het oppervlak aangetrokken, maar niet erg sterk, zodat de

gehechte bacteriën, als gevolg van de vloeistofstroom, over het oppervlak rollen

of glijden). Uit dit onderzoek bleek dat de water-affiniteit van het testoppervlak

een grote invloed had op het percentage bacteriën dat mobiel hecht. Op het

hydrofiele oppervlak bleek een substantieel deel van de bacteriën op een

mobiele manier te hechten, terwijl deze vorm nagenoeg niet aanwezig was op

het hydrofobe oppervlak. Geconcludeerd werd dat dit effect veroorzaakt wordt

door de zogenoemde Lewis acid-base interacties, die een maat zijn voor de

mogelijkheid van het vormen van waterstofbruggen tussen bacterie en water,

tussen oppervlak en water en tussen bacterie en oppervlak. Bij hechting aan het

hydrofiele oppervlak zijn deze interacties afstotend, waardoor de totale

aantrekkende kracht tussen bacterie en oppervlak te zwak is voor immobiele

hechting. Bij hechting aan het hydrofobe oppervlak zijn de Lewis acid-base

interacties aantrekkend, waardoor de hechting volledig immobiel is..

Tenslotte is, in Hoofdstuk 5, voor de S. epidermidis stammen op een

directe manier contacttijdafhankelijke interactiekrachten met het hydrofiele en

hydrofobe oppervlak gemeten, door gebruik te maken van AFM. Er was geen

eenduidige invloed van de water-affiniteit van het testoppervlak op de initiële

adhesiekrachten. Wel was er een invloed zichtbaar bij langere contacttijden

tussen de bacterie en het testoppervlak. Op het hydrofobe oppervlak werden de

adhesiekrachten namelijk nauwelijks sterker en ontstonden er slechts sporadisch

meer adhesiepieken in een enkele meting. Op het hydrofiele oppervlak echter,

ging het toenemen van de adhesiekracht bij langere contacttijden gepaard met

het ontstaan van meerdere adhesiepieken in een meting. Dit gedrag werd voor

alle onderzochte stammen gezien en met behulp van Poisson-analyse (een

methode uit de statistiek) kon de invloed van waterstofbrugvorming aangetoond

worden. Uit deze resultaten kon geconcludeerd worden dat op het hydrofobe

oppervlak hechting veroorzaakt wordt door het “hydrofobe effect” en dat dit

vrijwel direct plaatsvindt. Op het hydrofiele oppervlak wordt het sterker worden

Samenvatting

142

van de hechtingskracht veroorzaakt door de vorming van waterstofbruggen

tussen bacterie en oppervlak, nadat contact geforceerd is.

Tot nu toe zijn alleen parameters bepaald voor situaties waarin geen

specifieke interacties (zoals bijvoorbeeld binden van een bepaalde chemische

structuur aan een receptor van een bacterie) een rol spelen. Daarom is in

Hoofdstuk 6 onderzoek verricht naar de rol van specifieke receptoreiwitten

(bindingsplaatsen, BP’s) voor het eiwit fibronectine (Fn), zogenoemde FnBP’s,

die op Staphylococcus aureus aanwezig kunnen zijn. Het onderzoek toonde aan

dat de stam die de FnBP’s wel heeft, significant sneller en sterker aan een Fn-

coating hecht dan de stam zonder FnBP’s. In overeenstemming met deze

resultaten lieten studies met “isothermal titration calorimetry” (een manier om te

zien hoeveel warmte gepaard gaat met het binden van eiwitten aan receptoren op

bacteriën) zien dat bij deze stam de adsorptie-enthalpie van Fn hoger was dan bij

de stam zonder FnBP’s. Anders gezegd, er kwam meer warmte vrij als het eiwit

bindt met de bacterie die wel FnBP’s heeft. Tevens zijn er

desorptiesnelheidscoëfficiënten bepaald. Ook nu bleek dat de initiële

desorptiesnelheidscoëfficient snel enkele ordes kleiner werd met toenemende

contacttijd tussen bacterie en het testoppervlak (in dit geval een oppervlak van

fibronectine). Bij deze experimenten werd geen verschil gevonden tussen de

stam met FnBP’s en de stam zonder. Dat de bindingssterkte tussen bacterie en

oppervlak snel toeneemt, werd vervolgens met AFM bevestigd. Om te zien hoe

groot de rol van de FnBP’s op de gevonden parameters is, werd albumine (een

eiwit uit bloed) gebruikt om de specifieke interacties te blokkeren. Nadat óf de

Fn-coating, óf de bacteriën tijdens hun groei blootgesteld waren aan albumine,

bleek dat de adhesie van de stammen aan de oppervlakken sterk gereduceerd

werd. Dit wekte de suggestie dat het albumine niet alleen specifieke Fn-FnBP

bindingen blokkeert, maar ook de non-specifieke aantrekking onderdrukt omdat

ook de adhesie van de stam zonder FnBP’s gereduceerd was. Echter, na

blootstelling aan albumine lieten calorimetriemetingen zien dat er weinig effect

Samenvatting

143

was op de adsorptie-enthalpie van Fn aan bacteriën en ook AFM-krachten en

desorptiesnelheidscoëfficiënten waren nauwelijks verschillend.

Uit deze studies zijn twee belangrijke conclusies getrokken. De eerste is

dat AFM en calorimetrie in staat zijn om metingen te doen waarbij FnBP’s

betrokken zijn die wat dieper in de celwand liggen. Hierdoor is met deze

technieken geen verschil gevonden tussen de twee verschillende stammen

wanneer de specifieke interacties geblokkeerd werden door albumine. De

tweede conclusie is dat de desorptiesnelheidscoëfficiënt in deze gevallen

bepaald werd door bacteriën die op een non-specifieke manier gehecht waren.

Doordat bacteriën in de PPFC een milde landing op het testoppervlak hebben,

spelen de dieper gelegen receptoren in deze techniek geen rol en blijft de

desorptiesnelheidscoëfficiënt onafhankelijk van FnBP’s.

In de algemene discussie van dit proefschrift, Hoofdstuk 7, wordt

opgemerkt dat een directe correlatie tussen de adhesieparameters zoals bepaald

in de PPFC en met AFM bemoeilijkt wordt door de verschillende principes van

beide experimentele technieken. Metingen met de PPFC benaderen een

natuurlijke situatie het beste en geven daarom een goede indicatie voor

bacteriële adhesie zoals die plaatsvindt in een natuurlijke omgeving. Studies met

de PPFC laten zien dat waterstofbrugvorming tussen het oppervlak en het

omringende water een belangrijk mechanisme is in bacteriële adhesie en leiden

tot de conclusie dat hechting van bacteriën aan een hydrofoob oppervlak

gunstiger is. In experimenten met de AFM worden krachten bepaald die over het

algemeen in de praktijk niet bereikt worden. Dit komt doordat contact tussen de

bacterie en het oppervlak geforceerd wordt. Ook in deze techniek bleek

waterstofbrugvorming een belangrijk mechanisme voor adhesie. Echter, nu zijn

het de waterstofbruggen die direct tussen bacterie en oppervlak gevormd

worden. In tegenstelling tot studies met de PPFC, wordt met AFM

geconcludeerd dat bacteriële hechting aan het hydrofiele oppervlak gunstiger is.

Samenvatting

144

Een belangrijke conclusie van dit promotie-onderzoek is dan ook, dat

onderzoekers voorzichtig moeten zijn bij het gebruik van AFM om te bewijzen

dat de microbiële interacties op het ene oppervlak gunstiger zijn dan op het

andere oppervlak. Echter, uit beide experimentele technieken kan geconcludeerd

worden dat waterstofbrugvorming een belangrijk mechanisme in bacteriële

hechting aan oppervlakken is.

DANKWOORD

“So long, and thanks for all the fish” (Douglas Adams)

Dankwoord

146

Een promotie onderzoek is een onderneming die eigenlijk te omvangrijk is voor

één persoon. Hoewel ik, om te beginnen iedereen wil bedanken die op enige

wijze betrokken is geweest bij, en mij gesteund heeft in mijn promotie

onderzoek, wil ik de volgende mensen met name bedanken:

Allereerst mijn promotoren, prof. dr. ir. H.J. Busscher, prof dr. ir. W. Norde en

prof. dr. H.C. van der Mei. In veel dankwoorden wordt de “tandem” Henk &

Henny veelvuldig geroemd voor hun samenwerking (terecht overigens). Ik ben

blij hieraan toe te kunnen voegen, dat met Willem op de bagagedrager, jullie

voor mij een nog beter team zijn gebleken. Beste Henk, Willem en Henny, ik

heb in de afgelopen vier jaar veel van jullie geleerd. Bedankt voor de

mogelijkheid met jullie mee te fietsen, voor jullie inzet, input, vertrouwen,

kritische blik, goede gesprekken en nog veel meer.

Prof. dr. Y. Ren, prof. dr. ir. M. Cohen-Stuart en prof. dr. ir. M. van Loosdrecht

wil ik danken voor het beoordelen van het manuscript.

Hans Kaper en Joop de Vries wil ik bedanken voor hun hulp bij het praktische

werk. Zonder jullie waren de hoofdstukken 3 tot en met 6 niet mogelijk geweest.

Joop, heel veel dank voor al je geduld als ik weer eens aankwam met “Joop, de

AFM doet weer raar. Kun je even kijken?”. En ook voor je hulp bij de omslag

en het drukklaar maken van dit boekje.

Verder wil ik mijn kamergenoten, Astrid, Marco en Reza bedanken voor hun

gezelligheid en nodige afleiding. Daarbij heb ik erg veel gehad aan de

inhoudelijke discussies met jullie. In dit rijtje mag Pit, als “halve kamergenoot”

niet ontbreken. Na jouw promotie was het erg lastig weer iemand te vinden die

ik net zo leuk kon plagen (dat is me dan ook niet echt gelukt..). Bedankt voor je

gezelligheid en je goed getimede peptalk op de fiets!

Dankwoord

147

De “dames op het lab” wil ik bedanken voor de gesprekken tijdens het zoveelste

(flow) experiment. Op het lab staan met jullie maakt het experimentele werk

zoveel leuker! En Minie, gedeelde smart is inderdaad halve smart.

Beste Ina en Ellen, bedankt voor alle klusjes die jullie me uit handen hebben

genomen. Ellen, heel erg bedankt dat je mij veel frustratie hebt bespaard, door je

hulp met de lay-out van dit proefschrift.

Geen afdeling is compleet zonder goede koffie- en lunch- en andere pauzes.

Daarom wil ik iedereen van de disciplinegroep BME bedanken voor de

gezelligheid en de zo belangrijke “loze gesprekken” tijdens deze “kwartiertjes”.

Met name René, Daniëlle, Annet, Debbie, Theerthankar, Adam, Prashant en ook

Guru, Mihaela, Isa en Roelien, dank jullie wel voor de interessante en leuke

gesprekken. Debbie, ik ga de ziekenhuisdiners écht missen.

Beste Cynthia, Igor, Kalle, Laura en Anika. Zonder jullie steun en begrip tijdens

dit project had ik het echt veel zwaarder gehad. Vanaf nu komt er weer meer tijd

om leuke dingen te gaan doen. Beloofd!

Laura, dankjewel voor het zetje dat je me gegeven hebt toen ik bij jou mijn

afstudeeronderzoek deed. Jij bent echt een sturende factor geweest om zelf ook

een promotie onderzoek te gaan doen.

Hetzelfde geldt voor jou Anika, daarom vind ik het des te leuker dat we niet

alleen onze studie “samen” hebben gedaan, maar dat je ook mijn paranimf wil

zijn. Bedankt voor alle hulp rondom het proefschrift.

Dankwoord

148

Beste Eefje, jij was juist een sturende factor tijdens mijn promotie onderzoek. Je

had altijd tijd om me te helpen op het lab of om mij even de kans te geven stoom

af te blazen. Erg leuk dat ook jij mijn paranimf wil zijn. Bedankt voor alles!

Als laatste mijn directe familie. Beste Jarno, Linda en Maarten, bedankt voor

jullie begrip en hulp tijdens mijn promotie onderzoek. Jullie aanwezigheid thuis

hebben mijn Groningse start makkelijker gemaakt en mede daardoor gezorgd

voor een mooi eindresultaat. Ik kan jullie daar niet genoeg voor bedanken.

Zonder de niet aflatende steun en het vertrouwen van mijn ouders waren de

afgelopen jaren heel anders verlopen. Pa, ik geloof dat het wel eerlijk is om te

zeggen dat we allebei een roerige vier jaar achter de rug hebben. Ik ben blij dat

alles goed is gekomen en dat je mij ondanks alles hebt geholpen waar en

wanneer ik het nodig had. Moeders, ook jij bedankt voor alles dat je in de

afgelopen jaren voor me gedaan hebt. Door al die kleine dingen die je mij uit

handen hebt genomen, kon ik me veel beter op mijn onderzoek richten.

Dit alles was echter nooit tot stand gekomen zonder de stimulerende woorden

van mijn “tweede moeder”. Helaas heeft zij nooit van dit avontuur geweten,

maar ik weet zeker dat mijn oma dit geweldig mooi zou vinden. Aan haar is dit

werk dan ook opgedragen.

Allemaal bedankt voor vier onvergetelijke jaren.

Dankwoord

149

Curriculum vitae

150

Niels Peter Boks was born on the 3rd of April 1979 in Apeldoorn, The Netherlands. He

graduated from University Preparatory Education (VWO) in 1997 at the Christelijk Lyceum

in Apeldoorn. In that same year he started his university training in Chemical Engineering at

the faculty of Science and Technology of the University of Twente in Enschede. Specializing

in biomedical materials science, he graduated in the department of Polymer Chemistry and

Biomaterials (lead by prof. J. Feijen). From September 2004 to August 2008, he was

employed as a PhD-student at the department of BioMedical Engineering (lead by prof. H.J.

Busscher) of the University of Groningen / University Medical Center Groningen. The

research performed in that period is described in this dissertation.

List of publications:

N.P. Boks, W. Norde, H.C. van der Mei & H.J. Busscher, 2008. Forces involved in bacterial adhesion to hydrophilic and hydrophobic surfaces. Microbiology 154, 3122-3133. N.P. Boks, H.J. Kaper, W. Norde, H.J. Busscher, H.C. van der Mei, 2008. Residence time dependent desorption of Staphylococcus epidermidis from hydrophobic and hydrophilic substrata. Colloids and Surfaces B: Biointerfaces 67, 276-278. N.P. Boks, H.J. Busscher, H.C. van der Mei & W. Norde, 2008. Bond-strengthening in staphylococcal adhesion to hydrophilic and hydrophobic surfaces using AFM. Langmuir 24, 12990-12994. N.P. Boks, H.J. Kaper, W. Norde, H.C. van der Mei & H.J. Busscher, 2008. Mobile and immobile adhesion of staphylococcal strains to hydrophilic and hydrophobic surfaces. J Colloid Interf Sci (in press). C.-P. Xu, N.P. Boks, J. de Vries, H.J. Kaper, W. Norde, H.J. Busscher & H.C. van der Mei, 2008. Fibronectin interactions with Staphylococcus aureus with and without fibronectin-binding proteins and their role in adhesion and desorption. Appl Environ Microb (in press). A. Roosjen, N.P. Boks, H.C. van der Mei, H.J. Busscher & W. Norde, 2005. Influence of shear on microbial adhesion to PEO-brushes and glass by convective-diffusion and sedimentation in a parallel plate flow chamber. Colloid Surface B 46, 1-6. *

L. Buttafoco, N.P. Boks, P. Engbers-Buijtenhuijs, D.W. Grijpma, A.A. Poot, P.J. Dijkstra, I. Vermes & J. Feijen, 2006. Porous hybrid structures based on P(DLLA-co-TMC) and collagen for tissue engineering of small-diameter blood vessels, J Biomed Mater Res B Appl Biomater 79, 425-434. * * These publications are not the result of the research described in this dissertation.

Dankwoord

151

Curriculum vitae

152

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