Journal of Biomaterials and Nanobiotechnology, 2012, 3, 547-556
http://dx.doi.org/10.4236/jbnb.2012.324057 Published Online October 2012 (http://www.SciRP.org/journal/jbnb)
547
Study of the Adhesion of Clinical Strains of Staphylococcus
aureus on an Abiotic Surface Using the Biofilm Ring Test®
J. M. Liesse Iyamba1,2, N. B. Takaisi-Kikuni2, S. Dulanto1, J. P. Deh ay e 1
1Laboratoire de Chimie biologique et médicale et de Microbiologie pharmaceutique, Faculté de Pharmacie, Université libre de
Bruxelles, Brussels, Belgium; 2Laboratoire de Microbiologie Expérimentale et Pharmaceutique, Faculté des Sciences Pharmaceu-
tiques, Université de Kinshasa, Kinshasa, Democratic Republic of Congo.
Received June 14th, 2012; revised July 25th, 2012; accepted August 14th, 2012
ABSTRACT
Four methicillin-sensitive (MSSA) and 4 methicillin-resistant (MRSA) strains of Staphylococcus aureus were collected
and isolated at the Laboratory of Bacteriology of the Provincial General Reference Hospital of Kinshasa in the Democ-
ratic Republic of Congo. The microbial adhesion to solvents (MATS) test showed that the MRSA strains had a less hy-
drophobic membrane than the MSSA strains. Using the Biofilm Ring Test® (BFRT®) to investigate on the adhesion of
these bacterial strains to smooth surfaces, we observed that the MSSA strains adhered more rapidly than the MRSA
strains. The biomass of the produced biofilm measured by the Crystal violet staining method (CVSM) was more impor-
tant with MSSA than with MRSA strains. Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA)
inhibited the adhesion and the formation of a biofilm by MRSA strains; this inhibition was reversed by calcium, mag-
nesium and manganese. The MRSA strains adhered less to silicon tubing and the adhesion was inhibited by EGTA in 2
of the 4 MRSA strains and none of the MSSA strains. In conclusion, the MSSA and MRSA strains adhered on an
abiotic surface and formed a biofilm at distinct rates and with different sensitivities to ions. The results also confirm the
utility as well as the limits of the BFRT® to study the adhesion of bacteria on a surface.
Keywords: Biofilm Ring Test®; Crystal Violet; Cell Surface; Hydrophobicity; Adhesion; Catheter Tube
1. Introduction
Staphylococcus aureus is a human pathogen that causes
both chronic and nosocomial infections; many of which
are mediated by their ability to adhere to medical devices
and to form biofilms. This bacteria is responsible of a
large variety of diseases, including endocarditis, osteo-
myelitis, and foreign body infections [1]. A biofilm is de-
fined as aggregated, microbial cells surrounded by a
polymeric self-produced matrix, which may contain host
components [2]. According to the Center for Disease
Control and Prevention, 65% of human bacterial infec-
tions are associated to a biofilm [3]. Furthermore, biofilms
infections are a major problem in the clinic and cause
many deaths and high health costs, e.g. 12% to 25% of
patient mortality are attributable to catheter-related blood-
stream infections [4]. Biofilm-associated bacteria are ge-
nerally resistant to antibiotics [5], and to host immune
responses [6].
Two main stages are involved in S. aureus biofilm
formation [7]. In a first step, the cells adhere on an
abiotic or a biotic surface. The adherence of S. aureus to
foreign bodies depends on the cell surface characteristics
of the micro-organisms and the nature of foreign body
material and involves physicochemical forces such as
polarity, London-van der Waals forces and hydrophobic
interactions [8]. Divalent cationic ions (e.g. magnesium,
calcium) may enhance the attachment of bacteria to sur-
face by reducing electrostatic repulsion and stabilizing
interaction between the negatively charged surface of
bacteria and anionic substrates [9]. Cell surface hydro-
phobicity and initial adherence of micro-organisms to a
surface have been attributed to different bacterial surface-
associated adhesins. The attachment of S. aureus on a
biotic surface is likely to be mediated by cell-wall asso-
ciated proteins such as the microbial surface components
recognizing adhesive matrix molecules (MSCRAMM).
The second stage of biofilm development includes cells
multiplication and formation of a mature structure con-
sisting of many cell layers. This stage is associated with
the production of extracellular factors which may include
exo-polysaccharides, proteins and extracellular DNA
(eDNA) [10]. Excretion of polysaccharide intercellular
adhesion (PIA) polymers in Staphylococcus species and
the presence of divalent cations interact to form stronger

Study of the Adhesion of Clinical Strains of Staphylococcus aureus on an
Abiotic Surface Using the Biofilm Ring Test®
548
bonding between cells [11]. The detachment of cells
from established biofilms allows the spreading and the
colonization of new sites by planctonic bacteria [12].
Colorimetric microtiter plate systems are widely used
to determine bacterial adhesion [13]. Among these tech-
niques, the Crystal Violet staining method (CVSM) has
been modified to increase its accuracy and to allow the
quantification of the biomass of the biofilm in the entire
well [14]. Despite the fact that this method is suitable for
estimating the adherent cells stained after washing steps,
it requires cultures of at least 24 to 48 h. The Biofilm
Ring Test® (BFRT®), a newly described method, has
been recently proposed as an alternative to the CVSM for
studying bacterial adhesion [15]. This technique based on
the immobilization of magnetic beads by adherent cells
had not only been implemented to study the initial steps
of biofilm formation [16,17], but also to explore the
composition of Staphylococcus aureus and Leuconostoc
mesenteroides biofilm matrix [15,18]. The aim of this
study was to compare the adhesion of clinical methicil-
lin-resistant Staphylococcus aureus (MRSA) and methi-
cillin-sensitive Staphylococcus aureus (MSSA) strains
and to determine the effect of divalent cations on the
inhibition of bacterial adhesion by ethylene glycol-bis(2-
aminoethylether)-N,N,N' ,N'- tetraacetic acid (EGTA).
2. Materials and Methods
2.1. Origin of the Strains and Growth Conditions
The 8 strains used in this study were collected for diag-
nostic purposes in the Laboratory of Bacteriology of the
Provincial General Reference Hospital of Kinshasa (HGPRK)
in the Democratic Republic of Congo. Among the 4
MSSA strains, 2 strains (the 5668/B and 5741/B strains)
were isolated from blood samples and the 2 other strains
(the 1532/SW and 1620/SW) were from skin wounds.
Among the 4 MRSA strains, 2 strains (the 007/FV and
the 028/FV) were collected from a vaginal smear test, 1
strain (the 011/LP) from prostatic fluid and 1 strain
(027/U) from urine. The bacteria were grown and iso-
lated on Brain Heart Agar with 5% (v/v) sheep blood and
on Mannitol Salt Agar (Difco, BD Franklin Lakes, NJ,
USA). The identification of S. aureus was performed
with the latex agglutination test (Pastorex Staph-Plus,
Biorad, Marnes-la-Coquette, France). All MRSA strains
were positive for mecA gene (data not shown).
2.2. Characterization of Cell Surface Properties
of the Strains
The microbial adhesion to solvents (MATS) test was
performed to estimate, simultaneously, the hydrophobic-
ity and the electron-acceptor and electron-donor charac-
teristics of the bacterial membranes [19]. Briefly, the
bacteria strains were cultivated on TSA medium and in-
cubated for 24 h at 37˚C. TSB medium was inoculated
with 2 or 3 colonies and incubated for 24 h at 37˚C. The
bacteria were then harvested by centrifugation and
washed twice with 150 mM potassium phosphate. The
bacteria were suspended in the same buffer and adjusted
to a concentration of ~108 CFU/mL. An aliquot of this
suspension (2.4 mL) was mixed with 0.4 mL of one of 2
pairs of organic solvents and vigorously shaken by vor-
texing for 2 min. Each pair of polar and non-polar sol-
vents had similar Lifshitz-van der Waals surface tension
properties. The two pairs of solvents tested were 1)
chloroform (an acidic solvent) and hexadecane and 2)
ethyl acetate (a strong basic solvent) and hexane, its
n-alkane apolar control [19]. The mixture was allowed to
stand for 15 min to ensure complete separation of the two
phases. The OD of the aqueous phase was measured at
400 nm. The adhesion to each solvent was calculated by
the equation: % adhesion = (1 − A/A0) × 100, where A0
was the absorbance of the bacterial suspension (aqueous
phase) before mixing and A was the absorbance after
mixing. The experiment was repeated at least 3 times for
each strain.
2.3. Evaluation of Bacterial Adherence in a
Smooth Surface Using the BFRT®
The Biofilm Ring Test® (BFRT®) is based on the immo-
bilization of the magnetic beads by adherent cells. Con-
sequently, the more beads are entrapped by cells, the
fewer they are detectable after magnetization and, sub-
sequently, the more cells are attached to the surface [15].
Bacterial adhesion was assessed using an appropriate kit
commercially available (Biofilm Control, Saint Beauzire,
France). The toner solution (TON005) containing nega-
tively charged magnetic beads of 1 - 3 µm was mixed
with a calibrated Initial Bacterial Suspension (IBS) con-
taining ~107 bacteria/mL. Two hundred µL of this mix-
ture were placed in the wells of a 96-wells polystyrene
plate formed by the assembly of 12 individual 8-wells
strips (Strip Well MSW002B). After incubation time at
37˚C, wells of the strip were covered with a few drops of
contrast liquid (inert opaque oil used for the reading step)
and scanned with the plate reader to get Io image. Then,
the plate was placed for 1 min on the magnet (Blok test)
and scanned again to get I1 image. The adhesion capabil-
ity of each strain was expressed as the Biofilm Index
(BFI) calculated by the software and based on the com-
parison of the two images. When BFI ≥ 7, a spot corre-
sponding to the microbeads attracted by the magnet is
visible and indicates the absence of adhesion or biofilm.
When BFI ≤ 2, no spot can be observed after magnetiza-

Study of the Adhesion of Clinical Strains of Staphylococcus aureus on an
Abiotic Surface Using the Biofilm Ring Test®
549
tion indicating that microbeads are blocked by adherent
cells. Biofilm in formation results in microbeads partially
blocked 2 ≤ BFI ≤ 7. The experiment was repeated at
least 3 times for each strain.
2.4. Evaluation of the Formation of a Biofilm
with the CVSM
Polystyrene sterile strips were inoculated with 200 µL of
IBS and incubated for various times at 35˚C in a humid
atmosphere. A control well was inoculated with sterile
medium. Each strain was evaluated in triplicate. Medium
was removed from the wells which were washed 3 times
with 200 µL sterile distilled water. The strips were air-
dried for 45 min and the adherent cells were stained with
200 µL of 0.1% Crystal violet solution. After 45 min, the
dye was eliminated and the wells were washed 5 times
with 300 µL of sterile distilled water to remove excess
stain. The dye incorporated by the cells forming a biofilm
was dissolved with 200 µL of 33% (v/v) glacial acetic
acid and the absorbance of each well was read at 540 nm
in the microplate reader. The results were expressed as
variation of OD540 nm (OD540 nm
sample - OD540 nm control).
The experiment was repeated at least 3 times, for each
strain and incubation time.
2.5. Study of the Adhesion of the Bacteria on
Catheter Tubing
The bacteria were grown overnight and the OD600 nm ad-
justed to 1.00 ± 0.05. They were then diluted 250-fold. A
silicon tubing (2 cm long, 3 mm inner diameter) was in-
cubated with the bacteria at 37˚C for 18 h. At the end of
the incubation, the tubes were rinsed twice with water
before adding 1 mL phosphate-buffer solution (pH 7.2).
The cells were detached from the tubing by incubation in
an ultrasound bath at 25˚C for 5 min. After serial dilu-
tions of the bacterial suspension with PBS, the bacteria
were plated on Petri dishes containing 15 mL TSA me-
dium and the Petri dishes were incubated at 35˚C for 48 h
before counting the colonies. Dishes with less than 50
colonies or with more than 500 colonies were not count-
ed. Results are expressed as colonies forming units per
mL (CFU/mL).
2.6. Statistical Analysis
Results were analyzed with the Mann-Whitney non-pa-
rametric test. ***P < 0.005; **P < 0.01; *P < 0.05.
3. Results
3.1. Measure of the Adhesion of the Strains to
Solvents
The hydrophobicity and the electron-donor and electron-
acceptor properties of the cell walls of the various strains
were tested according to Bellon-Fontaine et al. [19]. The 4
MSSA strains migrated nearly totally (from 90% to 97%)
from the aqueous to the hexadecane phase establishing that
their membrane was very hydrophobic. The 4 MRSA
strains migrated slightly less (from 80% to 83%). The dif-
ference between MSSA (93.1% ± 1.4%) and MRSA
strains (82.1% ± 1.2%) was significant (P < 0.001, n = 12)
suggesting that MRSA strains were slightly less hydro-
phobic than MSSA strains (Figures 1(a) and (b)). The
bacteria were also extracted with chloroform. The extrac-
tion of MSSA strains (95% ± 0.6%) was better than the
extraction of MRSA strains (82.1% ± 1.5 %) (P < 0.001, n =
12) showing that the cell walls of MSSA strains were more
basic (electron-donor capacity) than cell walls from
MSSA strains
A TCC 259235668/S 5741/S1532/P 1620 /P
0
25
50
75
100
125
Chloroform
Hexadecane
Ethyl acetate
He xane
(a)
Strains
% bacteria extracted by the solvent
(a)
MRSA strains
A T CC 33 5 910011/LP027/U 028/FV007/FV
0
25
50
75
100
125
Chloroform
Hexadecane Hexane
Ethyl acetate
(b )
Strains
% bacteria extracted by the solvent
(b)
Figure 1. Study of the interaction of S. aureus with organic
solvents. The affinity of 4 MSSA strains (a) or 4 MRSA
strains (b) for chloroform, hexadecane, ethyl acetate or
hexane was measured. Results are expressed as percent-
ages of bacteria transferred from the aqueous phase to the
organic solvent. Values are the means ± s.e.m. of 3 ex-
periments.

Study of the Adhesion of Clinical Strains of Staphylococcus aureus on an
Abiotic Surface Using the Biofilm Ring Test®
550
MRSA strains. The comparison of the extraction of the
strains by ethyl acetate and hexane gave an estimate of
the electron-accepting property of the strains. The 2 sol-
vents extracted comparable percentages of the 4 MSSA
strains showing that the cell walls of these bacteria had
no electron-accepting propensity; ethyl acetate extracted
the 4 MRSA strains 3-times less than hexane and it could
be concluded that their walls were rather acidic.
3.2. Comparison between the BFRT® and the
CVSM to Evaluate the Adhesion of the
Bacteria
In a next experiment, the adhesion of the 8 strains on an
abiotic surface (the bottom of the wells of a multi-well
plate) was estimated using the BFRT® and the CVSM.
As shown in Figure 2(a), the 4 MSSA strains immobi-
lized the magnetic beads of the BFRT®. Two MSSA
strains (1620/SW and 1538/SW) fully blocked the beads
within 2 h (drop of the BFRT® from 17.0 ± 0.1 to 2.4 ±
0.3, n = 18). The drop of the BFI was slightly slower for
the 5668 and the 5741 strains (from 17.6 ± 0.2 to 9.3 ±
1.0 after 2 h). The BFI further dropped for the next 2
hours and reached 1.4 ± 0.1 after 4 h. The 4 MRSA stains
immobilized the beads at a slower rate (Figure 2b). After
2 hours, 3 strains (011/LP, 027/U and 028/FV) decreased
the BFI from 15.5 ± 0.9 to 9.1 ± 0.6, n =27). These
strains fully blocked the beads after 3 h (BFI: 2.1 ± 0.1, n =
27). One strain (007/FV) was much less effective and
significantly blocked the beads only after 4 h (7.3 ± 1.6,
n = 7). Similar experiments were performed in the wells
of a multi-well plate and the biomass adhering at the
bottom of the wells was assayed after staining with
Crystal violet. As shown in Figures 3(a) and (b), the
formation of a biomass became significant after 3 h and
differed among the strains. Some MSSA strains produced
a very significant biomass after 4 h (5668/B > 1620/SW >
1532/SW > 5741/B). Among the MRSA strains, the
007/FV strain did not produce any significant biofilm
after 4 h. From these results it could be concluded that
the results obtained with the BFRT® and the CVSM were
consistent but that the BFRT® was more efficient for the
study of short-term effects.
3.3. Effect of Ions on the Formation of a Biofilm
Divalent cations contribute to the interaction among bac-
teria or their interaction with components of the ex-
tracellular matrix [20]. The purpose of these experiments
was to test the effect of EGTA, a chelator of divalent
cations, on the adhesion of bacteria and the interaction
between EGTA and divalent cations. Preliminary ex-
periments were performed to test whether these ionic
conditions affected by themselves the mobility of the
MSSA strains
0 1 2 3 4
0
4
8
12
16
20
5668/B
5741/B
1532/SW
1620/SW
(a)
Ti me (hours )
BFI (arbitr a ry u n its )
(a)
MRS A stra i n s
0 12 3 4
0
4
8
12
16
20
007/FV
027/U
028/FV
011/LP
(b)
Time ( h ours)
BFI (arbi t rary units)
(b)
Figure 2. Time-course of the immobilization of magnetic
beads by clinical strains of S. aureus. MSSA strains (a) and
MRSA strains (b) of S. aureus were incubated at 35˚C in
the presence of magnetic beads. The immobilization of the
beads was measured after 1, 2, 3 or 4 h using the Biofilm
Ring Test®. Results are expressed as the Biofilm Index
(BFI). Data are the means ± s.e.m. of 3 experiments.
beads. As shown in Figure 4(a), EGTA (from 100 µM to
1 mM) had no effect on the BFI. One mM calcium,
magnesium and manganese decreased the BFI below 2.
Only 1 mM EGTA was tested on the adhesion of the
bacteria using the BFRT®. At this concentration, EGTA
has no effect on the viability and the doubling time of the
tested strains (data not shown). The bacteria were ex-
posed to the chelator for 6 h. As shown in Figure 4(b),
the metal-chelator had no effect on the drop of the BFI
provoked by the 4 MSSA strains. EGTA increased the
BFI measured in the presence of 3 MRSA strains (the
011/LP, 027/U and 028/FV strains). The 007/FV was not
affected by EGTA (Figure 4(c)). Considering the inter-
action of the ions with the BFRT®, the reversibility of the
inhibition by EGTA was tested using the CVSM. The 4
MRSA strains were cultured for 24 h in control condi-
tions or in the presence of 1 mM EGTA, in the absence

Study of the Adhesion of Clinical Strains of Staphylococcus aureus on an
Abiotic Surface Using the Biofilm Ring Test®
551
MSSA strains
0 1 2 3 4 5 6
0.0
0.2
0.4
0.6
0.8
1.0
1620/SW
5741/B
5668/B
1532/SW
(a)
Ti me (ho u r s )
Absorbance 540 nm
(a)
MRSA strains
0 1 2 3 4 5 6
0.0
0.2
0.4
0.6
0.8
1.0
007/FV
027/U
011/LP
028/FV
(b)
Ti me (ho u r s )
Absorbance 540 nm
(b)
Figure 3. Study of the formation of a biofilm by clinical
strains of S. aureus using the CVSM. MSSA strains (a) and
MRSA strains (b) of S. aureus were incubated at 35˚C. The
formation of a biofilm was measured after 1, 2, 3, 4 or 6 h
using the CVSM. Results are expressed as the absorbance
measured at 540 nm. Data are the means ± s.e.m. of 3 ex-
periments.
or in the presence of 1 mM Ca2+, Mg2+ or Mn2+. As
shown in Figure 5, EGTA significantly decreased the
biomass measured after 24 h with the 4 MRSA strains.
Adding a divalent cation to the medium (calcium, mag-
nesium or manganese) blocked the inhibition exerted by
EGTA.
3.4. Study of the Adhesion of the Microbial
Strains on Catheter Tube
Fragments of a catheter tube (2 cm long, 3 mm inner
diameter) were incubated for 24 h in the presence of cel-
lular suspensions from each strain (108 CFU/mL). After
the incubation, the catheter tubes were washed twice and
the adhering bacteria were detached from the tubing by
Effe ct of i onic con dit io n s on th e BFRT
0.00 0.25 0.50 0.751.00
0
5
10
15
20
C alc iu m
Magnesium
Mang anes e
EGTA
(a)
Concentration (mM)
BFI (arbitrary units)
(a)
Adhesion of MSSA strains
5668/B5741/B1532/SW 1620/SW
0
2
4
EGTA 1 m M n=6)
Control (n=6)
(b)
Strains
BF I (arb i trary unit s)
(b)
Adhesion of MRS A strains
011/LP027/U028/FV 007/FV
0.0
2.5
5.0
7.5
10.0
12.5
15.0
EGTA 1 mM
Control
999
12
666
5
n=
***
*** ***
(c)
Strains
BFI (arbitrary units)
(c)
Figure 4. Effect of EGTA on the adhesion of bacteria
measured with the BFRT®. (a) Magnetic beads were incu-
bated for 4 h in the presence of increasing concentrations of
EGTA, calcium, magnesium or manganese and in the ab-
sence of bacteria. At the end of the incubation, the BFI was
measured. Results are the means ± s.e.m. of 3 experiments;
(b) and (c) MSSA strains (middle panel) and MRSA strains
(lower panel) of S. aureus were incubated at 35˚C in the
presence of magnetic beads in the absence or in the pres-
ence of 1 mM EGTA. The immobilization of the beads was
measured after 6 h using the Biofilm Ring Test®. Results
are expressed as the Biofilm Index (BFI). Data are the
means ± s.e.m of 6 (MSSA strains) or of n (MSSA strains)
xperiments. ***: P < 0.005 when compared to control. e

Study of the Adhesion of Clinical Strains of Staphylococcus aureus on an
Abiotic Surface Using the Biofilm Ring Test®
552
011 /LP
CONT Ca2+ Mg2+ Mn2+
0.00
0.05
0.10
0.15
0.20
Control (n=3)
EGTA 1 mM (n=6)
*
(a )
Absorbance 540 nm
027/U
CONT Ca2+ Mg2+ Mn2+
0.00
0.05
0.10
0.15
0.20
Control (n= 3)
EGTA 1 mM (n=6)
*
(b )
Absorbance540 nm
(a) (b)
028/FV
CONT Ca2+ Mg2+ Mn2+
0.00
0.05
0.10
0.15
0.20
Con tr ol (n=3)
EGTA 1 mM (n=6)
*
(c)
Absorbance540 nm
007/FV
CONT Ca2+ Mg2+ Mn2+
0.00
0.05
0.10
0.15
0.20
Control
EGTA 1 mM
*
(d )
Absorbance540 nm
Control (n = 3)
EGTA 1 mM (n = 6)
(a) (d)
Figure 5. Reversibility of the inhibition by EGTA of the formation of a biofilm by MRSA strains. The 4 MRSA strains of S.
aureus were incubated at 35˚C in the control conditions or in the presence of 1 mM EGTA, in the absence or in the presence
of 1 mM calcium, magnesium or manganese. The formation of a biofilm was measured after 24 h using the CVSM. Results
are expressed as the absorbance measured at 540 nm. Data are the means ± s.e.m. of n experiments. *: P < 0.05.
sonication for 5 minutes in an ultrasound bath.
The MSSA strains adhered more on the tubing (1.095 ×
107 ± 5.37 × 106) than the MRSA strains (2.4 × 105 ±
6.75 × 104). Adding EGTA to the culture medium in-
creased the adhesion of the MSSA strains (1.176 × 108 ±
1.59 × 107) and decreased the adhesion of MRSA strains
(7.67 × 104 ± 2.24 × 104). The ANOVA analysis of the
results followed by the Bonferroni post test confirmed
that the adhesion of the MSSA strains was significantly
more important than the adhesion of MRSA strains.
EGTA had no significant effect on either group. Consid-
ering the large variations of the measurements, the results
for each strain were analyzed. As shown in Figures
6(a)-(d), the 4 MSSA strains were comparable and there
was no effect of EGTA on either strain. Two MRSA
strains (007/U and 028/FV) were not affected by EGTA.
The chelator inhibited the adhesion of the 2 other MRSA
strains (011/LP and 027/FV).
4. Discussion
In this work, we studied the interaction of clinical strains
of S. aureus with an abiotic surface. We show that the
BFRT® was a more rapid method than the CVSM to de-
tect the adhesion of the bacteria to the surface. MSSA
strains adhered more rapidly and the interaction of
MRSA strains with an abiotic surface was inhibited by
EGTA. Calcium, magnesium and manganese reversed
the inhibition exerted by EGTA.
Different methods have been used to evaluate the ad-
hesion of bacterial strains. Bacterial adhesion and the
early stage of biofilm formation can be studied using
reactors developed for the cultivation of biofilms like
flow cells [21,22], annular biofilm reactors [23], fowler
cells [24], modified Robbins devices [25], CDC biofilm
reactors [26,27]. Disadvantages of such flow cell devices
include limited sample area, and if operated on once-
through flow basis, the potential exists for gradients in
biofilm amount and fluid phase concentrations to develop
in longitudinal direction. The examination and charac-
terization of biofilms in medical devices, and human in-
fections can also be performed using microscopy tech-
niques (transmission electron, scanning electron, confocal

Study of the Adhesion of Clinical Strains of Staphylococcus aureus on an
Abiotic Surface Using the Biofilm Ring Test®
553
MS S A s t ra in s
5668/S 1620/P 1532/P 5741/S
0
2
4
6
8
10
Control EGTA 1 mM
(a)
Str a ins
Log CF U/ mL
(a)
MRSA stra i n s
011/LP007/U027/FV 028/FV
0
2
4
6
8
10
Control EGTA 1 mM
**
(b)
Str a in s
Log CFU/ mL
(b)
Figure 6. Adhesion of the clinical strains of S. aureus to a
catheter. The 4 MSSA strains (a) and the 4 MRSA strains
(b) were incubated for 18 h in the presence of a piece of
catheter. After washing, the bacteria were detached by
sonication and the number of colony forming units was
estimated. Results are the means ± s.e.m. of 4 measurements.
*: P < 0.05.
laser-scanning, epifluorescence, atomic force microscopy)
[28-31]. These methods are mostly qualitative and de-
scriptive, but they do not directly measure the adhesion
of bacterial populations [32]. The enumeration of bacte-
ria on plate counts after the removal of biofilms or
biofilm-associated bacteria by mechanical forces such as
vortexing or sonication is also widely used [33,34]. This
method gives indication only on the number of bacteria
inside the biofilm, and not on the number of bacteria ad-
hering on a surface. This slow and fastidious method is
also highly dependent on cell recovery which is difficult
to estimate and is not fitted to large scale screening ex-
periments. Microtiter plate methods have been also de-
veloped. Culture of the bacteria in the wells of the mi-
croplate followed by the estimation of the biomass re-
maining in the well after washing using the CVSM [35]
is a very convenient and widespread method to evaluate
biofilm adhesion. It has been modified to increase its
accuracy and to improve its reproducibility [13,14]. As
shown in this study, this method has a low sensitivity and
requires rather long incubation times to get significant
increase of the absorbance. The washing steps, used to
remove non-adherent cells, are also critical. The lack of
standardization of these washing steps makes difficult the
comparison of results obtained by different laboratories
[15]. The BFRT® is a newly described method for study-
ing biofilm formation. This technique is based on the
immobilization of magnetic beads by adherent cells [15,
17]. In this paper, we presented evidence that this method
could detect adhesion of S. aureus to the bottom of the
wells within a few hours, before any significant biofilm
could be detected with the CVSM. These observations
confirmed our previous results on S. aureus [18] or P.
aeruginosa [17]. The BFRT® could also demonstrate that
MSSA strains interacted more rapidly with the surface
than MRSA strains. The adhesion of the MRSA strains
was partly reverted by the presence in the culture me-
dium of EGTA, while the adhesion of the MSSA strains
was not affected by the metal-chelator. These results
were confirmed with the CVSM which illustrated that, at
later times, the MSSA strains formed a more important
biofilm than MRSA strains. Our results were in agree-
ment with those of O’Neill et al. [36]. These authors re-
ported that MRSA strains were less likely to form a
biofilm than MSSA strains and that their sensitivity to
salts differed. They concluded that the mechanism in-
volved in the formation of the biofilm was different
among MSSA and MRSA strains. Another explanation
for this difference between MSSA and MRSA strains
could be their distinct membrane properties. The MATS
test demonstrated that the two populations diverged: the
membranes of the MSSA strains were more hydrophobic
and had a higher propensity to donate electrons. These
properties should affect the adherence of the bacteria [8].
Cations (calcium, magnesium, manganese) interfered
with the behavior of the beads in the BFRT® and de-
creased the BFI. Similar artefactual interactions have
also been described with pronase [37] or with culture
media with high ionic strength [38]. Such interaction has
also been observed with antimicrobial cationic peptides
derived from cathelicidin and which, at neutral pH, have
many positive charges (C. Nagant, personal communica-
tion). These interactions might thus be a consequence of
some electrostatic interactions between the negatively
charged beads and the cations or between some compo-
nents of the medium. These results also illustrate the fact
that the mobility of the beads can be affected not only by
adhering bacteria but also by modifications of rheologi-
cal properties of the medium. The interaction of the
cations with EGTA was thus tested with the CVSM. The

Study of the Adhesion of Clinical Strains of Staphylococcus aureus on an
Abiotic Surface Using the Biofilm Ring Test®
554
ions had no effect on the formation of the biofilm by the
MRSA strains but the 3 cations reversed the inhibition by
EGTA. This lack of specificity suggested that the effect
of the ions was indirect. Considering the dissociation
constant of EGTA for the 3 cations [39], the concentra-
tion of free EGTA should be very low (in the micromolar
range) in solutions containing 1 mm EGTA and 1 mM
calcium, magnesium or manganese. The most likely ex-
planation is thus that, in the absence of any added diva-
lent cation, EGTA probably binds another endogenous
cation (iron [40] or zinc [41]) contributing to the forma-
tion of the biofilm. It has been recently reported that zinc
contributes to the rod-like structure of SasG, a protein
involved in the formation of a biofilm by S. aureus [42].
The results obtained in the wells of a microplate were,
at least, partly confirmed by measuring the adhesion of
the bacteria on catheter tubing. In agreement with the
results of the BFRT and the CVSM, the MRSA strains
adhered less than the MSSA strains. The 2 MRSA strains
with less adherence on the tubing in the presence of
EGTA were also the 2 strains mostly affected by EGTA
in the BFRT®. The conclusions of the studies comparing
the adhesion of MSSA and MRSA strains were not con-
sistent. Amaral et al. [43] reported that the adhesion of
clinical MRSA strains on bronchial epithelial cells was
higher than the adhesion of clinical MSSA strains. More
recently, Pozzi et al. [44] showed that the expression of a
gene responsible with resistance to methicillin was asso-
ciated with increased adhesion on a catheter [44]. At the
opposite, Aathitan et al. [45] reported that MRSA and
MSSA strains similarly interacted with liver epithelial
cells [45], whereas Karauzum et al. [46] observed that
MRSA strains were less adherent on human airway
epithelial cells than MSSA strains [46]. There is thus a
large panel of opinions on the ability of the two popula-
tions of strains to adhere on a surface.
In conclusion, the formation of a biofilm by MSSA
and MRSA strains proceeds at a different rate and is dif-
ferently affected by cations. This should help in the for-
mulation of washing solutions to clean material con-
taminated by MRSA-carriers. The BFRT® could contrib.-
ute to the search for these new cleaning solutions: it is a
very easy and efficient technique to study the initial steps
of the formation of a biofilm by S. aureus, at a time when
the growth of the bacterial population only marginally
affects the results. However, the results obtained with the
BFRT are also consistent with the investigation on
catheters. Further studies should also look for a correla-
tion between the BFRT® and the adhesion of S. aureus
on biotic surfaces like wounds. It should also be kept in
mind that the results of an assay might be indirectly af-
fected by modifications of the properties of the medium
provoked by the tested drugs.
5. Acknowledgements
This work was supported by grant No 3.4577.10 of the
Fonds National de la Recherche Scientifique of Belgium
and by a grant from the Coopération Technique Belge
(CTB). J.-M. Liesse Iyamba is a recipient of a Ph.D. Re-
search Fellowship from the CTB.
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