# Influence of Surface Properties on the Adhesion ofStaphylococcus epidermidis to Acrylic and Silicone

Authors: Cláudia Sousa; Pilar Teixeira; Rosário Oliveira
Journal: International Journal of Biomaterials
 (2009)

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## Abstract

The aim of the present study was to compare the ability of eightStaphylococcus epidermidis strains to adhere to acrylic and silicone, two polymers normally used in medical devices manufacture. Furthermore, it was tried to correlate that with the surface properties of substrata and cells. Therefore, hydrophobicity and surface tension components were calculated through contact angle measurements. Surface roughness of substrata was also assessed by atomic force microscopy (AFM). No relationship was found between microbial surface hydrophobicity and adhesion capability. Nevertheless, Staphylococcus epidermidis IE214 showed very unique adhesion behaviour, with cells highly aggregated between them, which is a consequence of their specific surface features. All strains, determined as being hydrophilic, adhered at a higher extent to silicone than to acrylic, most likely due to its more hydrophobic character and higher roughness. This demonstrates the importance of biomaterial surface characteristics for bacterial adhesion.


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## Body

## 1. Introduction

Staphylococcus epidermidis is a coagulase-negative
staphylococcus (CNS)  that often colonizes the skin and mucous membranes of the
human body, representing an important part of its normal microflora [1, 2].
However, these staphylococci have emerged in the last years as the most
frequently isolated pathogen in nosocomial sepsis, associated with
implanted medical devices [3, 4], namely,
prosthetic heart valves and joints, central venous catheters, urinary
catheters, contact lenses, and hip prostheses [5]. S. epidermidis has the ability to adhere
to biomaterials surface and develop as biofilm [6], which constitutes an
important virulence factor [7] and the most important pathogenic mechanism of
staphylococcal infection [8]. Therefore, initial adhesion
of bacteria to the biomaterial surface is thought to be a key step in the
colonization of indwelling medical devices. It is a complex process, affected
by numerous aspects, such as surface properties of bacteria, material surface
properties, and environmental factors [9]. The better understanding of these features
is of extreme importance for the development of effective adhesion control
mechanisms that will ultimately prevent biofilm formation and thus, the
infection of medical devices.During the
adhesion process, bacteria firmly adhere to the biomaterial surface through
physicochemical interactions [10]. These comprise cell surface hydrophobicity [11, 12] and charge [13] as well as the hydrophobicity, charge, roughness, and
chemical composition of the biomaterial surface itself [9]. Surface
hydrophobicity, in particular, has been described as one of the most important
properties involved in the adhesion phenomenon [14–16]. According to van
Oss and Giese [17], in biological systems, hydrophobic interactions are
normally the strongest of the long-range noncovalent interactions and can be
defined as the attraction among apolar, or slightly polar, cells or other
molecules themselves, when immersed in an aqueous solution. Biomaterial surface
roughness is another property relevant for the bacterial adhesion process, with
the irregularities of the polymeric surfaces normally promoting bacterial
adhesion and biofilm accumulation [18, 19]. This is due to the increased
surface area and depressions that provide more favourable and additional sites
for colonization [20], as such crevices protect bacterial cells from the shear
forces [21]. However, the accumulation of bacteria in such locations depends
largely on their size, cell dimension, and division mode [22]. In fact,
according to some authors [23, 24], a linear relation of bacterial adhesion
with surface roughness is not always verified. A small increase in roughness
can lead to a significant increase in bacterial adhesion, while a larger
increase in roughness can have no significant effect on cellular attachment.The aim of
the present work was to study the ability of eight strains ofS. epidermidis to adhere to acrylic and
to silicone, two materials commonly used in the manufacture of medical devices,
in relation to the surface properties of these materials.


## 2. Materials and Methods

### 2.1. Bacterial Strains

EightS. epidermidis strains were studied in
this work. S.
epidermidis 9142 is a known producer of the
surface polysaccharide intercellular adhesin (PIA),
which was identified as one of the main responsible factors for biofilm
formation [25]. The strain S. epidermidis 9142-M10 is an isogenic mutant with a transposon inserted in the ica
locus that encodes the proteins
involved in PIA production and thus does not form biofilm. The PIA-positive S. epidermidis 1457 was isolated from an infected central venous catheter, while S. epidermidis 1457-M10 is a PIA-negative isogenic mutant of S. epidermidis 1457, also obtained by insertion of a transposon
into the ica
locus [26]. S. epidermidis IE186, S. epidermidis IE214
and S.
epidermidis IE75 were previously
isolated from blood of patients with infective endocarditis, while S. epidermidis LE7 was isolated from the skin of a healthy individual. All strains were kindly
provided by Dr. G. B. Pier,
Channing Laboratory, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston.


### 2.2. Media and Growth Conditions

For all the assays,
cells were firstly grown for approximately 36 hours in plates of Tryptic Soy
Agar (TSA; Merck, Germany), and then for 24 hours in 15 mL of Tryptic Soy Broth
(TSB, Merck), at 37°C under a constant agitation of 120 rpm. After this
period, 50 μL of each suspension were transferred into 30 mL of fresh TSB and
incubated for 18 hours, under the same conditions. Then, the cells were
centrifuged for 5 minutes at 10 500  ×  g and 4°C, washed twice with a saline
solution (0.9% NaCl (Merck) in distilled water) and sonicated (Ultrasonic
Processor, Cole-Parmer, Ill, USA) during 10 seconds, with an amplitude of 22%
(previously optimized to avoid cell disruption). The cellular suspension was
adjusted to a final concentration of approximately 1  ×  109 cells/mL,
determined by optical density at 640 nm, prior to usage in the adhesion assays.


### 2.3. Substrate Preparation

2 cm  ×  2 cm squares of commercial acrylic, specifically Poly(methyl
methacrylate) (PMMA) (Repsol, Brønderslev, Denmark) and silicone (Leewood
Elastomer AB, Sweden), both with 2
mm in thickness, were used as substrata in the adhesion
assays. This acrylic is the synthetic polymer of methyl methacrylate,
containing small amounts of a UV-absorber and release agent and with a
molecular weight in the range 1.2–1.4 million Da. Concerning silicone, it consists mainly of cross-linked
polydimethylvinylmethylsiloxanes with the chain length of ca. 200 000 Da, but
also of low chain length polymers in the amount up to typically 2% and not
exactly defined amount of fumed silica.Prior to
use, the coupons were washed several times with sterile distilled water and let
to soak overnight. Next, they
were transferred to a new container with sterile distilled water and washed for
5 minutes under agitation, followed by a 30 minutes
immersion period in a 70% ethanol/sterile distilled water solution. Finally,
the coupons were aseptically and individually washed with ultra-pure sterile
water and let to dry overnight at 60°C.


### 2.4. Adhesion Assays

Adhesion
assays were performed as previously described [27]. Briefly, the acrylic and
silicone squares were placed in 6-well tissue-culture plates containing 4 mL of
bacterial suspension (1  ×  109 cells/mL) in saline solution (NaCl,
0.9% (w/v)). Initial
adhesion to each substrate was allowed to occur for 2
hours at 37°C, in a shaker rotating at 120 rpm. Negative controls were
obtained by placing the coupons in a saline solution without bacterial cells. Each coupon was then carefully removed and washed by
immersion, in order to remove loosely attached cells. This procedure was gently
undertaken and involved their transference to a glass beaker containing 50 mL of
distilled water, where they were kept for about 10 seconds. Afterwards, a new
transfer was made to an additional beaker with 50 mL of distilled water,
followed by a third transfer 10 seconds later. These washing steps were
carefully performed in order to remove loosely attached cells. The coupons were then let to dry at 37°C for about 1 hour.
All experiments were done in triplicate and repeated in four independent
assays.


### 2.5. Total Cell Counts of Adhered Bacteria

The dried coupons were
stained with a4′-6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, St Louis, Mo, USA) solution
(0.1 g/L) during 30 minutes. Subsequently, each coupon was rinsed with distilled
water in order to remove excess stain and let to air-dry in the dark for 30 minutes.
Adhered cells were visualised under an epifluorescence microscope (Carl Zeiss, Germany)
with a filter sensitive to DAPI fluorescence and coupled with a 3CCD video
camera. For each coupon, at least 20 images, with an 820  ×  560 resolution and 1000× magnification, were taken. Enumeration of adhered
cells was performed with automated enumeration software (SigmaScan Pro 5, SPSS
Inc., Chicago, Ill, USA)
and the results presented as number of adhered cells/cm2.


### 2.6. Scanning Electron Microscopy (SEM)

The coupons
with adhered bacteria were dehydrated by a 15-minute immersion in increasing
ethanol concentration solutions: 10, 25, 40, 50, 60, 70, 80, 90 and 100%  (v/v),
having then been placed in a sealed desiccator. Samples were then mounted on
aluminium stubs with carbon tape, sputter-coated with gold and observed with a
Leica Cambridge S-360 scanning electron microscope (Leo, Cambridge, UK).
In order to assess the extent of bacterial adhesion in each sample, three fields
were used for image analysis. All photographs were taken using a magnification of 3000×.


### 2.7. Substrata and Bacteria Hydrophobicity

Hydrophobicity
parameters of substrata and bacteria surface were determined through the
sessile drop contact angle technique [28], using an automated contact angle
measurement apparatus (OCA 15 Plus; Dataphysics,
Germany).
Cleaned and dried substratum surfaces were used for determining the
hydrophobicity parameters of acrylic and silicone. In the particular case of
bacteria, the measurements were performed on bacterial layers deposited on
membrane filters [28]. Briefly, a 30 mL suspension of S. epidermidis cells,
adjusted to a concentration of approximately
1  ×  109 cells/mL in
saline solution (NaCl, 0.9% (w/v)), was deposited onto a 0.45 μm cellulose
filter (Pall-Life Sciences, USA), previously wetted with 10 mL of distilled
water. To standardise the moisture content, the filters with the resultant lawn
of cells deposited were then let to dry onto Petri dishes containing 1% (w/v) agar
(Merck) and 10% (v/v) glycerol (Sigma-Aldrich), for at least 3.5 hours. All
measurements (at least 25 determinations for each material and bacterial
strain) were performed at room temperature and water, formamide and α-bromonaphtalene,
with known surface tension components [29], were used as reference liquids for
standardized contact angles measurements.Contact angle
measurements allowed the calculation of substrata and bacteria hydrophobicity
parameters, using the van Oss approach [17, 30]. According
to it, the absolute degree of hydrophobicity of a given material (i) is defined
in terms of the variation of the free energy of interaction (ΔG) between two
moieties of that material, when immersed in water (w), that is,  ΔGiwi.
When  ΔGiwi is negative, the free energy of interaction between molecules is attractive,
existing a smaller affinity for water than among molecules themselves, making
(i) (the microbial cell or material surface) hydrophobic. In an opposite way,
(i) is hydrophilic when  ΔGiwi is positive.


### 2.8. Atomic Force Microscopy (AFM)

Acrylic and silicone
surfaces topography was assessed by atomic force microscopy (AFM), using a
PicoPlus scanning probe microscope from Molecular Imaging (USA). Surface
imaging was performed in Tapping mode and the samples were analysed in air at
room temperature. The acrylic surfaces were analysed using a silicon (Si) tip
with a Spring Constant≅42 N/m, while for silicone surfaces the Si tip
had a Spring Constant ≅2.8 N/m. The roughness measurements were
performed under a scan range of
2.5  ×  2.5 μm, using the SPIP version 4.2.2.0 software. Measurements were made in three randomly
chosen areas in all samples.


### 2.9. Statistical Analysis

Results from all the assays were compared using one-way analysis of
variance (ANOVA) by applying Levene’s test of homogeneity of variances and the
Tukey multiple comparisons test, using software Statistical Package for the
Social Sciences Inc., (SPSS) (Chicago, Ill, USA). All tests were performed with a confidence level of 95%.


## 2.1. Bacterial Strains

EightS. epidermidis strains were studied in
this work. S.
epidermidis 9142 is a known producer of the
surface polysaccharide intercellular adhesin (PIA),
which was identified as one of the main responsible factors for biofilm
formation [25]. The strain S. epidermidis 9142-M10 is an isogenic mutant with a transposon inserted in the ica
locus that encodes the proteins
involved in PIA production and thus does not form biofilm. The PIA-positive S. epidermidis 1457 was isolated from an infected central venous catheter, while S. epidermidis 1457-M10 is a PIA-negative isogenic mutant of S. epidermidis 1457, also obtained by insertion of a transposon
into the ica
locus [26]. S. epidermidis IE186, S. epidermidis IE214
and S.
epidermidis IE75 were previously
isolated from blood of patients with infective endocarditis, while S. epidermidis LE7 was isolated from the skin of a healthy individual. All strains were kindly
provided by Dr. G. B. Pier,
Channing Laboratory, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston.


## 2.2. Media and Growth Conditions

For all the assays,
cells were firstly grown for approximately 36 hours in plates of Tryptic Soy
Agar (TSA; Merck, Germany), and then for 24 hours in 15 mL of Tryptic Soy Broth
(TSB, Merck), at 37°C under a constant agitation of 120 rpm. After this
period, 50 μL of each suspension were transferred into 30 mL of fresh TSB and
incubated for 18 hours, under the same conditions. Then, the cells were
centrifuged for 5 minutes at 10 500  ×  g and 4°C, washed twice with a saline
solution (0.9% NaCl (Merck) in distilled water) and sonicated (Ultrasonic
Processor, Cole-Parmer, Ill, USA) during 10 seconds, with an amplitude of 22%
(previously optimized to avoid cell disruption). The cellular suspension was
adjusted to a final concentration of approximately 1  ×  109 cells/mL,
determined by optical density at 640 nm, prior to usage in the adhesion assays.


## 2.3. Substrate Preparation

2 cm  ×  2 cm squares of commercial acrylic, specifically Poly(methyl
methacrylate) (PMMA) (Repsol, Brønderslev, Denmark) and silicone (Leewood
Elastomer AB, Sweden), both with 2
mm in thickness, were used as substrata in the adhesion
assays. This acrylic is the synthetic polymer of methyl methacrylate,
containing small amounts of a UV-absorber and release agent and with a
molecular weight in the range 1.2–1.4 million Da. Concerning silicone, it consists mainly of cross-linked
polydimethylvinylmethylsiloxanes with the chain length of ca. 200 000 Da, but
also of low chain length polymers in the amount up to typically 2% and not
exactly defined amount of fumed silica.Prior to
use, the coupons were washed several times with sterile distilled water and let
to soak overnight. Next, they
were transferred to a new container with sterile distilled water and washed for
5 minutes under agitation, followed by a 30 minutes
immersion period in a 70% ethanol/sterile distilled water solution. Finally,
the coupons were aseptically and individually washed with ultra-pure sterile
water and let to dry overnight at 60°C.


## 2.4. Adhesion Assays

Adhesion
assays were performed as previously described [27]. Briefly, the acrylic and
silicone squares were placed in 6-well tissue-culture plates containing 4 mL of
bacterial suspension (1  ×  109 cells/mL) in saline solution (NaCl,
0.9% (w/v)). Initial
adhesion to each substrate was allowed to occur for 2
hours at 37°C, in a shaker rotating at 120 rpm. Negative controls were
obtained by placing the coupons in a saline solution without bacterial cells. Each coupon was then carefully removed and washed by
immersion, in order to remove loosely attached cells. This procedure was gently
undertaken and involved their transference to a glass beaker containing 50 mL of
distilled water, where they were kept for about 10 seconds. Afterwards, a new
transfer was made to an additional beaker with 50 mL of distilled water,
followed by a third transfer 10 seconds later. These washing steps were
carefully performed in order to remove loosely attached cells. The coupons were then let to dry at 37°C for about 1 hour.
All experiments were done in triplicate and repeated in four independent
assays.


## 2.5. Total Cell Counts of Adhered Bacteria

The dried coupons were
stained with a4′-6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, St Louis, Mo, USA) solution
(0.1 g/L) during 30 minutes. Subsequently, each coupon was rinsed with distilled
water in order to remove excess stain and let to air-dry in the dark for 30 minutes.
Adhered cells were visualised under an epifluorescence microscope (Carl Zeiss, Germany)
with a filter sensitive to DAPI fluorescence and coupled with a 3CCD video
camera. For each coupon, at least 20 images, with an 820  ×  560 resolution and 1000× magnification, were taken. Enumeration of adhered
cells was performed with automated enumeration software (SigmaScan Pro 5, SPSS
Inc., Chicago, Ill, USA)
and the results presented as number of adhered cells/cm2.


## 2.6. Scanning Electron Microscopy (SEM)

The coupons
with adhered bacteria were dehydrated by a 15-minute immersion in increasing
ethanol concentration solutions: 10, 25, 40, 50, 60, 70, 80, 90 and 100%  (v/v),
having then been placed in a sealed desiccator. Samples were then mounted on
aluminium stubs with carbon tape, sputter-coated with gold and observed with a
Leica Cambridge S-360 scanning electron microscope (Leo, Cambridge, UK).
In order to assess the extent of bacterial adhesion in each sample, three fields
were used for image analysis. All photographs were taken using a magnification of 3000×.


## 2.7. Substrata and Bacteria Hydrophobicity

Hydrophobicity
parameters of substrata and bacteria surface were determined through the
sessile drop contact angle technique [28], using an automated contact angle
measurement apparatus (OCA 15 Plus; Dataphysics,
Germany).
Cleaned and dried substratum surfaces were used for determining the
hydrophobicity parameters of acrylic and silicone. In the particular case of
bacteria, the measurements were performed on bacterial layers deposited on
membrane filters [28]. Briefly, a 30 mL suspension of S. epidermidis cells,
adjusted to a concentration of approximately
1  ×  109 cells/mL in
saline solution (NaCl, 0.9% (w/v)), was deposited onto a 0.45 μm cellulose
filter (Pall-Life Sciences, USA), previously wetted with 10 mL of distilled
water. To standardise the moisture content, the filters with the resultant lawn
of cells deposited were then let to dry onto Petri dishes containing 1% (w/v) agar
(Merck) and 10% (v/v) glycerol (Sigma-Aldrich), for at least 3.5 hours. All
measurements (at least 25 determinations for each material and bacterial
strain) were performed at room temperature and water, formamide and α-bromonaphtalene,
with known surface tension components [29], were used as reference liquids for
standardized contact angles measurements.Contact angle
measurements allowed the calculation of substrata and bacteria hydrophobicity
parameters, using the van Oss approach [17, 30]. According
to it, the absolute degree of hydrophobicity of a given material (i) is defined
in terms of the variation of the free energy of interaction (ΔG) between two
moieties of that material, when immersed in water (w), that is,  ΔGiwi.
When  ΔGiwi is negative, the free energy of interaction between molecules is attractive,
existing a smaller affinity for water than among molecules themselves, making
(i) (the microbial cell or material surface) hydrophobic. In an opposite way,
(i) is hydrophilic when  ΔGiwi is positive.


## 2.8. Atomic Force Microscopy (AFM)

Acrylic and silicone
surfaces topography was assessed by atomic force microscopy (AFM), using a
PicoPlus scanning probe microscope from Molecular Imaging (USA). Surface
imaging was performed in Tapping mode and the samples were analysed in air at
room temperature. The acrylic surfaces were analysed using a silicon (Si) tip
with a Spring Constant≅42 N/m, while for silicone surfaces the Si tip
had a Spring Constant ≅2.8 N/m. The roughness measurements were
performed under a scan range of
2.5  ×  2.5 μm, using the SPIP version 4.2.2.0 software. Measurements were made in three randomly
chosen areas in all samples.


## 2.9. Statistical Analysis

Results from all the assays were compared using one-way analysis of
variance (ANOVA) by applying Levene’s test of homogeneity of variances and the
Tukey multiple comparisons test, using software Statistical Package for the
Social Sciences Inc., (SPSS) (Chicago, Ill, USA). All tests were performed with a confidence level of 95%.


## 3. Results

### 3.1. Adhesion to Acrylic and Silicone

The initial adhesion of theS.
epidermidis strains to the acrylic and silicone surfaces is presented in
Figure 1. As it can be seen, almost all S.
epidermidis strains adhered at a significantly (P<.05) higher
extent to the silicone substrate than to acrylic. The only exceptions were observed
for strains 1457-M10, IE75, and LE7, which also adhered more to silicone than
to acrylic but in a nonsignificant way (P>.05). In fact, the extent
of adhesion of S. epidermidis 9142 to
silicone was approximately 2.5 times greater than to acrylic. For strains
9142-M10, IE186, and IE214, this difference ranged between 1.8 and 2.0 times. Concerning
acrylic, strains IE214 and IE186 were the ones that most extensively, and
significantly (P<.05) adhered to the coupons. In opposition, strains
IE75 and LE7 showed the lowest levels of initial binding to this material,
being markedly different from all the other strains (P<.05).
Similarly, they also adhered least to silicone coupons (P<.05). S. epidermidis IE214 and S. epidermidis IE186 were the strains
showing the highest number of cells adhered (P<.05) to this
substrate, likewise to acrylic.Figure 1
Number of adhered cells
per cm2 onto acrylic
() and silicone
() coupons, after a 2-hour period of
contact for S. epidermidis strains
IE214, IE186, 9142, 9142-M10, 1457, 1457-M10, IE75, and LE7. The symbol (*) indicates the strains that adhered at a statistically higher extent to silicone
than to acrylic (P<.05).Scanning electron
microscope (SEM) images of bacteria adhered to acrylic (a) and silicone (b)
squares are presented in Figure2. The images reveal grape-like clusters of
variable dimensions. It is also visible the higher extent of adhesion to
silicone than to acrylic, especially for strains IE214, IE186, 9142, 9142-M10,
and 1457. In addition, it can be seen, both for acrylic and for silicone, how
strain IE214 cells (Figure 2(a)-A and Figure 2(b)-A) grew highly
aggregated, quite differently from the mode of growth of the remaining strains.
In fact, this strain formed flocculent suspensions in liquid medium. In Figure 2(b)-G, it must be noted (arrow) how cells adhered
to the silicone along the depression on its surface.SEM photomicrographs ofS.
epidermidis adhered to acrylic (a)
and silicone (b) surfaces. Strains: A—IE214; B—IE186; C—9142; D—9142-M10; E—1457; F—1457-M10; G—IE75; H—LE7. The arrow shows bacterial cells
adhered along a depression on silicone’s surface. Magnification ×3000, bar  =  10 μm.
(a)(b)


### 3.2. Substrata and Bacteria Hydrophobicity

Hydrophobicity of
substrata and bacteria was evaluated through contact angle measurements, using
the van Oss approach [17, 30]. Contact angles, surface tension parameters, and
hydrophobicity of acrylic and silicone are presented in Table 1. Water contact
angles can be used as a qualitative indication of the surface material
hydrophobicity, with higher values indicating a more hydrophobic surface. As it
can be seen, the water contact angles obtained for both surfaces are high, a
fact that is indicative of their hydrophobicity. The values of  ΔGiwi also showed that both materials are hydrophobic (ΔGiwi<0),
with silicone holding a more hydrophobic character. From Table 1, it can also
be seen that both acrylic and silicone surfaces are predominantly electron
donors (higher values of γ−), with low electron acceptor parameters (γ+). In fact, acrylic does not have an
electron acceptor parameter (γ+=0) but is only electron donor (γ−).Table 1
Water (θW), formamide (θF), and α-bromonaphtalene (θα−B) contact angles (in degrees), surface tension components, and
hydrophobicity (in mJ/m2) of the acrylic and silicone coupons
surface.
SubstratumContact angle  ±  SD (°)Surface tension components (mJ/m2)ΔGiwi (mJ/m2)θWθFθα−BγLWγ+γ−Acrylic85.3±2.264.1±1.224.5±1.240.50.04.5−62.5Silicone114.5±2.3104.3±2.481.4±3.514.70.41.7−67.1SD: standard deviation;γLW:
apolar Lifshitz-van der Waals surface free energy component; γ+: electron acceptor surface free energy component;
γ−: electron donor surface free energy component;  ΔGiwi:
degree of hydrophobicity.Cell surface
hydrophobicity parameters ofS.
epidermidis strains, as well as the contact angles obtained using the three
liquids tested, are presented in Table 2. The eight strains studied showed
similar values of water contact angles, lower than 65°, which in accordance to
Vogler [31] is indicative of a hydrophilic surface, ranging from 21.6° (strain
IE186) to 31.8° (S. epidermidis 1457). These values are quite similar to those obtained for formamide, also
polar, with exception of strain LE7 that presented a formamide contact angle
much lower than that of water. The contact angles determined by using the
apolar liquid, α-bromonaphtalene, showed small variation
between strains with values higher than 49.6° (S. epidermidis 1457-M10). Also, all strains showed positive values
of ΔGiwi and so, can be considered hydrophilic. In what concerns surface tension
components, all strains predominantly showed electron donation, with higher
values of electron donor parameters (γ−) compared to the low values
of the electron acceptor parameters (γ+). Strain IE214 showed the
highest values of electron acceptor and electron donor parameters of the
acid-base component of the surface tension, while strains IE186 and LE7
revealed the lowest values of electron acceptor and electron donor parameters,
respectively.Table 2
Water (θW), formamide (θF), and α-bromonaphtalene (θα−B) contact angles (in degrees), surface tension components, and
hydrophobicity (in mJ/m2) of the surface of S. epidermidis strains.
S. epidermidis strainContact angle  ±  SD (°)Surface tension components (mJ/m2)ΔGiwi   (mJ/m2)θWθFθα−BγLWγ+γ−IE21422.3±3.521±1.259±2.120.67.956.720.3IE18621.6±1.629.9±4.054.5±2.027.72.555.532.5914225.6±0.925.4±2.657.0±1.426.54.048.422.89142-M1021.8±1.219.0±1.954.7±1.027.64.348.422.0145731.8±1.031.4±2.553.2±1.528.42.745.922.81457-M1024.7±1.817.3±0.749.6±0.930.13.845.319.6IE7527.1±1.026.5±1.650.4±1.329.82.747.924.2LE723.7±0.79.4±0.652.3±1.328.85.043.616.5SD: standard deviation;γLW:
apolar Lifshitz-van der Waals surface free energy component; γ+: electron acceptor surface free energy
component; γ−: electron donor surface free energy component;  ΔGiwi:
degree of hydrophobicity.


### 3.3. Substrata Roughness

The surface topography
of acrylic and silicone was analyzed by AFM in tapping mode (Figure3).
Silicone surface displays higher roughness with an average value (Ra) of 4.237 nm and a maximum (Rmax) of 44.367 nm, in opposition to Ra  =  0.789 nm and Rmax  =  15.683 nm for acrylic surface. Ra indicates the average distance of the
roughness profile to the centre plane of the profile, while Rmax represents the
maximum height measured within the screened area.AFM images of acrylic (a) and silicone (b) surfaces with a scan range of 2.5μm  ×  2.5 μm (air Tapping mode). Axis x and y-nm; axis z−Å.
(a)(b)


## 3.1. Adhesion to Acrylic and Silicone

The initial adhesion of theS.
epidermidis strains to the acrylic and silicone surfaces is presented in
Figure 1. As it can be seen, almost all S.
epidermidis strains adhered at a significantly (P<.05) higher
extent to the silicone substrate than to acrylic. The only exceptions were observed
for strains 1457-M10, IE75, and LE7, which also adhered more to silicone than
to acrylic but in a nonsignificant way (P>.05). In fact, the extent
of adhesion of S. epidermidis 9142 to
silicone was approximately 2.5 times greater than to acrylic. For strains
9142-M10, IE186, and IE214, this difference ranged between 1.8 and 2.0 times. Concerning
acrylic, strains IE214 and IE186 were the ones that most extensively, and
significantly (P<.05) adhered to the coupons. In opposition, strains
IE75 and LE7 showed the lowest levels of initial binding to this material,
being markedly different from all the other strains (P<.05).
Similarly, they also adhered least to silicone coupons (P<.05). S. epidermidis IE214 and S. epidermidis IE186 were the strains
showing the highest number of cells adhered (P<.05) to this
substrate, likewise to acrylic.Figure 1
Number of adhered cells
per cm2 onto acrylic
() and silicone
() coupons, after a 2-hour period of
contact for S. epidermidis strains
IE214, IE186, 9142, 9142-M10, 1457, 1457-M10, IE75, and LE7. The symbol (*) indicates the strains that adhered at a statistically higher extent to silicone
than to acrylic (P<.05).Scanning electron
microscope (SEM) images of bacteria adhered to acrylic (a) and silicone (b)
squares are presented in Figure2. The images reveal grape-like clusters of
variable dimensions. It is also visible the higher extent of adhesion to
silicone than to acrylic, especially for strains IE214, IE186, 9142, 9142-M10,
and 1457. In addition, it can be seen, both for acrylic and for silicone, how
strain IE214 cells (Figure 2(a)-A and Figure 2(b)-A) grew highly
aggregated, quite differently from the mode of growth of the remaining strains.
In fact, this strain formed flocculent suspensions in liquid medium. In Figure 2(b)-G, it must be noted (arrow) how cells adhered
to the silicone along the depression on its surface.SEM photomicrographs ofS.
epidermidis adhered to acrylic (a)
and silicone (b) surfaces. Strains: A—IE214; B—IE186; C—9142; D—9142-M10; E—1457; F—1457-M10; G—IE75; H—LE7. The arrow shows bacterial cells
adhered along a depression on silicone’s surface. Magnification ×3000, bar  =  10 μm.
(a)(b)


## 3.2. Substrata and Bacteria Hydrophobicity

Hydrophobicity of
substrata and bacteria was evaluated through contact angle measurements, using
the van Oss approach [17, 30]. Contact angles, surface tension parameters, and
hydrophobicity of acrylic and silicone are presented in Table 1. Water contact
angles can be used as a qualitative indication of the surface material
hydrophobicity, with higher values indicating a more hydrophobic surface. As it
can be seen, the water contact angles obtained for both surfaces are high, a
fact that is indicative of their hydrophobicity. The values of  ΔGiwi also showed that both materials are hydrophobic (ΔGiwi<0),
with silicone holding a more hydrophobic character. From Table 1, it can also
be seen that both acrylic and silicone surfaces are predominantly electron
donors (higher values of γ−), with low electron acceptor parameters (γ+). In fact, acrylic does not have an
electron acceptor parameter (γ+=0) but is only electron donor (γ−).Table 1
Water (θW), formamide (θF), and α-bromonaphtalene (θα−B) contact angles (in degrees), surface tension components, and
hydrophobicity (in mJ/m2) of the acrylic and silicone coupons
surface.
SubstratumContact angle  ±  SD (°)Surface tension components (mJ/m2)ΔGiwi (mJ/m2)θWθFθα−BγLWγ+γ−Acrylic85.3±2.264.1±1.224.5±1.240.50.04.5−62.5Silicone114.5±2.3104.3±2.481.4±3.514.70.41.7−67.1SD: standard deviation;γLW:
apolar Lifshitz-van der Waals surface free energy component; γ+: electron acceptor surface free energy component;
γ−: electron donor surface free energy component;  ΔGiwi:
degree of hydrophobicity.Cell surface
hydrophobicity parameters ofS.
epidermidis strains, as well as the contact angles obtained using the three
liquids tested, are presented in Table 2. The eight strains studied showed
similar values of water contact angles, lower than 65°, which in accordance to
Vogler [31] is indicative of a hydrophilic surface, ranging from 21.6° (strain
IE186) to 31.8° (S. epidermidis 1457). These values are quite similar to those obtained for formamide, also
polar, with exception of strain LE7 that presented a formamide contact angle
much lower than that of water. The contact angles determined by using the
apolar liquid, α-bromonaphtalene, showed small variation
between strains with values higher than 49.6° (S. epidermidis 1457-M10). Also, all strains showed positive values
of ΔGiwi and so, can be considered hydrophilic. In what concerns surface tension
components, all strains predominantly showed electron donation, with higher
values of electron donor parameters (γ−) compared to the low values
of the electron acceptor parameters (γ+). Strain IE214 showed the
highest values of electron acceptor and electron donor parameters of the
acid-base component of the surface tension, while strains IE186 and LE7
revealed the lowest values of electron acceptor and electron donor parameters,
respectively.Table 2
Water (θW), formamide (θF), and α-bromonaphtalene (θα−B) contact angles (in degrees), surface tension components, and
hydrophobicity (in mJ/m2) of the surface of S. epidermidis strains.
S. epidermidis strainContact angle  ±  SD (°)Surface tension components (mJ/m2)ΔGiwi   (mJ/m2)θWθFθα−BγLWγ+γ−IE21422.3±3.521±1.259±2.120.67.956.720.3IE18621.6±1.629.9±4.054.5±2.027.72.555.532.5914225.6±0.925.4±2.657.0±1.426.54.048.422.89142-M1021.8±1.219.0±1.954.7±1.027.64.348.422.0145731.8±1.031.4±2.553.2±1.528.42.745.922.81457-M1024.7±1.817.3±0.749.6±0.930.13.845.319.6IE7527.1±1.026.5±1.650.4±1.329.82.747.924.2LE723.7±0.79.4±0.652.3±1.328.85.043.616.5SD: standard deviation;γLW:
apolar Lifshitz-van der Waals surface free energy component; γ+: electron acceptor surface free energy
component; γ−: electron donor surface free energy component;  ΔGiwi:
degree of hydrophobicity.


## 3.3. Substrata Roughness

The surface topography
of acrylic and silicone was analyzed by AFM in tapping mode (Figure3).
Silicone surface displays higher roughness with an average value (Ra) of 4.237 nm and a maximum (Rmax) of 44.367 nm, in opposition to Ra  =  0.789 nm and Rmax  =  15.683 nm for acrylic surface. Ra indicates the average distance of the
roughness profile to the centre plane of the profile, while Rmax represents the
maximum height measured within the screened area.AFM images of acrylic (a) and silicone (b) surfaces with a scan range of 2.5μm  ×  2.5 μm (air Tapping mode). Axis x and y-nm; axis z−Å.
(a)(b)


## 4. Discussion

S. epidermidis is strongly associated with infections related to implants and medical devices,
such as joint prosthesis, prosthetic heart valves, vascular catheters and
contact lenses [3, 32, 33]. Given the fact that acrylic and silicone are
materials normally used in the production of some of these devices, it is of major
importance to study the adhesion of S.
epidermidis to these polymers. Thus, the primary intention of this study
was to attempt to correlate the adhesion capability of 8 S. epidermidis strains with the hydrophobicity parameters of cells
and these materials surfaces. Surface roughness of acrylic and silicone was
also assessed.As the present results
indicated, allS. epidermidis strains
adhered at a higher extent to silicone substrate than to acrylic (Figure 1).
These results are in accordance with other studies that refer silicone rubber as being especially prone to being colonized by Candida,
streptococci, Pseudomonas species, and also by staphylococci and other
CNS, depending on the site of implantation [34–36]. Taking into
consideration water contact angle values (114.5±2.3°) and the hydrophobicity
degree parameter,  ΔGiwi  =
−67.1 mJ/m2, the silicone assayed was found to be more hydrophobic than
acrylic (water contact angle  =  85.3±2.2°;  ΔGiwi  =
−62.5 mJ/m2) (Table 1), despite this difference was more pronounced if
only water contact angles values were considered. These results are in
accordance with values previously obtained [12, 37] and clearly demonstrate the
importance of the material hydrophobic effect in initial adhesion, since
acrylic and silicone have both a hydrophobic character. A higher surface hydrophobicity
of silicone is probably responsible for the highest levels of initial binding
to this substrate. This fact is corroborated by the work of Oliveira et al. [12] where the attachment of S. epidermidis to four polymeric
materials (including silicone), commonly used in indwelling medical devices, was
assayed. All materials were considered hydrophobic (ΔGiwi<0)
and an increase in the degree of hydrophobicity was linearly correlated with
the number of attached cells. A point also to be noted is that acrylic stands
solely as an electron-donor (γ−). Given the fact that a microorganism may adhere to a substratum via the
hydrophobic effect, that is, if hydrophobic areas are available for interactions
with hydrophobic sites on substrata [38], the lower densities of apolar
areas in acrylic (γ+=0) help to justify the preferential adhesion
of S. epidermidis cells to a more
hydrophobic material such as silicone.In what concerns cell surface hydrophobicity parameters determined, all strains were considered to be hydrophilic (ΔGiwi>0)
(Table 2). The most hydrophilic S.
epidermidis strain, IE186, (ΔGiwi  =  32.5 mJ/m2) was the second most adherent strain to both materials, while
the least adherent, strain LE7, was the one with the weakest hydrophilic
character. Thus, contrary to what was found for materials hydrophobicity, no
correlation was found between cell surface hydrophobicity of the S. epidermidis strains and their ability
to adhere to both hydrophobic surfaces. This fact is corroborated by previous
studies [37, 39] and suggests that other cell surface factors can as well
contribute to the initial attachment to biomaterials surfaces, such as the
production of exopolysaccharides like extracellular polysaccharide adhesins and  autolysins with adhesive properties like AtlE [40] and Aae [41]. In fact, some
authors attribute cell surface hydrophobicity to covalently bound cell-wall-associated
proteins [42–45]. Furthermore, it was observed (Table 2) that all cell
surfaces were predominantly electron donors (higher values of γ−), with low
electron acceptor parameters (γ+). This polar character can be due to the
presence of residual water of hydration or polar groups [46]. However, the high
value of the electron acceptor parameter of strain IE214 can justify its
highest number of cells adhered to both materials by increasing the
interactions between the electron-donor groups of the substrata and the
electron-acceptor groups of cells. These slightly higher values of surface
tension parameters comparing to the other strains can also indicate that
acid-base interactions between cells of this strain are more favoured than in the
other S. epidermidis strains studied.
Therefore, when the first S. epidermidis IE214 cells approach the surface, they have good conditions to adhere to it, but
as long as more cells approximate, they tend to adhere to another close cell
instead to the surface itself. This leads to the formation of prominent cell
aggregates that can be seen in Figure 2, which were still firmly adhered to the
surface. The polysaccharide intercellular adhesin (PIA), a polymer of N-acetyl
glucosamine [25] synthesised by enzymes encoded by the ica
operon [47], is crucial to the cell-to-cell adhesion process and biofilm
accumulation [48, 49]. According
to the hemagglutination ability [37], which reflects the level of PIA expression, S. epidermidis IE214 is a strong
producer of PIA (hemagglutination titer of 1:16). Therefore, the high levels of
PIA production by S. epidermidis IE214, along with its physicochemical properties,
namely, surface tension, aid
to support the unique behaviour of S.
epidermidis IE214, compared to the remaining strains. These specific
physicochemical properties are most probably due to IE214 being the only strain
capable of producing a 148 kDa cell wall protein (AtlE) (data not shown), which
has been described as determinant in the adhesion to unmodified polymer
surfaces [40].In addition to hydrophobicity
and surface tension parameters, the material surface roughness is another
factor that has been pointed out as capable of influencing bacterial adhesion
to a given material [50]. This is probably due to the
fact that rough surfaces have greater surface areas and that depressions in the
roughened surfaces provide more favourable sites for colonisation [51].
In fact, according to van Hoogmoed et
al. [52], there is a microorganism’s preference for adherence to
scratches or grooves, which could be seen in Figure 2(b)-G. The AFM results obtained showed that the average
roughness is higher for silicone than for acrylic. However, it is difficult to
ascertain the possible effect of this parameter in cocci adhesion, since for
both surfaces the roughness is at a nanoscale, meaning that there are no
microcrevices in the surfaces to act as niches for the microbial cells. Nevertheless,
according to Katainen et al. [53],
while in particles smaller than the surface features the interaction is limited
to one contact between the particle and a single asperity, being the strength
of adhesion determined by this only contact, particles larger than the surfaces
features (which is the present case for silicone) have several contacts with
the surface. This thus allows a higher level of interaction leading to a major
influence on the adhesion phenomenon.Therefore, a
stable bacterial adhesion to a biomaterial requires a high degree of
hydrophobicity, as well as a certain degree of roughness between other
physicochemical properties of the substratum [10]. Silicone is widely used as a biomaterial but it
has the disadvantages of being more hydrophobic and rougher than acrylic, thus
becoming a more prone material to S.
epidermidis adherence.


## 5. Conclusions

Bacterial adhesion to
the less hydrophobic material (acrylic) was significantly lower than to the
more hydrophobic (silicone). These results showed the importance of the
hydrophobic effect of the biomaterial surface in initial adhesion. The higher roughness of silicone seems also to exert some
effect in bacterial adhesion. On the other hand, bacteria surface physicochemical
properties seem to have less effect in their binding to substrata, highlighting
the importance of other cell surface factors to the initial adhesion process.
Nevertheless,S. epidermidis strain
IE214 revealed completely distinct adherence behaviour compared to the remaining
strains, probably as a consequence of its unique surface features, as displayed
by its flocculence ability.



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*Source: 718017-2009-01-25.xml*