Efficacy of silver-releasing rubber for the prevention of Pseudomonas
aeruginosa biofilm formation in water
KRISTOF DE PRIJCK, HANS NELIS & TOM COENYE
Laboratorium voor Farmaceutische Microbiologie, Universiteit Gent, Gent, Belgium
10
Abstract
The aim of the present study was to evaluate the efficacy of silver-releasing rubber in preventing
Pseudomonas aeruginosa biofilm formation in water. Biofilm formation by P. aeruginosa under
various conditions in an in vitro model system were compared for silver-releasing and conventional
rubber. Under most conditions tested, the numbers of sessile cells attached to the silver-releasing
rubber were considerably lower with reference to conventional rubber, although the effect
diminished with increasing volumes. The release of silver also resulted in a decrease in planktonic
cells. By exposing both materials simultaneously to conditions for biofilm growth, it became
obvious that the antibiofilm effect is due to a reduction in the number of planktonic cells, rather
than to contact-dependent killing of sessile cells. Our data demonstrate that the use of silver-
20
releasing rubber reduces P. aeruginosa biofilm in water and reduces the number of planktonic cells
present in the surrounding solution.
Key words : Biofilm, silver, Pseudomonas aeruginosa, disinfection
Running title : Inhibition of P. aeruginosa biofilm formation by silver
Correspondence : Tom Coenye, Laboratorium voor Farmaceutische Microbiologie, Universiteit
Gent, Harelbekestraat 72, 9000 Gent, Belgium. E-mail : Tom.Coenye@UGent.be
30
-1-
Introduction
Biofilms are consortia of micro-organisms that are formed on various surfaces, including industrial
surfaces and various household surfaces. Biofilm formation is a multi-stage process in which
microbial cells adhere to the surface, and the subsequent production of an exopolysaccharide matrix
results in a more irreversible attachment (Donlan, 2001 ; Donlan & Costerton, 2002 ; Hall-Stoodley et
al. 2004). Depending on the conditions, biofilms can further develop into complex and differentiated
40
communities. Biofilms can be a serious threat to human health, as sessile cells are often extremely
resistant to antimicrobial treatment and biofilms are known to be involved in indwelling medical
device-related infections, endocarditis, otitis media, periodontitis and various airway infections
(Lewis, 2001 ; Donlan, 2001 ; Donlan & Costerton, 2002 ; Fux et al. 2005). In addition, the impact of
biofilms on industrial processes can hardly be overestimated as losses caused by biofilm formation
during food processing, in drinking water distribution systems and in various industries are
significant (Wong, 1996 ; Cloete et al. 1998 ; Szewzyk et al. 2000 ; Fleming et al. 2002).
Pseudomonas aeruginosa is an important human pathogen, causing a wide range of diseases,
including potentially life-threatening pulmonary infections in cystic fibrosis patients (Govan &
Deretic, 1996). Recent data have shown that P. aeruginosa can form biofilms on many different
50
surfaces, including whirlpools, waterlines in dental units and various parts of drinking water
systems (see for example Price & Ahearn, 1988 ; Barbeau et al. 1996 ; Chaidez & Gerba, 2004 ;
Kohnen et al. 2005). Its presence can be associated with complaints about taste, odour and turbidity
and may pose a threat to the health of susceptible individuals (WHO, 2004).
Silver (Ag) has a long history of use as antimicrobial agent, and is currently still used for the
prophylaxis of conjunctivitis of the newborn and the topical treatment of burn wounds (Weber &
Rutala, 2001). Other medical applications include the use of silver impregnated urinary or vascular
catheters and bandages for trauma and diabetic wounds (Weber & Rutala, 2001 ; Silver, 2003). The
non-medical applications of silver include its use in textile products (sleeping bags, sport socks),
paint and as a disinfectant for particular water systems (e.g. hospital water systems) (Rogers et al.
60
1995 ; Weber & Rutala, 2001 ; Silver, 2003). Silver-containing disinfectants for surface
decontamination have also been described (Brady et al. 2003 ; Surdeau et al. 2006). The exact
mechanism of action of silver is not known, but it is thought that, due to its reactive nature, silver
will interact with thio, amino, imidazole, carboxylate, and phosphate groups in biological
molecules. The binding of silver ions to bacterial DNA results in DNA condensation and blocking
of replication (Feng et al. 2000). In addition, silver can interact with proteins involved in cellular
oxidation processes as well as the respiratory chain (Feng et al. 2000 ; Weber & Rutala, 2001).
-2-
More recently, it was demonstrated that silver ions can interact with the bacterial ribosome and may
exhibit their bactericidal action by inhibiting the production of essential proteins (Yamanaka et al.
2005). Chaw et al. (2005) proposed that silver can also exert a specific antibiofilm effect by
70
destabilising the biofilm matrix. This was shown for Staphylococcus epidermidis biofilms and was
thought to be the result of binding to electron donor groups of biological molecules, leading to
reduction in the number of binding sites for H-bonds and electrostatic and hydrophobic interactions.
There are numerous studies in which silver-impregnated or –coated medical materials have been
compared with their native counterparts in terms of preventing device-related infections, often with
apparently conflicting results. For example, a meta-analysis on the use of silver-impregnated central
venous catheters revealed that their use results in significant reduction of risk of bloodstream
infection (Weber & Rutala, 2001) while a large-scale study (1309 patients) failed to demonstrate
efficacy of a silver-oxide coated urinary catheter in prevention of catheter-associated bacteriuria
(Riley et al. 1995). Studies with P. aeruginosa and silver-coated or silver-releasing devices showed
80
that the use of modified materials often resulted in reduced P. aeruginosa biofilm formation
(Liedberg et al. 1990 ; Stickler et al. 1996 ; Kumon et al. 2001 ; Balazs et al. 2004), although in
some cases a lack of efficacy in preventing P. aeruginosa biofilm formation was noted as well
(Biedlingmaier et al. 1998 ; Kampf et al. 1998 ; Berry et al. 2000 ; Jarret et al. 2002).
The goal of the present study was to investigate whether the incorporation of a silver-releasing
compound in rubber would result in reduced P. aeruginosa biofilm formation on this material.
Materials and methods
90
Surfaces, strains and growth conditions
The surface tested (provided by Milliken Europe, Gent, Belgium) was a heat-cured rubber
containing a zirconium phosphate-based ceramic ion-exchange resin. The latter is responsible for
the release of silver ions in exchange for monovalent cations like K+ and Na+. Rubber without the
silver-loaded ion exchange resin was used as a control surface. Disks were cilinder-shaped and were
6.8 mm in diameter and 2.6 mm in height (total surface area appr. 120 mm²). The test strain used
was Pseudomonas aeruginosa ATCC 9027 (a clinical isolate recovered from an ear infection and
used in many standardised susceptibility tests). Other tests strains included in this study were P.
aeruginosa ATCC 27853 (a clinical isolate recovered from blood), ATCC 15442 (isolated from an
animal room water bottle), CIP A22 (isolated from a wound) and reference strain PAO-1. Strains
-3-
100
were routinely cultured aerobically on Tryptic Soy Agar (TSA) (Oxoid, Drongen, Belgium) at
37°C, unless otherwise mentioned.
Determination of the minimal inhibitory concentration (MIC) of silver
MIC determinations were carried out in modified asparagine broth (MAB), containing 3% (w/v)
DL-asparagine, 0.1% (w/v) KH2PO4 and 0.05% MgSO4.7H2O (pH 6.9 – 7.2), by making serial
dilutions of a silver nitrate (AgNO3) solution in the wells of a round-bottom 96-well microtiter plate
(TPP, Trasadingen, Switzerland) and adding 100 µl of a standardised cell suspension. The final Ag+
concentrations ranged from 0.00128 µg ml-1 to 128 µg ml-1. Following 24h incubation at 37°C,
growth was assessed by determining the absorbance at 690 nm using a Wallac Victor2 (PerkinElmer
110
Life And Analytical Sciences, Waltham, MA, USA) microtiter plate reader.
Biofilm formation on silver-releasing and conventional rubber in microtiterplates
Biofilms were formed on disks in 24-well microtiter plates (TPP) (for 700 µl and 2 ml volumes) or
in Petri dishes (for 5 ml and 10 ml volumes). Disks were placed in P. aeruginosa suspensions and
biofilms were allowed to be formed. During biofilm formation at room temperature the microtiter
plate or petri dish was placed in a humidified environment (to minimise evaporation) on a rotary
shaker (300 rpm) (Titramax 1000, Heidolph, Nurnberg, Germany). Unless otherwise mentioned, an
inoculum density of 106 CFU ml-1 was used. Biofilm experiments were carried out in MAB and in
milliQ water. In order to determine the efficacy of silver-releasing rubber in preventing biofilm
120
formation in commercial waters, we also tested five different commercial waters (labelled A
through E).
Enumeration of planktonic and sessile cells
After biofilm formation, each disk was transferred to 10 ml 0.9% (v/w) NaCl. Tubes were subjected
three times to 30 s of sonication (Branson 3510, 42 kHz, 100 W, Branson Ultrasonics Corp.) and 30
s of vortex mixing to remove the biofilm cells from the disks. From these suspensions serial tenfold
dilutions were made and the number of CFU per disk was calculated by plating on TSA and
counting colonies on the plates following incubation. For the enumeration of planktonic cells,
supernatant was diluted 100-fold in Tryptic Soy Broth (TSB) (Oxoid, Drongen, Belgium). From
130
this suspension serial tenfold dilutions were made and the number of CFU per ml was calculated by
plating on TSA and counting colonies on the plates following incubation. Parallel enumerations
using membrane filtration (0.22 µm pore diameter) (Microfil, Millipore Corp., Bedford, CT, USA)
-4-
showed that the supernatant was diluted enough for the silver not to interfere with the actual
enumeration.
Determination of the amount of silver released
The amount of silver released was determined by Inductively Coupled Plasma-Optical Emission
Spectrometry (ICP-OES) using a Vista-MPX ICP-OES (Varian, Palo Alto, CA, USA). Prior to the
analysis the samples were filtered by filtration (0.22 µm pore diameter) (Microfil) to remove
140
microbial cells. We made several attempts to quantify the amounts of silver released using ICPOES but we consistently observed that the levels of silver were below the detection limit (appr. 1
µg l-1) of the assay. We assume that this was due to the fact that the silver is bound to cellular
components (e.g. proteins) : as the microbial cells had to be removed from the supernatant prior to
the analysis, an accurate quantification of the silver was not possible.
Interpretation of the data and statistical analysis
We used the standard deviation of a measurement across repetitions to indicate repeatability and
based on this repeatability standard deviation, the coefficient of variation was calculated (Pitts et al.
2001). Currently no standardised tests or guidelines are available to test the efficacy of
150
antimicrobial surfaces under the conditions used in the present study and for that reason we defined
efficient treatments as those resulting in at least 99.9% reduction (3 log) of P. aeruginosa
microorganisms compared to the control. Whenever appropriate, statistical tests were performed
using the SPSS 12.0 software package (SPPS).
Results
Minimal inhibitory concentration of silver against P. aeruginosa
The MIC of silver for P. aeruginosa ATCC 9027 was determined using a broth microdilution
160
method. In order to determine the potential effect of the presence of rubber on the MIC, MICs were
determined in the absence and presence of a conventional rubber disk. As can be seen from Fig. 1,
the MIC was appr. 0.26 µg Ag+ ml-1. MICs were appr. the same in MAB and water (data not
shown). In addition, the presence of a rubber disk had no meaningful influence on the MIC,
indicating that it did not interfere with the antimicrobial effect of silver. In order to determine
whether the susceptibility of P. aeruginosa ATCC 9027 towards silver was representative for the
susceptibility of other P. aeruginosa strains, MICs were also determined for P. aeruginosa ATCC
-5-
27853, ATCC 15442, CIP A22 and PAO-1, and were found to be in the same range as for ATCC
9027 (data not shown). These MIC values are also in agreement with previously determined values
(Berger et al. 1976). [INSERT FIGURE 1 HERE]
170
Repeatability assessment of biofilm formation
As expected, P. aeruginosa readily formed biofilms on rubber disks immersed in different growth
media, including TSB, MAB and water. In order to determine the reproducibility of our assay,
biofilms were formed on conventional and silver-releasing disks (belonging to two different
production batches) by three different operators, on multiple occasions. Experiments were carried
out in 700 µl MAB and the bacterial biomass was quantified following 24 h of incubation (Table I).
For rubber disks, the repeatability standard deviations were 0.20 CFU per disk for sessile cells and
0.21 CFU per ml for planktonic cells. Slightly higher values (0.23 CFU per disk for sessile cells and
0.50 CFU per ml for planktonic cells) were obtained for silver-releasing disks. Repeatability
180
standard deviations for observed log reductions were also low (0.30 CFU per disk for sessile cells
and 0.54 CFU per ml for planktonic cells). [INSERT TABLE I HERE]
Effect of silver incorporation on P. aeruginosa biofilm formation
In order to determine the effect of the release of silver ions on the numbers of sessile and planktonic
cells, biofilms were formed on conventional and silver-releasing rubber under various conditions.
An overview of the results is shown in Tables II and III. When using MAB as growth medium there
was considerably less biofilm formation (at least 99.9% reduction) on the silver-releasing disks at
all time points sampled for the 700 µl, 2 ml and 5 ml experiments (Table II). [INSERT TABLE Ii
HERE] The silver released from the disks drastically reduced the numbers of planktonic cells as
190
well. When a larger volume was tested (10 ml) the efficacy dropped, especially with longer
incubation periods (> 24 h). When water was used as growth medium, similar results were obtained,
with prolonged incubation (72 hours and more) of disks in a small (up to 5 ml) volume even
resulting in near-sterility of the disk and/or the surrounding solution (Table III). [INSERT TABLE
III HERE] All experiments were carried out with rather high inocula (appr. 106 CFU ml-1) and using
MAB as growth medium, higher reductions were obtained with more dilute inocula (105, 104, 103
or 102 CFU ml-1) than with the standard inoculum (Fig. 2). [INSERT FIGURE 2 HERE] The use of
silver-releasing disks led to sterile or near-sterile surfaces and solutions as neither sessile nor
planktonic cells could be recovered. When lower inocula (≤ 105 CFU ml-1) were used with water as
growth medium, similar reductions were observed as with the standard inoculum (106 CFU ml-1)
-6-
200
(Fig. 2). With these inocula, neither sessile nor planktonic cells could be recovered, indicating
sterile (or near-sterile) surfaces and solutions.
Mechanism of the antibiofilm effect
In order to determine which mechanism underlies the antibiofilm effect of silver-releasing rubber
we determined the effect of simultaneous incubation of two disks in the same well of a microtiter
plate. As can be seen from Table IV, the presence of a silver-releasing disk also resulted in reduced
biofilm formation on the conventional rubber disk, suggesting that surface contact-dependent killing
is not the mechanism by which the silver exerts its action. This is confirmed by the consistently
observed reductions in planktonic cell numbers and by the decreasing efficacy in higher volumes
210
(Tables II and III). [INSERT TABLE IV HERE]
Application of silver-releasing rubber in commercial waters
We determined whether silver-releasing rubber could be used to control biofilm formation in water.
For this test we used five different commercial waters available on the Belgian market, which
varied significantly in their composition (Table V). As can be seen from Fig. 3, the presence of a
silver-releasing disk resulted in a marked decrease in numbers of both sessile and planktonic cells
when commercial waters were used as growth media. [INSERT TABLE V HERE] [INSERT
FIGURE 3 HERE]
220
Discussion
The aim of the present study was to evaluate the efficacy of silver-releasing rubber in preventing P.
aeruginosa biofilm formation in various conditions, using a microtiter plate model system. The
results from our experiments clearly show that our biofilm model system can provide repeatable
assays of the efficacy of silver-releasing rubber disks against P. aeruginosa biofilms, as relatively
small repeatability standard deviations were obtained for log reduction measurements (Table I)
which are within the range of standard deviations observed for standard suspension and surface
disinfection assays (0.2 – 1.2) (Tilt & Hamilton 1999). It was also obvious that the use of silver230
releasing rubber results in a marked decrease in biofilm formation on the surface, as well as in a
marked reduction in the number of planktonic cells in the surrounding suspension, in a volume-,
time- and inoculum-dependent way (Table II, Fig. 2).
-7-
There are two possible mechanisms of action that could explain the observed reductions in
cell numbers. A first possible mode of action is “contact-dependent killing”, in which silver ions
continuously released from the surface kill bacterial cells adhering to this surface. A second
possible mechanism is that the number of sessile cells is reduced because the release of silver ions
into the culture medium reduces the number of planktonic cells. We tried to determine this
mechanism by determining the effect of simultaneous incubation of two disks. The rationale behind
these experiments is that when the antibiofilm effect would arise from contact-dependent killing,
240
there would be a significant difference in biofilm biomass on a conventional and a rubber-releasing
disk that have been incubated together. However, if the killing of planktonic cells is the underlying
reason for the antibiofilm effect, no meaningful differences should be observed between the
biomass on both disks, and our data (Table IV) strongly suggest that the latter is the case.
We also tested the applicability of silver-releasing rubber in commercial waters with
different compositions and our data clearly indicated that its use also results in a marked decrease in
biofilm build-up under those conditions (Fig. 3). In addition, there appeared to be no correlation
between the chemical composition of the waters tested and the observed reduction in sessile and
planktonic cells, indicating that inorganic ions (including chloride) and/or other compounds present
in these waters did not interfere with the antimicrobial activity of the released silver ions.
250
In conclusion, our study presents an evaluation of the effect of a silver-releasing rubber in a
microtiter plate model system against P. aeruginosa biofilms and the data clearly demonstrate that,
compared to conventional rubber, the use of the silver-releasing rubber prevents the build-up of
these biofilm in water, under various conditions. We also showed that the antibiofilm effect is due
to a reduction in the number of planktonic cells, rather than to a contact-dependent killing.
Together, the present study indicates that silver-releasing rubber may be applicable in various
situations where surfaces come into contact with small volumes of water (e.g. tubing, valves and
faucets of water dispensers) to reduce or even prevent biofilm formation. As such its use may be a
valuable addition to existing cleaning/disinfection procedures.
260
Acknowledgements
This work was funded by a grant of the Research Foundation – Flanders (to TC) and by Milliken
Europe. We thank Ine Patou, Gudrun Lafaut and Gloria Wullepit for excellent technical assistance.
-8-
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Table I. Repeatability assessment of biofilm formation on conventional and silver-releasing rubber
disks. Results are expressed in log CFU per disk (average±standard deviation) (for sessile cells), log
CFU per ml (average±standard deviation) (for planktonic cells) or % (coefficient of variation). n = 3
for each operator. Conditions : 700 µl MAB, 24 hours of incubation.
Conventional rubber
Sessile
Planktonic
cells
cells
Silver-releasing rubber
Sessile
Planktonic
cells
cells
Batch A
Operator I
Operator II
Operator III
8.35±0.14
8.44±0.07
8.44±0.33
9.02±0.11
8.70±0.07
9.34±0.19
3.35±0.09
3.39±0.26
3.36±0.18
3.52±0.13
3.02±0.34
3.49±0.20
Batch B
Operator I
Operator II
Operator III
7.85±0.07
7.85±0.07
7.95±0.14
8.84±0.18
8.90±0.14
8.89±0.18
2.94±0.11
3.04±0.16
2.85±0.16
3.53±0.34
3.80±0.31
3.58±0.74
Biomass
Average
Repeatability SD
Coefficient of variation
8.15
0.20
2.5
8.95
0.21
2.4
3.16
0.23
7.3
3.49
0.50
14.3
Log reduction
Average
Repeatability SD
Coefficient of variation
-
-
4.99
0.30
6.1
5.46
0.54
9.9
- 12 -
Table II. Effect of silver-releasing rubber on P. aeruginosa ATCC 9027 biofilm growth and planktonic growth using MAB as growth medium.
Log average number of CFU per disk (for sessile cells) or log CFU per ml (for planktonic cells) ± standard deviation are shown.
Volume
(ml)
Incubation
n
time (hours)
0.7
0.7
2.0
2.0
2.0
5.0
5.0
10.0
10.0
24
48
24
48
148
24
48
24
48
46
10
16
4
4
3
5
3
4
Conventional rubber
Sessile
Planktonic
cells
cells
8.05±0.47
8.75±0.27
8.21±0.22
8.39±0.08
8.58±0.40
9.16±0.26
8.04±0.12
8.32±0.16
8.19±0.24
9.26±0.23
7.29±0.16
7.02±0.18
8.10±0.15
8.38±0.35
6.96±0.44
6.94±0.27
8.25±0.12
8.33±0.09
- 13 -
Silver-releasing rubber
Sessile
Planktonic
cells
cells
4.17±0.83
4.35±1.14
3.77±1.43
3.93±0.36
4.82±0.71
5.26±0.42
3.12±0.30
3.01±0.11
2.31±0.21
3.41±0.17
3.06±0.17
2.85±0.22
3.12±0.49
2.31±0.25
4.80±0.38
3.96±0.31
7.05±0.99
5.66±1.60
Log reduction
Sessile Planktonic
cells cells
3.88 4.40
4.44 4.46
3.76 3.91
4.92 5.31
5.88 5.85
4.23 4.17
4.98 6.07
2.16 2.98
1.20 2.67
Table III. Effect of silver-releasing rubber on P. aeruginosa ATCC 9027 biofilm growth and planktonic growth using water as growth medium.
Log average number of CFU per disk (for sessile cells) or log CFU per ml (for planktonic cells) ± standard deviation are shown.
Volume
(ml)
Incubation
n
time (hours)
0.7
0.7
0.7
0.7
5.0
5.0
10.0
10.0
24
48
72
96
24
48
24
48
10
10
3
3
3
5
3
4
Conventional rubber
Sessile
Planktonic
cells
cells
5.72±0.62
6.65±0.16
5.82±0.42
4.84±1.56
5.57±0.08
7.17±0.67
5.44±0.09
4.89±0.56
6.19±0.34
3.87±0.12
6.03±0.27
3.92±0.17
6.09±0.28
3.69±0.63
6.13±0.24
3.79±0.46
- 14 -
Silver-releasing rubber
Sessile
Planktonic
cells
cells
2.38±0.34
2.26±0.60
3.59±0.29
2.52±1.57
<1
<1
<1
<1
3.07±0.10
<1
3.82±0.02
<1
3.45±1.11
<1
4.77±0.21
1.65±0.49
Log reduction
Sessile Planktonic
cells cells
3.34 4.39
2.23 2.32
≥4.57 ≥6.17
≥4.44 ≥3.89
3.12 ≥2.87
2.21 ≥2.92
2.64 ≥2.69
1.36 2.14
Table IV. Effect of combined incubation of two disks on biofilm and planktonic growth. Log average number of CFU per disk (for sessile cells)
or log CFU per ml (for planktonic cells) ± standard deviation are shown (n = 12). Conditions : 700 µl MAB, 24 hours of incubation. NA, not
applicable
Conventional + silver-releasing rubber disk
Two conventional rubber disks
Two –silver-releasing rubber disks
- 15 -
Sessile cells on
Conventional Silver-releasing
rubber
rubber
Planktonic
cells
5.74±0.19
8.22±0.08
NA
6.50±0.28
9.29±0.09
<1
5.54±0.33
NA
<1
Table V. Composition of the commercial waters tested. Concentrations are expressed as mg l-1 and were provided by the producers. -, not
mentioned on label.
Dry rest at 180°C
Ca2+
Na+
K+
NO3Mg2+
HCO3ClSO42-
A
B
C
D
E
208
70
2
4
2
210
-
164
86
21
1.6
78
312
144
-
2513
549
14
4
4.3
119
384
11
1530
33
5
1
2
1
15
5
4
39
69
6
25
287
63
29
- 16 -
Figure 1. Determination of the minimal inhibitory concentration of silver for P. aeruginosa ATCC
9027 in the presence (squares) or absence (triangles) of a conventional rubber disk.
- 17 -
Figure 2. Influence of the inoculum density on the reduction of P. aeruginosa ATCC 9027 sessile
(grey) and planktonic (open) cell numbers in MAB and milliQ water in the presence of a silverreleasing disk with reference to a conventional rubber disk (700 µl volume, 24 hours of incubation).
Reductions are shown as log average number of CFU per disk (for sessile cells) or log CFU per ml
(for planktonic cells) (± standard deviation).
- 18 -
Figure 3. Reduction of P. aeruginosa ATCC 9027 sessile (grey) and planktonic (open) cell numbers
in five commercial waters (A – E) in the presence of a silver-releasing disk with reference to a
conventional rubber disk (700 µl volume, 24 hours of incubation). Reductions are shown as log
average number of CFU per disk (for sessile cells) or log CFU per ml (for planktonic cells) (±
standard deviation).
- 19 -