Efficacy of silver/hydrophilic poly(p-xylylene) on preventing bacterial growth and

biofilm formation in urinary catheters

Hamideh Heidari Zare, Viktorija Juhart, Attila Vass, and Gerhard FranzDieter Jocham

Citation: Biointerphases 12, 011001 (2017); doi: 10.1116/1.4974197

View online: http://dx.doi.org/10.1116/1.4974197
View Table of Contents: http://avs.scitation.org/toc/bip/12/1
Published by the American Vacuum Society
Efficacy of silver/hydrophilic poly(p-xylylene) on preventing bacterial growth
and biofilm formation in urinary catheters
Hamideh Heidari Zare,a) Viktorija Juhart, Attila Vass, and Gerhard Franz
Laboratory for Surface Refinement and Thin Film Technology, Munich University of Applied Sciences,
Munich D-80335, Bavaria, Germany
Dieter Jocham
University Hospital of the State of Schleswig-Holstein at L€
ubeck, L€
ubeck D-23538, Schleswig-Holstein,
Germany
(Received 4 October 2016; accepted 5 January 2017; published 18 January 2017)
Catheter associated urinary tract infections (CAUTI), caused by several strains of bacteria, are a
common complication for catheterized patients. This may eventually lead to a blockage of the
catheter due to the formation of a crystalline or amorphous biofilm. Inhibiting bacteria should
result in a longer application time free of complaints. This issue has been investigated using an
innovative type of silver-coated catheter with a semipermeable cap layer to prevent CAUTI. In this
work, two different types of silver catheters were investigated, both of which were capped with
poly(p-xylylene) (PPX-N) and exhibited different surface properties that completely changed their
wetting conduct with water. The contact angle of conventionally deposited PPX-N is approximately
80 . After O2 plasma treatment, the contact angle drops to approximately 30 . These two systems,
Ag/PPX-N and Ag/PPX-N-O2, were tested in synthetic urine at a body temperature of 37  C. First,
the optical density and the inhibition zones of both bacteria strains (Escherichia coli and
Staphylococcus cohnii) were examined to confirm the antibacterial effect of these silver-coated
catheters. Afterward, the efficacy of silver catheters with different treatments of biofilm formed by
E. coli and S. cohnii were tested with crystal violet staining assays. To estimate the life cycles of
silver/PPX-catheters, the eluted amount of silver was assessed at several time intervals by anodic
stripping voltammetry. The silver catheter with hydrophilic PPX-N coating limited bacterial growth
in synthetic urine and prevented biofilm formation. The authors attribute the enhanced bacterio-
static effect to increased silver ion release detected under these conditions. With this extensive pre-
paratory analytic work, the authors studied the ability of the two different cap layers (without
silver), PPX-N and oxygen plasma treated PPX-N, to control the growth of a crystalline biofilm by
measuring the concentrations of the Ca2þ and Mg2þ ions after exposure of the catheters to saturated
urine for 24 h. The higher concentrations of Ca2þ and Mg2þ in the precipitates on the PPX-N cathe-
ters indicates that the hydrophilic PPX-N coating is superior to the simple PPX-N coating, with
regard to the formation of a crystalline biofilm. Moreover, hydrophilic PPX-N as a cap layer may
promote wettability and increase silver ion release rate and thus reduce the adhesion of suspended
crystals to the catheter. Reduced bacterial growth and reduced adhesion may help to prevent
CAUTI. V C 2017 Author(s). All article content, except where otherwise noted, is licensed under a

Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

[http://dx.doi.org/10.1116/1.4974197]

I. INTRODUCTION (CAUTI) is Escherichia coli. Other common organisms are

With a share of 24%, urinary tract infections represent the
most common nosocomial infection in hospital and home-
care settings. Of these infections, 70%–80% are associated
with use of an indwelling urethral catheter.1 Indwelling uri-
nary catheterization is grouped into short-term (<7 days)
and long-term applications (>28–30 days). Approximately
10%–30% of patients undergoing short-term catheterization
and virtually all patients with long-term catheterization
develop catheter-associated bacteriuria.2,3 In cases involving
short-term catheterization, the most common infecting
organism of catheter associated urinary tract infection
a)
Electronic mail: h-zare@gmx.de; www.gerhard-franz.org
Enterococci, Pseudomonas, Enterobacter, Staphylococcus
aureus or epidermidis, Klebsiella, Staphylococcus cohnii
ssp. urealyticus, and Serratia.4,5 In the long-term catheteri-
zation setting, common uropathogens include E. coli,
Pseudomonas aeruginosa, and Proteus mirabilis.4
CAUTI often leads to the development of a biofilm on the
long-term indwelling urethral catheter, which can shield the
bacteria from antibiotics and promote further bacterial adhe-
sion. The urease which is produced by bacteria reacts with
urea to form ammonia and carbon dioxide. The resulting pH
increase leads to precipitation of calcium or magnesium
phosphate crystals on the catheter. Inorganic salts are colo-
nized by bacteria, which may even block the catheter
lumen.6 To prevent morbidity and mortality associated with

011001-1 Biointerphases 12(1), March 2017 1934-8630/2017/12(1)/011001/10 C Author(s) 2017.

V 011001-1
011001-2 Heidari Zare et al.: Efficacy of silver/hydrophilic PPX-N
infection of urinary catheters, we therefore need to develop
methods to inhibit bacterial growth on catheters.7 Numerous
groups have tested the efficacy of bactericidal or bacterio-
static coatings with the antibiotics ciprofloxacin, gentamicin,
norfloxacin, and nitrofural,8,9 as well as silver oxide, tita-
nium oxide, or combinations thereof.10–12
Other attempts have involved antimicrobial peptides con-
jugated to copolymer brushes,13 silver-silicone-hydrogels,14
poly(vinyl-pyrrolidone)-coating,15 and heparin16 to reduce
bacterial growth and adhesion to the catheter surface in vitro.
However, not all methods were effective. Due to the develop-
ment of resistance against antibiotics, the high cost of deliv-
ery or synthesis, and potential side effects, antibiotics should
be avoided for surface modification of biomaterials.17 For the
near future, this is one of the most challenging concerns. In
recent years, silver nanoparticles at low concentrations have
been successfully used in biomedical instruments due to their
unique physicochemical properties such as nanometric sizes,
high surface area, and high reactivity. Thanks to its oligody-
namic activity, silver can damage bacterial cell walls and
membranes and inhibit bacterial genome replication of a
broad spectrum of bacteria and fungi. We therefore coated
urethral catheters with synthetic silver nanoparticles. To
improve adhesion of silver nanoparticles to the surface, cathe-
ters were coated with poly(p-xylylene) or parylene (PPX-N),
a biocompatible polymer.18 In a previous work, we have
already optimized the silver thickness to compare PPX-N and
silver particle morphology and tested minimum inhibitory
concentration of silver ions in synthetic urine against E. coli
and S. cohnii.19 The aim of this study is to evaluate the anti-
bacterial efficacy of silver/PPX catheters in a simulated body
fluid (synthetic urine), including the power to prevent genera-
tion of a biofilm. The antibacterial activity of the catheters
was studied with quantitative (growth curve) and qualitative
methods (inhibition zone) against E. coli and S. cohnii. The
adhesion of bacteria to treated surfaces was studied through
crystal violet staining assays, while the growth of bacteria
was evaluated by optical density (OD). Afterward, with the
aim of preventing crystalline biofilm formation, the concen-
trations of Ca2þ and Mg2þ were measured on top of catheters
capped by a layer PPX-N and modified PPX-N, respectively.
The most recent results obtained with inductively coupled
plasma optical emission spectroscopy (ICP-OES) proved the
constant release rate of silver through very thin cap layers of
PPX-N. However, ICP-OES cannot discriminate between sil-
ver nanoparticles and Agþ ions and measures the sum of both
of them. As Behra et al. have noted, the activity of silver
nanoparticles is far lower than the impact of isolated Agþ
ions.20 To distinguish between Ag nanoparticles and Agþ
ions, we used anodic stripping voltammetry (ASV).
II. MATERIALS AND METHODS
A. Materials
Silicone catheters [16 Ch./Fr. ( ¼ 5:3 mm), 18 Ch./Fr.
( ¼ 6 mm)] were obtained from Urokink Industries AG
(Halberstadt, Germany). E. coli K12 and S. cohnii subsp.
Biointerphases, Vol. 12, No. 1, March 2017
011001-2
urealyticus strain CK27 were selected as representatives of
Gram-negative and Gram-positive bacteria, respectively. The
bacterial strains were obtained as freeze-dried cultures from the
Leibniz Institute DSMZ-German Collection of Microorganisms
and Cell Cultures (Braunschweig, Germany) and were grown
in Mueller-Hinton growth medium (Carl Roth, Karlsruhe,
Germany). The organic and inorganic components, silver
nitrate solution 0.1 mol/l 6 0.2%, and silver ICP standard solu-
tion 10 000 mg/l 6 0.2 were purchased from Carl Roth
(Karlsruhe, Germany). Perchloric acid 70% (HClO4) and 0.1 M
KNO3 (Merck, Darmstadt, Germany) were also used. The syn-
thetic urine (AU) medium was prepared from inorganic constit-
uents (sodium chloride 5.2 g/l, sodium hydrogen carbonate
2.1 g/l, sodium sulfate 3.2 g/l, ammonium chloride 1.3 g/l,
potassium dihydrogen phosphate 0.95 g/l, potassium hydrogen
phosphate 1.2 g/l, calcium chloride 0.37 g/l, magnesium sulfate
0.49 g/l, and iron sulfate 0.0012 g/l), as well as organic constitu-
ents (urea 10 g/l, uric acid 2,6,8-trihydroxypurine 0.07 g/l, cre-
atinine 0.8 g/l, peptone from casein 1.0 g/l, yeast extract
0.005 g/l, lactic acid 0.1 g/l, and citric acid 0.4 g/l). All compo-
nents were mixed, and the pH was adjusted to 6.5. Finally, the
synthetic urine was sterilized through filters with a pore size of
0.2 lm from Carl Roth (Karlsruhe, Germany).
B. Preparation of the silver/PPX-N system
The catheter surfaces were activated by immersing them in
a solution of 30% nitric acid for 30 min as described previ-
ously.19 To coat the catheters with silver, 5 ml aqueous silver
nitrate (0.1 M) was mixed with 0.05 g sodium hydroxide
resulting in a brown solid precipitate of aqueous Ag2O. By
adding 0.7 ml ammonia solution 25% (13.3 mol/l), the silver
oxide completely dissolved by forming [Ag(NH3)2]þ as the
main component of Tollens’s reagent. By adding maltose
solution (0.8 mol/l) to this solution (ratio 1:1), colloidal silver
particles were formed due to reduction of silver ions into
metallic silver. To promote silver deposition on the silicon
catheters, the reaction took place at 70  C in a water bath.
This reaction is actually performed by pumping the Tollens’s
reagent into the catheter by means of a peristaltic pump TV/
TVS (medorex e.K., N€orten-Hardenberg, Germany) as
described previously.19 As the final step, the catheter was
immersed in a water bath (70  C) for 5 min. After the deposi-
tion process, the silver coating solution was pumped out, and
the catheters were washed three times with deionized water.
Silver-coated catheters were dried overnight at room tempera-
ture. To control and prolong the rate of silver release, the sil-
ver film was coated with PPX-N, an inert and biocompatible
polymer, by means of chemical vapor deposition.21 The film-
building gas (monomeric p-xylylene) was diluted by argon to
yield a layer thickness of approximately 200 nm. The thick-
ness of the PPX film was measured by a mechanical profilom-
eter (Tencor Alpha Step 200).
C. Plasma treatment of PPX-N surfaces
After preparation of silver/PPX-N catheters, the PPX-N
cap layer was modified using capacitatively coupled plasma
011001-3 Heidari Zare et al.: Efficacy of silver/hydrophilic PPX-N
driven by a radio-frequency power supply of 13.56 MHz
(Plasmalab lP, Oxford Instruments, Yatton, UK). The silver/
PPX-N catheters were placed into a reaction chamber and
evacuated to lower than 104 Torr by means of a turbomo-
lecular pump. Specifically, the catheters were treated at a
power of 160 W and a pressure of 100 mTorr for 3 min. We
excluded reduction of the layer thickness through the plasma
treatment by measuring the layer thickness on microscope
slides before and after exposure to the O2 plasma using pro-
filometry (measured layer thickness typically before and
after plasma exposure: 900 nm 6 20 nm; for comparison,
ashing of photoresist is executed in the same reactor at
300 W microwave power and a discharge pressure of 1000
mTorr with an etch rate of 50 nm/min).
D. Characterization of PPX-N surfaces
The wetting behavior of the PPX-N surface was evaluated
by measuring the contact angle applying the drop shape anal-
ysis system from Kr€uss (DA 10 MK2, Hamburg, Germany).
Substrates were microscope slides of glass which were first
coated with PPX-N and then further processed with different
methods (untreated or O2 plasma treatment). Using the ses-
sile drop method, the static angles were obtained for water
by applying a droplet to the surface and measuring the con-
tact angle directly after treatment. All measurements were
performed at room temperature (23 6 3  C) in triplicate.
The wetting behavior (against water) can be discriminated
into these categories:22 contact angle 0  H < 90 : partial
wetting, 90  H  180 : poor to very poor wetting, and
H ¼ 0 perfect or complete wetting, no droplets can be
found.
E. Microbiological testing
The antibacterial activity of Ag/PPX system was evalu-
ated against E. coli and S. cohnii using three methods: inhi-
bition zone assay, growth curve analysis, and biofilm
formation. All catheter samples were sterilized by exposure
to UV light in a tissue culture hood (20 min).
1. Zone of inhibition test
The efficacy of the silver-treated silicon in reducing bacte-
rial viability was tested with a spot modification of standard
agar diffusion tests. The treated silicon squares (15  15 mm)
were separated into three different categories: Ag without cap
layer, Ag/PPX-N and Ag/hydrophilic PPX-N. The antibiotic
control was a single sheet of filter paper immersed with 10 ll
of penicillin–streptomycin 100 (PAA, C€olbe, Germany).
These were placed with the coated side up on a petri dish
with Mueller-Hinton agar. Then, 13 ml of 0.7% agarose in
Mueller-Hinton medium were mixed with 108 CFU/ml bacte-
ria and poured over the sample onto the agar plate.
Afterward, this was incubated for 18 h at 37  C. The zone
without bacterial growth surrounding the samples was mea-
sured to quantify the antibacterial capability, according to the
aforementioned standard diffusion test SNV 195920-1992. If
the width of the bacterial inhibition zone was longer than
Biointerphases, Vol. 12, No. 1, March 2017
011001-3
1 mm, then the sample had good antibacterial activity. If it
was shorter than 1 mm, then its quality was fairly good.
However, if the surface of the sample was free of bacterial
growth, then the sample was labeled as sufficient. If the sam-
ple was covered by bacteria, then its antibacterial activity was
evaluated as “limited.”23
2. Growth curve of bacteria toward silver-treated
catheters
The bacterial growth in synthetic urine was measured in six
replicates in 48-well plates. First, 5 mm-long pieces of treated
catheters were incubated in 1 ml of synthesized urine for 24 h
at 37  C. The samples were then removed, and 450 ll of the
supernatant (containing released silver ions) was transferred to
another sterile 48-well plate and inoculated with 103 CFU/ml
of E. coli and S. cohnii. The optical density of bacterial growth
was measured continuously at 37  C (600 nm, 218 rpm) using
a photometer (TECAN Infinite M200 PRO Nano Quant,
Tecan Trading AG, Switzerland) every 20 min for 21 h.
3. Biofilm formation by crystal violet staining assay
Biofilm formation was quantified in 48-well plates,
according to the method by Alt et al.24 The 5 mm catheters
were incubated in synthetic urine for 24 h at 37  C. Then,
270 ll of supernatant was transferred into new sterile
well plates and inoculated with 103 CFU/ml of E. coli and
S. cohnii. After 24 h of incubation at 37  C without agitation,
the bacteria formed a biofilm, the supernatant was removed,
and the wells were washed with deionized water to remove
the nonadherent cells. This procedure was repeated three
times. Adherent cells of the biofilm were stained with 1%
crystal violet for 15 min at room temperature, and unbound
dye was removed by three washes with water. Finally, the
adherent cells were dissolved with 33% acetic acid. The opti-
cal density of the solution was measured with a photometer at
595 nm and was compared to the bacteria-only controls.
F. Chemical Analysis
1. In vitro assay for inorganic incrustation of catheters
To simulate the inorganic (salt) depositions during a uri-
nary tract infection, two nonsaturated aqueous stock solu-
tions A þ B were mixed, which together form insoluble salts
(Tables I and II)27 caused by supersaturation.
Equal volumes of solutions A and B were mixed and incu-
bated in flask 1 at a water bath temperature of 40  C. This
oversaturated solution was called synthetic urine, which was
used to simulate urinary tract infection by addition of urease.
Due to the breakdown of urea into basic ammonia and CO2
by urease, the pH rose after mixing from 6.8 to 9 within 16 h.
This was accompanied by deposition of brushite (CaHPO42
H2O), hydroxylapatite carbonate, [Ca10(PO4)5CO3(OH)2],
and struvite (MgNH4PO46 H2O).
For each run, two catheters with different coatings were
put into a second reaction flask (flask 2) equilibrated in the
water bath at 40  C under identical chemical and physical
011001-4 Heidari Zare et al.: Efficacy of silver/hydrophilic PPX-N 011001-4

TABLE I. Solution A, pH: 6.5. carbon <5 ppb). For the recording of calibration curves, stan-
dard silver solutions were freshly prepared by appropriate
Component Concentration (g/l)
dilutions of a 0.1 M AgNO3 standard solution. The silver
Urease 2.9 solution was protected from daylight. During the measure-
CaCl22 H2O 1.3 ments, the electrolyte was stirred with a magnetic stirring bar
MgCl26 H2O 1.3 at constant speed.
NaCl 9.2 The measurements were initiated by checking the work-
Na2SO4 4.6 ing electrode background by recording a cyclic voltammo-
Sodium citrate 1.3
gram in the range from 150 to 500 mV at the scan rate of
KCl 3.2
100 mV/s. During the deposition step, silver ions were
reduced at a constant potential of 550 mV for 1000 s. This
conditions. The urine supply flask 1 was connected with step was followed by a stripping reaction, which consisted of
reaction flask 2. A peristaltic pump removed 60 ml/h from an oxidation of the deposited silver (see Fig. 1). The solution
resistance between the two electrodes was determined using
flask 2, leading to an equal influx of fresh synthetic urine
an AC signal (5 kHz, 5 mV) and was thereafter compensated
from flask 1. Thus, the catheter was maintained in identical
for using the analogous potentiostat feedback scheme. The
urine volume and temperature (2 l, 37  C) with constant flow
resulting effective solution resistance was less than 5 X in
of fresh synthetic urine. After 58 h of constant flux, the cath-
each experiment. The Agþ concentration was determined
eters were dried at room temperature for 24 h. Afterward, the
from the peak height obtained in the stripping-step using the
catheters were sliced into pieces of 5 cm in length, and the
calibration curve.26
deposited salt on the catheter surface was dissolved by 1 M
Four pieces of 2 cm silver/hydrophilic PPX-N catheters
HCl solution. The aqueous solutions of Ca2þ and Mg2þ [18 Ch./Fr. ( ¼ 6 mm)] were placed in 50 ml polypropyl-
could then be subjected to quantitative analysis. ene tubes and fully immersed in 10 ml of 1:10 diluted syn-
thetic urine with deionized water and incubated with mild
2. Measuring silver ion release by anode stripping stirring (120 rpm, 37  C). After 24 h, the silver concentration
voltammetry
of the supernatant was measured and the coated catheters
Anode stripping voltammetry (ASV) is a widely applied were incubated in fresh synthetic urine for two further con-
electrochemical method for the detection of metal ions25 and secutive sampling runs, which were terminated on days
was used here to quantitatively determine the released silver 8 and 22. The urine samples were analyzed for silver ion
ions. The experiments were performed at room temperature release in the following manner: 5 ml of the solution was
in a home-built Teflon cell using a computer-controlled added to 5000.50 ll of the supporting electrolyte, and ASV
potentiostat ECI 200 (Nordic Electrochemistry, Copenhagen, measurements were performed. All measurements were con-
Denmark). The auxiliary electrode was a platinum mesh and ducted under the same conditions.
the reference electrode was an Ag/AgCl/KCl system (Schott,
Mainz, Germany). All potentials in this work were communi- III. RESULTS AND DISCUSSION
cated with respect to the reference electrode used. A glassy We protected the silver coating on the catheter surface
carbon (GC) disk (5 mm diameter) was used as a working using a layer of PPX-N. As an organic polymer, PPX-N gen-
electrode. GC was polished to a mirror finish with alumina erates a hydrophobic layer with water-repellent properties,
oxide paste 0.05 lm (Buehler-Met, IL) and washed thor- which hinders the diffusion of hydrated silver ions from the
oughly with 70% HClO4 (Merck, Darmstadt, Germany) and silver depot to the aqueous solution (synthetic urine). We
deionized water. Before each measurement, the working elec- asked whether increasing the hydrophilic properties of the
trode was characterized by cyclic voltammetry at a low scan surface would increase the silver ion release rate. The hydro-
rate to check the quality of the GC-electrodes. The supporting philicity can be increased by two independent principles:
electrolyte used in all experiments was 50 ml 0.1 M KNO3
(1) enlargement of O-containing groups at the surface
adjusted to pH 3 using 50 ll 65 HNO3. All solutions were pre-
(C¼O, CH-OH, CH-O-OH) or
pared using deionized water (r < 3 lS/cm, total organic
(2) increase in the roughness.28
TABLE II. Solution A, pH: 5.6.
A. Layer characterization of PPX-N after plasma
Component Concentration (g/l) treatment
Urea 50 1. Roughness and thickness
KH2PO4 5.6
The catheters themselves are too rough to detect a rough-
Creatinine 2.2
ening or smoothening of their surface. Therefore, we tested
Sodium oxalate 0.004
NH4Cl 2.0
this question on microscope slides coated with PPX-N,
Albumin 0.005 which were further treated with O2 (Sec. II C). AFM meas-
urements showed 50 nm roughness before and after O2

Biointerphases, Vol. 12, No. 1, March 2017

011001-5 Heidari Zare et al.: Efficacy of silver/hydrophilic PPX-N 011001-5

FIG. 1. Determining the Agþ concentration with anode stripping voltammetry (ASV): step 1 (left): quality check of the WE, step 2 (middle): deposition, and
step 3 (right): stripping.

treatment, suggesting that any effects of plasma treatment surfaces (Fig. 2). The liquid drops spread out on the PPX-N
are not driven by increased roughness. surface after treatment, indicating improved wetting of the
The measured profilometric values of the layer thickness Ag/PPX coating on the catheter surface.
before and after O2 treatment were indistinguishable.
Therefore, the activation is likely restricted to the topmost C. Enhanced inhibition of bacterial growth
region of the polymeric film. of silver coating with hydrophilic PPX-N
It is evident that a presumptive increase of the antibacte- How the increased wetting affects the antibacterial prop-
rial conduct by generation of reactive oxygen species is erties of the Ag/PPX system was quantified by measuring
negligible. the inhibition zone without bacterial growth surrounding the
catheter samples in a diffusion agar assay with coated silicon
B. Increased wettability of PPX-N after plasma squares. Four test samples on silicon squares were measured:
treatment
The wettability of the PPX-N and O2-plasma treated
PPX-N on microscope slides were characterized using the
contact angle method. All the samples were tested in nine
replicates. Figure 2 shows the height contact angle of PPX-N
surfaces before and after oxygen treatment. After 3 min of
plasma exposure, the contact angle H significantly decreased
from 84.18 to 29.68 , confirming the increased hydrophilic-
ity caused by the formation of polar oxygen-containing
groups and not by the changes in roughness. Bi et al. have
shown by x-ray photoelectronic spectroscopy that O2 treat-
ment of PPX under similar exposure conditions causes the
origin of two new peaks at 287.8 and 289.3 eV in the decon-
voluted C 1s spectrum, which they attributed to the carbon
atoms in the free carbonyl group (C¼O) and carbonate group
(O2C¼O), respectively.28
This improved wettability was also confirmed for longitu- FIG. 2. Plasma treatment increased the hydrophilic properties of the PPX-N
coated microscope slides. Quantitative representation of contact angle
dinal catheter sections, although contact angle measurement
PPX-N as depicted. Photographs show isolated water droplets on top of
is not possible in this setting. Photographs provide an over- PPX-N catheters. The water droplets spread out on O2-plasma treated cathe-
view of the interior with hydrophobic and hydrophilic ters n ¼ 9, p < 0.001 (t-test).

Biointerphases, Vol. 12, No. 1, March 2017

011001-6 Heidari Zare et al.: Efficacy of silver/hydrophilic PPX-N
silver coating (Ag), silver coating with PPX-N (Ag/PPX-
N), and silver coating with O2-plasma treatment PPX-N
(Ag/hydrophilic PPX-N). As antibiotic control, we used fil-
ters immersed in penicillin–streptomycin 100 (PAA,
C€olbe, Germany). Figure 3 shows the formation of an inhi-
bition zone around these three specimens against E. coli
and S. cohnii. In Fig. 3(a), the performance against E. coli
is shown. All three samples inhibited bacterial growth to a
certain extent. Among the catheter samples silver/hydro-
philic PPX-N was best and comparable to the antibiotic
control. Against S. cohnii, only hydrophilic Ag/PPX was
sufficiently effective [quantification in Fig. 3(b)].
This inhibition of bacterial growth confirmed that suffi-
cient amounts of silver ions diffused into the agar from
coated surfaces. While all three conditions inhibited the
growth of E. coli to some extent, the quantitative evaluation
of the width of inhibition zones showed that the silver/hydro-
philic PPX-N had the largest inhibition zone toward E. coli
(5.3 6 0.18 mm) and against S. cohnii (1.35 6 0.4 mm). The
silver coating without the cap layer and Ag/PPX coating
against E. coli showed less efficient inhibition of bacterial
growth. Against S. cohnii, only the silver/hydrophilic PPX-N
showed an inhibitory effect, which is presumed to be caused
by the highest silver release rate of the three different sand-
wich layers.
The inhibition zone against E. coli is larger than against
S. cohnii. This is probably due to the different biological and
physiological responses of Gram-positive and negative bac-
teria to the effects of silver ions. A Gram-positive bacterium
with peptidoglycan is more negatively charged than a Gram-
negative one. Therefore, more positively charged silver may
be bound to peptidoglycan by Gram-positive bacteria.
This may allow fewer silver ions to reach the cytoplasmic
membrane.29
The inhibition zone test prompts the conclusion that a
hydrophilic coating enhances the antibacterial effect of silver
against both strains of bacteria. The Ag/hydrophilic PPX-N
seems to have a higher silver release rate than Ag/PPX-N,
which may due to better wetting of its hydrophilic surface.
FIG. 3. Inhibition zone test. Bacterial growth around samples with different coatings after 18 h against E. coli and S. cohnii was prevented to a different degree
for four test samples on silicon squares (15  15 mm): silver coating (Ag), silver coating with PPX-N (Ag/PPX-N), and silver coating with plasma treatment
PPX-N (Ag/hydrophilic PPX-N). The antibiotic control was a filter immersed in 100 penicillin–streptomycin. Left: Inhibition zone test in the Petri plate
against E. coli. Right: Quantification of the width (mm) of the inhibition zones on M€ uller Hilton agar spread with 108 CFU/ml of E. coli and S. cohnii. The
experiments were performed in four replicates, one-way ANOVA with Bonferroni’s multiple comparison test:  p < 0:01;  p < 0:001, and error bars denote
the standard deviation (SD).
Biointerphases, Vol. 12, No. 1, March 2017
011001-6
These results show that hydrophilic coating improves the
antibacterial effect of silver against both bacteria strains.
D. Influence of silver/PPX-N catheter of the bacteria
growth rate
To quantify the antibacterial effect of the different silver-
treated catheters, we observed the growth of bacterial cul-
tures in the presence of coated catheters (1 cm in length) for
the two types Ag/PPX-N and Ag/hydrophilic PPX-N. The
growth of E. coli and S. cohnii with 103 CFU/ml was mea-
sured in synthetic urine using continuous densitometry dur-
ing incubation at 37  C (every 18 min for 20 h).
The curves present the optical density of bacterial growth
against time (Fig. 4). Inoculated urine was used as a
bacteria-only control, and as antibiotic control, we applied
inoculated urine with penicillin–streptomycin 100 (PAA,
C€olbe, Germany). All samples were tested in six replicates
in 48-well plates. The Ag/PPX-N coating system delayed the
growth of both bacterial strains. The Ag/hydrophilic PPX-N
coating system blocked the growth of both E. coli and
S. cohnii nearly as efficiently as penicillin–streptomycin
used as antibiotic control. Consistent with the inhibition
zone experiments, hydrophilic treatment of the PPX-layer
boosted the antibacterial effect of silver coating.
E. Prevention of biofilm formation with the hydrophilic
silver-PPX-N system
These two analytic experiments clearly show that a sand-
wich system consisting of silver/hydrophilic PPX-N inhibits
the growth curves of bacteria. To test how our coating sys-
tem affects the adhesion of E. coli and S. cohnii to the cathe-
ter and the biofilm formation, we used the crystal violet
staining method to quantify the adherence of the cells.
Three different types of catheter were tested (PPX-N, sil-
ver/PPX-N, and silver/hydrophilic PPX-N). As in the two
experiments described above, urine inoculated with penicil-
lin–streptomycin (1) was used as antibiotic control. All
samples were tested in six replicates. After incubating the
011001-7 Heidari Zare et al.: Efficacy of silver/hydrophilic PPX-N
FIG. 4. Growth curves of E. coli (left) and S. cohnii (right) as determined from OD600 measurements in presence of different coatings. Four groups were ana-
lyzed, silver/PPX-N and plasma treatment silver-PPX-N catheters, 1 penicillin–streptomycin as antibiotic control and a bacteria-only control. The synthetic
urine was inoculated with 103 CFU/ml, n ¼ 6, two-way ANOVA shows a significant difference between the curves (p < 0.0001).
silver-coated catheters in synthetic urine, we inoculated the
Agþ containing supernatant with both bacterial strains in
48-well plates. Under these conditions, the biofilm formed
on well plates that were stained with crystal violet. The opti-
cal density of the crystal violet stain (OD ¼ 595 nm) is a
measure for biofilm formation (Fig. 5).
We considered OD > 0.2 to be inactive and those with
OD < 0.2 to represent the antibacterial activity of our sample
[penicillin–streptomycin (1) as antibiotic control]. As
expected, biofilm growth was highest in the supernatant of
untreated catheters and lowest in penicillin–streptomycin.
Compared to the supernatant of the uncoated catheters,
the supernatant of Ag/PPX-N catheters slightly inhibited bio-
film formation. The Ag/PPX-N coating is presumed not to
release enough silver ions to inhibit the bacteria growth
completely, resulting in sluggish biofilm formation. In con-
trast, the supernatant of the silver/hydrophilic PPX-N cathe-
ters resulted in impressive reduction of biofilm formation,
similar to the antibiotic control. This supports the efficacy of
FIG. 5. Sandwich layer silver/hydrophilic PPX-N reliably prevents the bio-
film formation of E. coli and S. cohnii at the surface, the sandwich layer sil-
ver/PPX-N to a lesser extent. Penicillin–streptomycin was used as control.
six replicates, one-way ANOVA with Bonferroni’s multiple comparison
test:  p < 0:001;  p < 0:01.
Biointerphases, Vol. 12, No. 1, March 2017
011001-7
the hydrophilic coating in preventing biofilm formation in
addition to inhibiting growth of both bacterial strains.
F. Reduced incrustation with hydrophilic PPX-coating
Bacterial adhesion is often associated with incrustations
of Ca2þ and Mg2þ salts that may promote further bacterial
growth. To simulate the formation of inorganic deposits, we
took advantage of the urease reaction. Adding urease to
supersaturated synthetic urine provokes a rise in pH and
results in crystalline deposits on the catheter surface. Two
differently treated catheter surfaces were investigated in the
absence of silver to discriminate between the two catheter
coatings (hydrophobic PPX-N or hydrophilic PPX-N) with
respect to prevention of crystalline biofilm formation. After
58 h incubation time of the urease urine mix with the cathe-
ters with PPX-N coating or modified PPX-N coating in five
replicates, we measured the inorganic encrusting on the cath-
eter surface by means of quantitative analysis of Ca2þ and
Mg2þ cations. By this in vitro analysis, experimental in vivo
results should be confirmed, which were found by Grases
et al. by inspection of encrusted stents during the incubation
time.30
From Fig. 6, it is evident that the amounts of the two cati-
ons Mg2þ and Ca2þ in the precipitates clearly depend on the
quality of the coating after incubation time in the presence
of urease. The catheters with PPX-N coating exhibit a Ca2þ
concentration of 150 6 0.86 mg/l and Mg2þ concentration of
55.83 6 0.52 mg/l. After O2 plasma treatment of the surface
to increase the hydrophilic properties, the concentrations
dropped by a factor of 2 (Ca2þ: 65.81 6 2.68 mg/l and
Mg2þ: 17.12 6 0.66 mg/l). Thus, the hydrophilic PPX-N
coating is very effective in resisting the encrustation process.
G. Silver ion release from hydrophilic Ag/PPX-N
in synthetic urine
In a previous work, silver release behaviors from differen-
tially coated silver catheters [16 Ch./Fr. ( ¼ 5:3 mm), two
pieces of 2 cm] in synthetic urine, were quantitatively deter-
mined for 22 days by ICP-OES.19 We showed that the silver/
011001-8 Heidari Zare et al.: Efficacy of silver/hydrophilic PPX-N
FIG. 6. Analysis of the Ca2þ and Mg2þ cation precipitates as a function of
reduced incrustation on the catheter after a 58 h incubation in synthetic urine
with a simulated urinary tract infection, treated with two different coatings
(PPX-N and hydrophilic PPX-N), n ¼ 5, the Ca2þ and Mg2þ concentrations
in the two conditions are significantly different (p < 0.001, t-test).
hydrophilic PPX-N catheters release more silver than Ag/
PPX-N catheters (Fig. 7).
Therefore, this coating system is most suited for long-
term antibacterial applications. In all cases, the quantitative
measurement of the silver release is initiated by exposing a
piece of a treated catheter to a synthetic urine. It must be
considered that not only Agþ ions but also silver nanopar-
ticles migrate through the PPX cap layer.31
Silver nanoparticles can interact with biological fluids as
metallic particles (Ag0) or can be oxidized to Agþ.20
However, ICP-OES cannot discriminate between these two
species; therefore, release of both species by measuring the
sample solutions by this method cannot be excluded. As
Behra et al. have noted, the antibacterial activity of silver
nanoparticles is far lower than the impact of isolated Agþ
ions.20 To determine the antibacterial activity of silver cor-
rectly, a method is required which measures the concentration
FIG. 7. Enhancement of silver release rate by O2 plasma treatment, measured
by ICP-OES, two pieces of 2 cm catheters [16 Ch./Fr. ( ¼ 5:3 mm)], n ¼ 5,
two-way ANOVA with Bonferroni’s post-test:  p < 0:001;  p < 0:01.
Biointerphases, Vol. 12, No. 1, March 2017
011001-8
of silver ions selectively. One such method is ASV,32 which
was used in this work to verify specifically the silver ion
release from Ag/hydrophilic PPX-N coating. We immersed
catheter samples in synthetic urine and collected the silver-
containing supernatant after 1, 8, and 22 days.
To investigate the efficiency under conditions similar to
those in human body, special attention was given to the
selection of electrolytes used for the recording of calibration
curves. The supporting electrolyte used in all experiments
was a solution consisting of 50 ml of 0.1 M KNO3 and 5 ll
HNO3 (65%). Five milliliters of diluted urine and standard
silver solutions were added to the supporting electrolyte for
the recording of calibration curves. As already reported by
Jacobs, the peak current depends on the silver concentration,
pH, and electrical conductivity of the medium, deposition
time, and measurement parameters.33 Furthermore, under
certain conditions, dissolved silver can be complexed by
organic and inorganic salts contained in urine. The complex-
ing ability of Agþ decreases the diffusion coefficient and
hence the rate of Agþ deposition at accumulation potential,34
which impairs detection of Agþ by ASV. For this reason, we
decided to perform our measurements at conditions where
no formation of Agþ complexes with ions of ammonium
–
(NH3), phosphate (PO3 4 ) and acetate (CH3COO ) is possi-
ble. Said conditions were determined by analyzing various
potential-pH diagrams for the silver systems.35–38
The calibration curve for the silver ion concentration in
urine is used for the determination of Agþ concentration in
the range of 2–150 lg/l (i.e., approximately 2 orders of mag-
nitude, Fig. 8) and was obtained by plotting the intensity of
the maximum current versus the concentration of silver ions.
The calibration curve shows a linear dependence with the
regression equation of I ¼ 4:1E  6cðAgþ Þ þ 0:16 and a
correlation coefficient value R2 ¼ 0.99.
By application of ASV, the silver ion release from Ag/
hydrophilic PPX-N coated catheter in diluted urine was veri-
fied. Figure 9 shows the concentrations of silver ions
released from Ag/hydrophilic PPX-N. These data were
FIG. 8. Calibration curve obtained for silver ion concentrations in synthetic
urine with a concentration range of 2–150 lg/l.
011001-9 Heidari Zare et al.: Efficacy of silver/hydrophilic PPX-N
FIG. 9. Silver ion concentrations released from Ag/hydrophilic PPX-N in
diluted urine using ASV, four pieces of 2 cm catheters [18 Ch./Fr.
( ¼ 6 mm)], n ¼ 3, one-way ANOVA with Bonferroni’s multiple compari-
son test,  p < 0:001;  p < 0:05.
compared with the results of our previous work, specifically
with the total silver amount in the same electrolyte measured
by ICP-OES.19
There exists a large difference between the total silver
concentration (as measured by ICP-OES) and the sole silver
ion content (measured by ASV). Hence, ASV is superior to
ICP-OES for measuring only silver ions exclusively as anti-
bacterial agents in silver-coated catheters. This difference is
due to the presence of metallic silver in the urine sample.
Schamberger et al. showed that thin layers of PPX exhibited
a thickness-correlated porosity or averaged hole density,
which was determined with electrical impedance spectros-
copy.39 However, the averaged hole density was a product of
the number of holes and their diameter. Therefore, small
Ag0 nanoparticles could even pass the cap layer via channels
with a large diameter. In our recent work,19 it was shown
that the minimum inhibitory concentration of silver ions for
E. coli and S. cohnii amounted to 30 lg/l. The silver ion
release rate was determined to be approximately 200 lg/l.
This correlated with the value of minimum inhibitory con-
centration and established the excellent antibacterial effi-
ciency for long-term applications.
IV. CONCLUSION AND OUTLOOK
Innovative antibacterial urinary catheters were obtained
by direct deposition of silver nanoparticles generated from
Tollens’s reagent at slightly elevated temperatures. This
deposit acted as a source for Agþ ions whose diffusion rate
was controlled by a cap layer of PPX-N. This layer also
improved the adhesion of the silver layer, which in fact con-
sisted of small Ag0 nanoparticles. To ensure the wettability
of the PPX-N surface in this system, it was treated with radio
frequency-generated oxygen plasma. This step enhances the
silver release rate and therefore the antibacterial effect of the
catheter measured by inhibition zone and biofilm formation.
The efficacy of this hydrophilic PPX-N to act as a barrier
with a tunable diffusion constant for Agþ ions was
Biointerphases, Vol. 12, No. 1, March 2017
011001-9
investigated by bacterial growth curves and tests of the inhi-
bition zone against E. coli and S. cohnii against the “normal”
PPX-N. Moreover, treating the cap layer also significantly
reduced the adhesion of crystalline biofilms on the surfaces,
which was demonstrated by measuring the concentrations of
the two ions Ca2þ and Mg2þ on the catheter surfaces. By
applying the method of anodic stripping voltammetry, which
is selectively sensitive to Agþ alone (in contrast to optical
emission spectroscopy), we have shown that sufficient
amount of silver ions could diffuse through the porous cap
layer to combat bacteria effectively, thereby prolonging the
lifecycle of the silver catheters. The hydrophilization of
the PPX-N cap layers generates two benefits compared to
PPX-N coating. First, the enhanced release rate of silver ions
inhibits bacterial growth and thus formation of an organic
biofilm. Second, the hydrophilic coating inhibits deposition
of an inorganic biofilm compared to untreated PPX-N. Thus,
our hydrophilic PPX-N silver nanoparticle coating is a sig-
nificant improvement toward safer and longer-lasting urinary
catheters without any disadvantages for patients.
ACKNOWLEDGMENTS
The antibacterial investigations were conducted in the
Laboratory for Microbiology by Karlheinz Trebesius and
Julia Wilke and in the Laboratory of Biochemistry by J€urgen
Gilch and Stefan Diemer. The improvements of incrustation
were performed by Steffen Hackenberg in the operating unit
Chemie of Hochschule M€unchen. The financial support from
Germany’s Federal Secretary of Education and Research
under Nos. 1715X04 and 1753X08 and of the State of
Bavaria is gratefully acknowledged.
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