Professional Documents
Culture Documents
JVST 18
JVST 18
infection of urinary catheters, we therefore need to develop urealyticus strain CK27 were selected as representatives of
methods to inhibit bacterial growth on catheters.7 Numerous Gram-negative and Gram-positive bacteria, respectively. The
groups have tested the efficacy of bactericidal or bacterio- bacterial strains were obtained as freeze-dried cultures from the
static coatings with the antibiotics ciprofloxacin, gentamicin, Leibniz Institute DSMZ-German Collection of Microorganisms
norfloxacin, and nitrofural,8,9 as well as silver oxide, tita- and Cell Cultures (Braunschweig, Germany) and were grown
nium oxide, or combinations thereof.10–12 in Mueller-Hinton growth medium (Carl Roth, Karlsruhe,
Other attempts have involved antimicrobial peptides con- Germany). The organic and inorganic components, silver
jugated to copolymer brushes,13 silver-silicone-hydrogels,14 nitrate solution 0.1 mol/l 6 0.2%, and silver ICP standard solu-
poly(vinyl-pyrrolidone)-coating,15 and heparin16 to reduce tion 10 000 mg/l 6 0.2 were purchased from Carl Roth
bacterial growth and adhesion to the catheter surface in vitro. (Karlsruhe, Germany). Perchloric acid 70% (HClO4) and 0.1 M
However, not all methods were effective. Due to the develop- KNO3 (Merck, Darmstadt, Germany) were also used. The syn-
ment of resistance against antibiotics, the high cost of deliv- thetic urine (AU) medium was prepared from inorganic constit-
ery or synthesis, and potential side effects, antibiotics should uents (sodium chloride 5.2 g/l, sodium hydrogen carbonate
be avoided for surface modification of biomaterials.17 For the 2.1 g/l, sodium sulfate 3.2 g/l, ammonium chloride 1.3 g/l,
near future, this is one of the most challenging concerns. In potassium dihydrogen phosphate 0.95 g/l, potassium hydrogen
recent years, silver nanoparticles at low concentrations have phosphate 1.2 g/l, calcium chloride 0.37 g/l, magnesium sulfate
been successfully used in biomedical instruments due to their 0.49 g/l, and iron sulfate 0.0012 g/l), as well as organic constitu-
unique physicochemical properties such as nanometric sizes, ents (urea 10 g/l, uric acid 2,6,8-trihydroxypurine 0.07 g/l, cre-
high surface area, and high reactivity. Thanks to its oligody- atinine 0.8 g/l, peptone from casein 1.0 g/l, yeast extract
namic activity, silver can damage bacterial cell walls and 0.005 g/l, lactic acid 0.1 g/l, and citric acid 0.4 g/l). All compo-
membranes and inhibit bacterial genome replication of a nents were mixed, and the pH was adjusted to 6.5. Finally, the
broad spectrum of bacteria and fungi. We therefore coated synthetic urine was sterilized through filters with a pore size of
urethral catheters with synthetic silver nanoparticles. To 0.2 lm from Carl Roth (Karlsruhe, Germany).
improve adhesion of silver nanoparticles to the surface, cathe-
ters were coated with poly(p-xylylene) or parylene (PPX-N), B. Preparation of the silver/PPX-N system
a biocompatible polymer.18 In a previous work, we have The catheter surfaces were activated by immersing them in
already optimized the silver thickness to compare PPX-N and a solution of 30% nitric acid for 30 min as described previ-
silver particle morphology and tested minimum inhibitory ously.19 To coat the catheters with silver, 5 ml aqueous silver
concentration of silver ions in synthetic urine against E. coli nitrate (0.1 M) was mixed with 0.05 g sodium hydroxide
and S. cohnii.19 The aim of this study is to evaluate the anti- resulting in a brown solid precipitate of aqueous Ag2O. By
bacterial efficacy of silver/PPX catheters in a simulated body adding 0.7 ml ammonia solution 25% (13.3 mol/l), the silver
fluid (synthetic urine), including the power to prevent genera- oxide completely dissolved by forming [Ag(NH3)2]þ as the
tion of a biofilm. The antibacterial activity of the catheters main component of Tollens’s reagent. By adding maltose
was studied with quantitative (growth curve) and qualitative solution (0.8 mol/l) to this solution (ratio 1:1), colloidal silver
methods (inhibition zone) against E. coli and S. cohnii. The particles were formed due to reduction of silver ions into
adhesion of bacteria to treated surfaces was studied through metallic silver. To promote silver deposition on the silicon
crystal violet staining assays, while the growth of bacteria catheters, the reaction took place at 70 C in a water bath.
was evaluated by optical density (OD). Afterward, with the This reaction is actually performed by pumping the Tollens’s
aim of preventing crystalline biofilm formation, the concen- reagent into the catheter by means of a peristaltic pump TV/
trations of Ca2þ and Mg2þ were measured on top of catheters TVS (medorex e.K., N€orten-Hardenberg, Germany) as
capped by a layer PPX-N and modified PPX-N, respectively. described previously.19 As the final step, the catheter was
The most recent results obtained with inductively coupled immersed in a water bath (70 C) for 5 min. After the deposi-
plasma optical emission spectroscopy (ICP-OES) proved the tion process, the silver coating solution was pumped out, and
constant release rate of silver through very thin cap layers of the catheters were washed three times with deionized water.
PPX-N. However, ICP-OES cannot discriminate between sil- Silver-coated catheters were dried overnight at room tempera-
ver nanoparticles and Agþ ions and measures the sum of both ture. To control and prolong the rate of silver release, the sil-
of them. As Behra et al. have noted, the activity of silver ver film was coated with PPX-N, an inert and biocompatible
nanoparticles is far lower than the impact of isolated Agþ polymer, by means of chemical vapor deposition.21 The film-
ions.20 To distinguish between Ag nanoparticles and Agþ building gas (monomeric p-xylylene) was diluted by argon to
ions, we used anodic stripping voltammetry (ASV). yield a layer thickness of approximately 200 nm. The thick-
ness of the PPX film was measured by a mechanical profilom-
II. MATERIALS AND METHODS eter (Tencor Alpha Step 200).
A. Materials
Silicone catheters [16 Ch./Fr. ( ¼ 5:3 mm), 18 Ch./Fr. C. Plasma treatment of PPX-N surfaces
( ¼ 6 mm)] were obtained from Urokink Industries AG After preparation of silver/PPX-N catheters, the PPX-N
(Halberstadt, Germany). E. coli K12 and S. cohnii subsp. cap layer was modified using capacitatively coupled plasma
driven by a radio-frequency power supply of 13.56 MHz 1 mm, then the sample had good antibacterial activity. If it
(Plasmalab lP, Oxford Instruments, Yatton, UK). The silver/ was shorter than 1 mm, then its quality was fairly good.
PPX-N catheters were placed into a reaction chamber and However, if the surface of the sample was free of bacterial
evacuated to lower than 104 Torr by means of a turbomo- growth, then the sample was labeled as sufficient. If the sam-
lecular pump. Specifically, the catheters were treated at a ple was covered by bacteria, then its antibacterial activity was
power of 160 W and a pressure of 100 mTorr for 3 min. We evaluated as “limited.”23
excluded reduction of the layer thickness through the plasma
treatment by measuring the layer thickness on microscope 2. Growth curve of bacteria toward silver-treated
slides before and after exposure to the O2 plasma using pro- catheters
filometry (measured layer thickness typically before and The bacterial growth in synthetic urine was measured in six
after plasma exposure: 900 nm 6 20 nm; for comparison, replicates in 48-well plates. First, 5 mm-long pieces of treated
ashing of photoresist is executed in the same reactor at catheters were incubated in 1 ml of synthesized urine for 24 h
300 W microwave power and a discharge pressure of 1000 at 37 C. The samples were then removed, and 450 ll of the
mTorr with an etch rate of 50 nm/min). supernatant (containing released silver ions) was transferred to
another sterile 48-well plate and inoculated with 103 CFU/ml
D. Characterization of PPX-N surfaces
of E. coli and S. cohnii. The optical density of bacterial growth
The wetting behavior of the PPX-N surface was evaluated was measured continuously at 37 C (600 nm, 218 rpm) using
by measuring the contact angle applying the drop shape anal- a photometer (TECAN Infinite M200 PRO Nano Quant,
ysis system from Kr€uss (DA 10 MK2, Hamburg, Germany). Tecan Trading AG, Switzerland) every 20 min for 21 h.
Substrates were microscope slides of glass which were first
coated with PPX-N and then further processed with different 3. Biofilm formation by crystal violet staining assay
methods (untreated or O2 plasma treatment). Using the ses-
Biofilm formation was quantified in 48-well plates,
sile drop method, the static angles were obtained for water
according to the method by Alt et al.24 The 5 mm catheters
by applying a droplet to the surface and measuring the con-
were incubated in synthetic urine for 24 h at 37 C. Then,
tact angle directly after treatment. All measurements were
270 ll of supernatant was transferred into new sterile
performed at room temperature (23 6 3 C) in triplicate.
well plates and inoculated with 103 CFU/ml of E. coli and
The wetting behavior (against water) can be discriminated
S. cohnii. After 24 h of incubation at 37 C without agitation,
into these categories:22 contact angle 0 H < 90 : partial
the bacteria formed a biofilm, the supernatant was removed,
wetting, 90 H 180 : poor to very poor wetting, and
and the wells were washed with deionized water to remove
H ¼ 0 perfect or complete wetting, no droplets can be
found. the nonadherent cells. This procedure was repeated three
times. Adherent cells of the biofilm were stained with 1%
E. Microbiological testing crystal violet for 15 min at room temperature, and unbound
dye was removed by three washes with water. Finally, the
The antibacterial activity of Ag/PPX system was evalu- adherent cells were dissolved with 33% acetic acid. The opti-
ated against E. coli and S. cohnii using three methods: inhi-
cal density of the solution was measured with a photometer at
bition zone assay, growth curve analysis, and biofilm
595 nm and was compared to the bacteria-only controls.
formation. All catheter samples were sterilized by exposure
to UV light in a tissue culture hood (20 min). F. Chemical Analysis
1. Zone of inhibition test 1. In vitro assay for inorganic incrustation of catheters
The efficacy of the silver-treated silicon in reducing bacte- To simulate the inorganic (salt) depositions during a uri-
rial viability was tested with a spot modification of standard nary tract infection, two nonsaturated aqueous stock solu-
agar diffusion tests. The treated silicon squares (15 15 mm) tions A þ B were mixed, which together form insoluble salts
were separated into three different categories: Ag without cap (Tables I and II)27 caused by supersaturation.
layer, Ag/PPX-N and Ag/hydrophilic PPX-N. The antibiotic Equal volumes of solutions A and B were mixed and incu-
control was a single sheet of filter paper immersed with 10 ll bated in flask 1 at a water bath temperature of 40 C. This
of penicillin–streptomycin 100 (PAA, C€olbe, Germany). oversaturated solution was called synthetic urine, which was
These were placed with the coated side up on a petri dish used to simulate urinary tract infection by addition of urease.
with Mueller-Hinton agar. Then, 13 ml of 0.7% agarose in Due to the breakdown of urea into basic ammonia and CO2
Mueller-Hinton medium were mixed with 108 CFU/ml bacte- by urease, the pH rose after mixing from 6.8 to 9 within 16 h.
ria and poured over the sample onto the agar plate. This was accompanied by deposition of brushite (CaHPO42
Afterward, this was incubated for 18 h at 37 C. The zone H2O), hydroxylapatite carbonate, [Ca10(PO4)5CO3(OH)2],
without bacterial growth surrounding the samples was mea- and struvite (MgNH4PO46 H2O).
sured to quantify the antibacterial capability, according to the For each run, two catheters with different coatings were
aforementioned standard diffusion test SNV 195920-1992. If put into a second reaction flask (flask 2) equilibrated in the
the width of the bacterial inhibition zone was longer than water bath at 40 C under identical chemical and physical
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
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).
silver coating (Ag), silver coating with PPX-N (Ag/PPX- These results show that hydrophilic coating improves the
N), and silver coating with O2-plasma treatment PPX-N antibacterial effect of silver against both bacteria strains.
(Ag/hydrophilic PPX-N). As antibiotic control, we used fil-
ters immersed in penicillin–streptomycin 100 (PAA, D. Influence of silver/PPX-N catheter of the bacteria
C€olbe, Germany). Figure 3 shows the formation of an inhi- growth rate
bition zone around these three specimens against E. coli To quantify the antibacterial effect of the different silver-
and S. cohnii. In Fig. 3(a), the performance against E. coli treated catheters, we observed the growth of bacterial cul-
is shown. All three samples inhibited bacterial growth to a tures in the presence of coated catheters (1 cm in length) for
certain extent. Among the catheter samples silver/hydro- the two types Ag/PPX-N and Ag/hydrophilic PPX-N. The
philic PPX-N was best and comparable to the antibiotic growth of E. coli and S. cohnii with 103 CFU/ml was mea-
control. Against S. cohnii, only hydrophilic Ag/PPX was sured in synthetic urine using continuous densitometry dur-
sufficiently effective [quantification in Fig. 3(b)]. ing incubation at 37 C (every 18 min for 20 h).
This inhibition of bacterial growth confirmed that suffi- The curves present the optical density of bacterial growth
cient amounts of silver ions diffused into the agar from against time (Fig. 4). Inoculated urine was used as a
coated surfaces. While all three conditions inhibited the bacteria-only control, and as antibiotic control, we applied
growth of E. coli to some extent, the quantitative evaluation
inoculated urine with penicillin–streptomycin 100 (PAA,
of the width of inhibition zones showed that the silver/hydro-
C€olbe, Germany). All samples were tested in six replicates
philic PPX-N had the largest inhibition zone toward E. coli
in 48-well plates. The Ag/PPX-N coating system delayed the
(5.3 6 0.18 mm) and against S. cohnii (1.35 6 0.4 mm). The
growth of both bacterial strains. The Ag/hydrophilic PPX-N
silver coating without the cap layer and Ag/PPX coating
coating system blocked the growth of both E. coli and
against E. coli showed less efficient inhibition of bacterial
S. cohnii nearly as efficiently as penicillin–streptomycin
growth. Against S. cohnii, only the silver/hydrophilic PPX-N
used as antibiotic control. Consistent with the inhibition
showed an inhibitory effect, which is presumed to be caused
zone experiments, hydrophilic treatment of the PPX-layer
by the highest silver release rate of the three different sand-
wich layers. boosted the antibacterial effect of silver coating.
The inhibition zone against E. coli is larger than against
E. Prevention of biofilm formation with the hydrophilic
S. cohnii. This is probably due to the different biological and
silver-PPX-N system
physiological responses of Gram-positive and negative bac-
teria to the effects of silver ions. A Gram-positive bacterium These two analytic experiments clearly show that a sand-
with peptidoglycan is more negatively charged than a Gram- wich system consisting of silver/hydrophilic PPX-N inhibits
negative one. Therefore, more positively charged silver may the growth curves of bacteria. To test how our coating sys-
be bound to peptidoglycan by Gram-positive bacteria. tem affects the adhesion of E. coli and S. cohnii to the cathe-
This may allow fewer silver ions to reach the cytoplasmic ter and the biofilm formation, we used the crystal violet
membrane.29 staining method to quantify the adherence of the cells.
The inhibition zone test prompts the conclusion that a Three different types of catheter were tested (PPX-N, sil-
hydrophilic coating enhances the antibacterial effect of silver ver/PPX-N, and silver/hydrophilic PPX-N). As in the two
against both strains of bacteria. The Ag/hydrophilic PPX-N experiments described above, urine inoculated with penicil-
seems to have a higher silver release rate than Ag/PPX-N, lin–streptomycin (1) was used as antibiotic control. All
which may due to better wetting of its hydrophilic surface. samples were tested in six replicates. After incubating the
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).
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 the hydrophilic coating in preventing biofilm formation in
Agþ containing supernatant with both bacterial strains in addition to inhibiting growth of both bacterial strains.
48-well plates. Under these conditions, the biofilm formed
on well plates that were stained with crystal violet. The opti- F. Reduced incrustation with hydrophilic PPX-coating
cal density of the crystal violet stain (OD ¼ 595 nm) is a Bacterial adhesion is often associated with incrustations
measure for biofilm formation (Fig. 5). of Ca2þ and Mg2þ salts that may promote further bacterial
We considered OD > 0.2 to be inactive and those with growth. To simulate the formation of inorganic deposits, we
OD < 0.2 to represent the antibacterial activity of our sample took advantage of the urease reaction. Adding urease to
[penicillin–streptomycin (1) as antibiotic control]. As supersaturated synthetic urine provokes a rise in pH and
expected, biofilm growth was highest in the supernatant of results in crystalline deposits on the catheter surface. Two
untreated catheters and lowest in penicillin–streptomycin. differently treated catheter surfaces were investigated in the
Compared to the supernatant of the uncoated catheters, absence of silver to discriminate between the two catheter
the supernatant of Ag/PPX-N catheters slightly inhibited bio- coatings (hydrophobic PPX-N or hydrophilic PPX-N) with
film formation. The Ag/PPX-N coating is presumed not to respect to prevention of crystalline biofilm formation. After
release enough silver ions to inhibit the bacteria growth 58 h incubation time of the urease urine mix with the cathe-
completely, resulting in sluggish biofilm formation. In con- ters with PPX-N coating or modified PPX-N coating in five
trast, the supernatant of the silver/hydrophilic PPX-N cathe- replicates, we measured the inorganic encrusting on the cath-
ters resulted in impressive reduction of biofilm formation, eter surface by means of quantitative analysis of Ca2þ and
similar to the antibiotic control. This supports the efficacy of
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.
18 30
H. Heidari Zare, St. Sudhop, F. Schamberger, and G. Franz, F. Grases, A. I. Villacampa, O. S€ ohnel, E. K€
onigsberger, and P. M. May,
Biointerphases 9, 31002 (2014). Cryst. Res. Technol. 32, 707 (1997).
19 31
H. Heidari Zare, O. D€ uttmann, A. Vass, G. Franz, and D. Jocham, E. de Giglio, D. Cafagna, S. Cometa, A. Allegretta, A. Pedico, L. C.
Biointerphases 11, 31002 (2016). Giannossa, L. Sabbatini, M. Mattioli-Belmonte, and R. Iatta, Anal.
20
R. Behra, L. Sigg, M. J. D. Clift, F. Herzog, M. Minghetti, B. Johnston, A. Bioanal. Chem. 405, 805 (2013).
32
Petri-Fink, and B. Rothen-Rutishauser, J. R. Soc. Interface 10, 20130396 D. R. Atherton, “Anodic stripping voltammetry at a glassy carbon elec-
(2013). trode for the determination of platinum species derived from cis-
21
G. Franz and F. Schamberger, J. Vac. Sci. Technol., A 31, 61602 (2013). diamminedichloroplatinum(II),” Ph.D. thesis (University of Florida,
22
H.-D. D€orfler, Grenzfl€ achen und kolloid-disperse Systeme (Springer, Gainesville, 1984).
33
Berlin/Heidelberg, 2002), p. 77. E. S. Jacobs, Anal. Chem. 35, 2112 (1963).
23 34
M. Pollini, F. Paladini, M. Catalano, A. Taurino, A. Licciulli, A. L. Baldrianova, I. Svancara, A. Economou, and S. Sotiropoulos, Anal.
Maffezzoli, and A. Sannino, J. Mater. Sci. 22, 2005 (2011). Chim. Acta 580, 24 (2006).
24 35
V. Alt, T. Bechert, P. Steinrucke, M. Wagener, P. Seidel, E. Dingeldein, E. P. Delahay, M. Pourbaix, and P. V. Rysselberghe, J. Electrochem. Soc. 98,
Domann, and R. Schnettler, Antimicrob. Agents Chemother. 48, 4084 (2004). 65 (1951).
25 36
R. G. Compton and C. E. Banks, Understanding Voltammetry, 2nd ed. Electroless Plating. Fundamentals and Applications, edited by G. O.
(Imperial College, London, 2011). Mallory and J. B. Hajdu (American Electroplaters and Surface Finishers
26
J. A. V. Fraunhofer and C. H. Banks, Potentiostat and Its Applications Society, Inc., Orlando, FL, 1990).
37
(Butterworths, London, 1972). J. H. Sluyters, M. D. Wijnen, and H. J. V. D. Hul, Electrochim. Acta 5, 72
27
L. Kleinen, U. B€ode, K. Schenk, H. Busch, J. Bradenahl, and St. C. (1961).
38
M€ uller, Plasma Processes Polym. 4, S386 (2007). O. N. Starovoytov, N. S. Kim, and K. N. Han, Hydrometallurgy 86 114
28
X. Bi, B. P. Crum, and W. Li, J. Microelectromech. Syst. 23, 628 (2014). (2007).
29 39
K. Kawahara, K. Tsuruda, M. Morishita, and M. Uchida, Dent. Mater. 16, F. Schamberger, A. Ziegler, and G. Franz, J. Vac. Sci. Technol., B 30,
452 (2000). 51801 (2012).