Biomaterials 116 (2017) 69e81

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Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Anti-adhesive antimicrobial peptide coating prevents catheter

associated infection in a mouse urinary infection model
Kai Yu a, 1, Joey C.Y. Lo b, 1, Mei Yan a, Xiaoqiang Yang a, Donald E. Brooks a, c,
Robert E.W. Hancock d, Dirk Lange b, **, Jayachandran N. Kizhakkedathu a, c, *
a
Centre for Blood Research, Department of Pathology & Laboratory Medicine, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
b
Department of Urologic Sciences, University of British Columbia, Vancouver, British Columbia V5Z 1M9, Canada
c
Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
d
Department of Microbiology and Immunology, Centre for Microbial Diseases and Immunity Research, University of British Columbia, Vancouver, British
Columbia V6T 1Z4, Canada

a r t i c l e i n f o a b s t r a c t

Article history: Catheter-associated urinary tract infections (CAUTIs) represent one of the most common hospital ac-
Received 2 September 2016 quired infections with significant economic consequences and increased patient morbidity. CAUTIs often
Received in revised form start with pathogen adhesion and colonization on the catheter surface followed by biofilm formation.
14 November 2016
Current strategies to prevent CAUTIs are insufficiently effective and antimicrobial coatings based on
Accepted 24 November 2016
antimicrobial peptides (AMPs) hold promise in curbing CAUTIs. Here we report an effective surface
Available online 24 November 2016
tethering strategy to prepare AMP coatings on polyurethane (PU), a common biomedical plastic used for
catheter manufacture, by using an anti-adhesive hydrophilic polymer coating. An optimized surface
Keywords:
Catheter-associated urinary tract infections
active AMP, labeled with cysteine at the C-terminus (RRWRIVVIRVRRC), was used. The coated PU surface
Urinary infection model was characterized using ATR-FTIR, XPS and atomic force microscopy analyses. The tethered peptides on
Antimicrobial peptide the PU catheter surface displayed broad spectrum antimicrobial activity and showed long term activity
Polymer brush coating in vitro. The surface coating prevented bacterial adhesion by up to 99.9% for both Gram-positive and
Biocompatibility -negative bacteria, and inhibited planktonic bacterial growth by up to 70%. In vivo, the coating was tested
in a mouse urinary catheter infection model; the AMP-coated PU catheter was able to prevent infection
with high efficiency by reducing the bacteria adhesion on catheter surface by more than 4 logs (from
1.2
106 CFU/mL to 5
101 CFU/mL) compared to the uncoated catheter surface, and inhibit planktonic
bacterial growth in the urine by nearly 3 logs (1.1
107 CFU/mL to 1.47
104 CFU/mL). The AMP-brush
coating also showed good biocompatibility with bladder epithelial cells and fibroblast cells in cell cul-
ture. The new coating might find clinical applications in preventing CAUTIs.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction limiting the long-term use of these devices are catheter-associated

Urinary catheters are one of the most commonly inserted de-
vices in healthcare, with up to 16% of acute-care inpatients having
an indwelling urethral catheter inserted at some time during their
hospitalization [1]. One of the most common complications
* Corresponding author. Department of Pathology and Laboratory Medicine,
Centre for Blood Research, University of British Columbia, Canada.
** Corresponding author. Department of Urologic Sciences, University of British
Columbia, Vancouver, British Columbia V5Z 1M9, Canada.
E-mail addresses: dirk.lange@ubc.ca (D. Lange), jay@pathology.ubc.ca
(J.N. Kizhakkedathu).
1 being reported annually in the USA [8] with associated costs of
Those authors contribute equally.
urinary tract infections (CAUTIs), which account for up to 23% of
infections in the intensive care units (ICU) and 40% of infections
hospital-wide [2e4]. The vast majority of these infections,
(approximately 70e80%) occur after catheter insertion [5,6]. The
scope of the problem is indicated by the fact that up to 23% of short-
term catheterized patients (i.e., after 2e4 days) develop asymp-
tomatic bacteriuria triggered by bacterial adhesion to the device
surface, which increases to 100% of patients with long-term
indwelling times [7]. While the majority of these patients may
not go on to develop active infections, the presence of a significant
number of bacteria on the device and in the urine significantly
increases the risk with more than 400,000 urinary tract infections

http://dx.doi.org/10.1016/j.biomaterials.2016.11.047
0142-9612/© 2016 Elsevier Ltd. All rights reserved.
70 K. Yu et al. / Biomaterials 116 (2017) 69e81
around $400 million per year [9,10].
The significant incidence of urinary tract infections associated
with urethral catheters stems from the fact that they provide direct
access for bacteria from the outside environment into the favour-
able environment of the urinary tract. Once inside the urinary tract,
the internal and external surface of the catheter facilitates bacterial
adhesion, colonization and the eventual development of a highly
resistant biofilm [11,12]. Biofilms are a defined stress-triggered
growth state of bacteria on surfaces and they are between 10 and
1000-fold more adaptively resistant to most antibiotics primarily
due to altered expression of multiple resistance genes [13]. As a
result, bacterial biofilms are very difficult to eradicate allowing
them to form a continuous nidus for infection that is usually only
eliminated by the constant removal and exchange of the indwelling
device itself, resulting in significant costs to the healthcare system
and morbidity to the patient.
Current strategies to prevent CAUTIs including interventions for
limiting bacterial buildup on the catheter surface include avoiding
long term indwelling periods through frequent exchanges or
intermittent catheterization, improving the techniques for device
insertion thereby limiting the introduction of bacteria into the
urinary tract and developing specific protocols for the management
of postoperative urinary catheters [14,15]. Considering that exten-
sive efforts to improve in these areas has not resulted in a notice-
able difference in the rate of urinary tract infections associated with
indwelling catheters, the use of anti-infective catheters is still
considered to be potentially the most effective solution for this
significant clinical problem. Previous attempts to develop such
catheters have included antimicrobial coatings using silver, nitro-
furazone, or other antimicrobial materials, but all of these have
shown to have significant limitations. These include poor overall
biocompatibility, instability, short-term antimicrobial activity and
efficacy against only a limited spectrum of bacterial species [16], all
of which limit the use of such coatings for both the short- and long-
term catheterization [17,18]. Furthermore, the use of antibiotic-
based coatings is further limited by the significant rise in anti-
biotic resistance [19]. Even if new antibiotics are developed, their
use as effective antibiotic-based catheter coatings would be limited
by the rapid rate at which bacterial species can acquire and/or
develop suitable antibiotic resistance mechanisms.
Antimicrobial peptides (AMPs) represent an attractive alterna-
tive for the development of antimicrobial indwelling device coat-
ings, since they have shown to have broad-spectrum activity
against a multitude of bacterial species, fungi and viruses, and have
been shown to be biocompatible in various models [20e22].
Furthermore, given the variety of effective peptide sequences and
multiple targets, resistance mechanisms towards them are limited.
Over the years, a larger variety of substrates have been successfully
coated with AMPs including nanoparticles [23e26], contact lenses
[27], titanium disks or titanium coated silicon surfaces [28e31] and
catheter-like surfaces [32e35]. While surface immobilization has
been successful, the AMPs in such coatings often show lower
antimicrobial activity compared to their soluble version, possibly
due to the non-specific chemistry of the immobilization techniques
utilized, resulting in inadequate surface concentrations or altered
orientations of peptide molecules on the surface and/or associated
host cell toxicities [34e36]. Previous in vitro studies have shown
that grafted coatings consisting of AMPs on catheter-relevant sur-
faces result in modest efficacy especially under conditions repre-
sentative of those encountered clinically, such as the urinary
environment [34e36]. While some in vivo studies have been per-
formed to test the efficacy of AMP coatings, they have been limited
to subcutaneous models using AMPs attached only to nanoparticles
or titanium surfaces [25,28,30]. As such, studies testing the efficacy
of AMP coatings in the urinary tract are lacking, and testing these
coatings in more clinically-relevant in vivo models is necessary to
determine whether AMP-based coatings have a utility in decreasing
CAUTIs, especially on material surfaces relevant to urinary cathe-
ters, which has never been tested.
To this end, we developed a very effective antimicrobial coating
consisting of highly active AMPs attached to PU catheter material
surfaces using a novel polymer-based tethering strategy that not
only had non-fouling characteristics but also provided specific
flexible binding sites for peptide conjugation. The antimicrobial
activity of tethered peptides was tested against different Gram-
positive and -negative bacteria in-vitro in both bacterial culture
medium and artificial urine. To verify in vivo efficacy, the optimized
coating was evaluated in a clinically relevant CAUTI mouse model
and demonstrated excellent antimicrobial and antibiofilm activity.
To test biocompatibility, the cytotoxicity of the AMP coated PU
surface was evaluated against human bladder epithelial cells and
fibroblasts.
2. Materials and methods
2.1. Materials
N,N-Dimethylacrylamide (DMA) (Aldrich, 99%) was distilled
under reduced pressure before use. N-(3-Aminopropyl) meth-
acrylamide hydrochloride (APMA) (98%) was from Polysciences,
USA and was used as supplied. 1, 1, 4, 7, 10, 10-Hexamethyl tri-
ethylene tetramine (HMTETA) (97%), methyl 2- chloropropionate
(97%), CuCl (99%), CuCl2 (99%), allylamine (98%), thioglycerol (97%)
were purchased from Sigma-Aldrich (Oakville, ON). Cysteine
labeled peptide E6 (RRWRIVVIRVRRC) was synthesized by Can-
Peptide Corp (>95% purity by HPLC) (Montreal, Canada) and was
used as supplied. Iodoacetic acid N-hydroxysuccinimide ester was
synthesized using a procedure similar to that reported in the
literature [37]. All other reagents were purchased from Sigma-
Aldrich and used without further purification. SurFlash® I.V. Cath-
eter Cat. #SS*FF1832 (18 Gauge and 24 Gauge) made of poly-
urethane (PU), were purchased from Terumo. For in vitro studies, an
18 Gauge catheter was used with a longitudinal slit made to better
expose the inner surface of the catheter for coating purposes. A 24
Gauge catheter was used for the in vivo study. A longitudinal slit
was also made to the section 4 mm below the tip of the PU catheter.
Polyurethane sheet with a thickness of 0.78 mm, purchased from
Professional Plastics, WA, USA, was used for cell toxicity evaluation.
2.2. Synthesis of anti-adhesive antimicrobial brush coating on
polyurethane substrates
2.2.1. Allylamine plasma treatment and initiator coupling
The PU catheters and sheets were pretreated with nitrogen at a
flow rate of 50 SCCM (standard cubic centimeter per minute) and at
a pressure of 350 mTorr. A plasma power (75 W) was used for 3 min
in a M4L Plasma Processing System from PVA Tepla (Corona, Cali-
fornia, USA). The surface was then treated using allylamine at 200
Watt plasma power at a pressure of 300 mTorr and a flow rate of 80
SCCM for 10 min. The allylamine modified devices were cleaned by
ultrasonication in Milli-Q water for 10 min, and dried under argon.
The allylamine modified devices were immersed in a solution of 2-
chloro-N-glycidyl propionamide in Milli-Q water (1 wt%) at 40  C
overnight. The initiator modified devices were then cleaned by
ultrasonication in Milli-Q water for 10 min, and dried under argon.
2.2.2. Preparation of PDMA-co-APMA brush coating on PU catheter
or PU sheet
The copolymer brush coating was prepared using aqueous sur-
face initiated atom transfer radical polymerization (SI-ATRP).
 K. Yu et al. / Biomaterials 116 (2017) 69e81
Copper (II) chloride (CuCl2, 1 mg), copper (I) chloride (CuCl, 12 mg)
and HMTETA (60 mL) were added successively into a glass tube
followed by the addition of 12 mL Milli-Q water. The solution was
degassed with three freeze-pump-thaw cycles, then transferred
into the glove box. The catalyst solution (6 mL) was thoroughly
mixed before adding DMA (630 mL) and APMA (225 mg). The
modified devices were immersed in the polymerization mixture.
Soluble methyl 2-chloropropionate (20 mL from a stock solution of
40 mL in 5 mL methanol) was added immediately to the reaction
mixture, and the polymerization was allowed to proceed at RT
(22  C) for 24 h. The device or substrate was then rinsed thoroughly
with Milli-Q water and sonicated in water for 10 min. The soluble
polymer formed along with the surface grafted polymer was
collected and purified by dialysis (molecular weight cut off: 1000)
against water for 1 week with daily exchange of water. The grafting
density (s) for the polymer layer on the catheter surface was esti-
mated by using the equation, s¼(hrNA)/Mn [38], where Mn is the
number average molecular weight of free polymer in the solution
along with surface grafted polymer, NA is the Avogado's number, h
is the polymer layer thickness measured by AFM, r is the density of
polymer (we assumed the density of polymer is equally to 1 g/cm3).
2.2.3. Peptide conjugation onto polymer brush modified PU catheter
or PU sheet
The brush modified devices were immersed in 20 mL PBS buffer
(137 mM sodium chloride, 2.7 mM potassium chloride, 10 mM
disodium hydrogen phosphate, 1.8 mM potassium dihydrogen
phosphate). Iodoacetic acid N-hydroxysuccinimide ester (25 mg)
was added and the pH was adjusted between 7 and 7.5. The reac-
tion was allowed proceed overnight. The devices or substrates were
then rinsed in PBS buffer, sonicated in water for 5 min and dried
under argon flow. The linker (iodoacetic acid N-hydrox-
ysuccinimide ester) modified devices were immersed in the pep-
tide solution in 100 mM phosphate buffer (61 mM disodium
hydrogen phosphate, 39 mM potassium dihydrogen phosphate) at
0.6 mg/mL overnight followed by incubation with excess of 1-
thioglycerol (10 mL/mL) for another day. The peptide immobilized
devices or substrates were thoroughly rinsed with 100 mM phos-
phate buffer and milli-Q water consecutively, and dried under
argon flow.
2.3. Surface characterization of AMP-brush coating on the surface
of catheter or PU sheet
ATR-FTIR spectra of AMP-brush devices were collected on a
Nexus 670 FT-IR ESP (Nicolet Instrument Corp., Waltham, MA) with
a MCT/A liquid nitrogen cooled detector, a KBr beam splitter, and a
MkII Auen Gate single-reflection attenuated total reflectance (ATR)
accessory (Specac Inc., Woodstock, GA). Spectra were recorded at
4 cm1 resolution, and 128 scans were collected. Water Contact
Angle Measurements: A water droplet (6 mL) was placed on the
surface and an image of the droplet was taken with a digital camera
(Retiga 1300, Q-imaging Co.). The contact angle was analyzed using
Northern Eclipse software. Over three different sites were tested for
each sample. X-ray photoelectron spectroscopy (XPS) was per-
formed using a Leybold LH Max 200 surface analysis system (Ley-
bold, Cologne, Germany) equipped with a Mg Ka source at a power
of 200 W. Atomic force microscopy measurements were performed
on a commercially available multimode system with an atomic
head of 130
130 mm2 scan range which used a NanoScope IIIa
controller (Digital Instruments, Santa Barbara, CA). A commercially
manufactured V-shaped silicon nitride (Si3N4) cantilever with gold
on the back for laser beam reflection (Veeco, NP-S10) was used. The
collection of approach and retraction force curves was performed in
PBS buffer (pH 7.4). On tip approach, the onset of region of constant
71
compliance was used to determine the zero distance. The rate of
tip-sample approach or retraction was set as 0.5 mm/s. The raw AFM
force data (cantilever deflection vs. displacement data) was con-
verted into force vs. separation following the principle of Ducker
et al. by using custom Matlab v.5.3 (Math Works, Natick, MA)
software [39]. The software converts the cantilever deflection vs.
linear voltage displacement transformer signal into restoring force
vs. tip-substrate separation using user input trigger and spring
constant values.
2.4. Antimicrobial activity testing of AMP-tethered 18G PU
catheters in LB medium
The antimicrobial activity of the AMP-tethered 18G PU catheter
was assessed using a static microtitre plate assay. Briefly, bacteria
(Pseudomonas aeruginosa luminescence tagged strain PAO1
Tn7:Plac-lux, Staphylococcus aureus luminescence tagged strain
Xen36 Lux, and S. saprophyticus strain (ATCC 15305) were grown in
Luria broth (LB; 10 g tryptone, 5 g yeast extract, and 10 g NaCl per
litre) from freezer stocks at 37  C O/N. The cultures were sub-
cultured, grown and used at 37  C and tested at approximately
5
105 CFU/mL as determined by OD600 readings using the equa-
tion 0.1 OD600 ¼ 108 CFU/mL. All coated and uncoated PU catheter
pieces (each N ¼ 5) were disinfected by submerging in 1 mL of 70%
ethanol for 5 min. The ethanol was then removed, and samples
were each rinsed with 1 mL of sterile LB for a total of 3 times. Once
LB from the last rinse was removed, 1 mL of the prepared bacterial
culture (~5
105 CFU/mL) was added to each sample in Eppendorf
tubes, and all tubes were gently tapped on the lab bench to ensure
all samples were fully submerged and were placed at 37  C at 50
RPM on shaker. At 6 h post-incubation, 100 mL of bacterial culture
was transferred to new, sterile tubes serially diluted and spot plated
for CFUs of planktonic growth. Each catheter piece was then rinsed
with 1 mL sterile PBS for 3 times were transferred into Eppendorf
tubes containing 500 mL sterlile PBS and were sonicated in water
bath for 10 min. After sonication, each sample was vortexed at high
speed for 10 s, then serially diluted and spot plated for CFU
measurements.
2.5. Antimicrobial activity testing of AMP-tethered 18G PU
catheters in artificial urine
Coated and uncoated samples were suspended in 70% ethanol
for 5 min. The ethanol was removed, and samples were rinsed in
sterile artificial urine (AU; composition in g/L: peptone L37, 1; yeast
extract, 0.005; lactic acid, 0.1; citric acid, 0.4; NaHCO3, 2.1; urea, 10;
uric acid, 0.07; creatinine, 0.8; CaCl2,2H2O, 0.37; NaCl, 5.2; FeS-
O4,2H2O, 0.0012; MgSO4,7H2O, 0.49; Na2SO4,2H2O, 3.2; KH2PO4,
0.95; K2HPO4, 1.2; NH4Cl, 1.3) for a total of 5 times. The last rinse
was removed, and each sample was introduced to AU containing
approximately 1 mL of 5
105 CFU/mL bacteria at per sample in
Eppendorf tubes. All samples were incubated at 37  C on a 360
rotator. The medium was refreshed every 24 h by replacing half of
the existing medium with fresh, sterile AU. At 4 h, 24 h, and 7 d
post-incubation, samples (N ¼ 6 per condition) were plated for
both planktonic growth and bacterial adhesion of PU catheter
surface. To plate for planktonic growth, bacterial culture was seri-
ally diluted and plated on LB agar for CFUs. All plates were incu-
bated at 37  C overnight or until visible colonies form. To plate for
bacterial adhesion, each sample was rinsed in sterile AU for a total
of 3 times. Rinsed samples were then transferred to 300 mL of AU
and sonicated for 10 min in a water bath. Samples were vortexed at
high speed for 10 s, then serially diluted and plated on LB agar for
CFUs. Plates were incubated at 37  C overnight or until visible
colonies form.
72 K. Yu et al. / Biomaterials 116 (2017) 69e81
2.6. Evaluation of antimicrobial activity of AMP-tethered 24G PU
catheters in vivo in a mouse urinary infection model
All procedures were performed with ethical approval from the
University of British Columbia animal care committee. A total of 21
male C57BL/6 mice (Harlan®) at 10 weeks of age were included in
experiments. One mouse developed pain after introducing the
bacteria in the bladder and didn't survive throughout the entire
study. Nine mice were included in the control group and 11 mice for
the treated group. Prior to animal procedures, uncoated and coated
24G catheters were modified under strict aseptic conditions.
Briefly, the needle portion of the catheter was temporarily
removed, and a 4 mm section from the tip of the PU catheter was
cut off using sterile blades. For uncoated samples, the 4 mm piece
and the remaining catheter portion was re-assembled back onto
the original needle (Supplementary Fig. S2). For coated samples, the
4 mm sections were rinsed in 70% ethanol for 5 min, and then
rinsed in sterile PBS for 3 times prior to being assembled back onto
the needle.
All mice were administered inhalational anesthesia with 3%
isoflurane for initial induction. Once anaesthetized, isoflurane was
set to 2.5%, and animals were positioned dorsally. The abdominal
area was shaved, and the area around the mouse bladder was
secured by using a plastic belt in a heating pad set at 38  C. Sterile
ultrasound gel was applied, and a Vevo 770® HighResolution Im-
aging System was used to locate and visualize the bladder. The
modified 24G PU catheter, mounted on the original needle, was
positioned at a 30-degree angle just above the pubic bone with the
bevel directed to the anterior. Once the needle had been properly
aligned and visualized on the ultrasound machine, it was carefully
inserted towards the bladder. As soon as the 4 mm catheter
segment was confirmed to be entirely inside the bladder via ul-
trasound imaging (Supplementary Fig. S3), the needle was removed
while the ‘pusher’ was pushed slightly inward. This dislodged the
short PU catheter segment into the lumen of the bladder, such that
once the ‘pusher’ was removed, the only thing that remained inside
the mouse bladder was the implanted 4 mm catheter piece.
One day after catheter implantation, all mice were anaes-
thetized and either P. aeruginosa or S. aureus (5
105 CFU/mL in
50 mL PBS) was percutaneously injected into the bladder lumen
using a 30 gauge needle under ultrasound guidance, utilizing
separate needles for each mouse. Once successfully injected, mice
were kept anaesthetized with 1% isoflurane for 1 h on a heating pad
to allow time for bacteria to adhere onto the implanted catheter.
IVIS images were taken, and the mice were then recovered from
anesthesia. The starting amount of bacterial inoculum was
confirmed by performing serial dilutions followed by CFU counts.
The Xenogen in vivo Imaging System (IVIS® Lumina, CA, USA) was
used to image for the presence of the luminescently-tagged bac-
teria by measuring luminescence levels detected within the mouse
body immediately after mice were instilled with bacteria (Day 1);
since bacterial luminescence requires bacteria to be energized, only
live bacteria produced light. Mice were again imaged for lumines-
cence at 7 days post-instillation (Day 7). Bioluminescence from the
region of interest (ROI) was expressed as total photon flux (pho-
tons/s). Background photon flux was defined from an ROI of the
same size drawn over the head of each mouse.
At 7 days post-instillation, all mice were sacrificed by CO2
asphyxiation. Urine samples were collected from the bladder (if
present), and the amount of bacteria in the urine was quantified via
serial dilutions and CFU counts. Indwelling catheters were
collected, rinsed in 200 mL of sterile PBS and transferred to 200 mL of
fresh PBS prior to sonication at 50/60 Hz for 10 min in an ultrasonic
water bath (No. 21811-820, VWR®) to aid biofilm dispersal. Samples
were then vortexed at high speed for 10 s, and bacterial numbers
were determined by serial dilutions and CFU counts. There were 1
and 2 cases of unsuccessful infection with P. aeruginosa in each
group as the CFU reading on both catheter surface and in urine was
0. In the case of S. aureus infections, in addition to the CFU mea-
surements, some of the explanted catheter samples were fixed in
2.5% glutaraldehyde (200 mL) for 1 h, then dehydrated using
gradient ethanol/water solutions (200 mL for each) (50%, 70%, 90%,
100%) for 10 min each. The samples were died in the ambient
condition, sputtered with gold with a thickness ~10 nm and viewed
using a scanning electron microscope (SEM, Hitachi S-3000 N) to
visualize biofilm formation.
2.7. Cytotoxicity of AMP-tethered PU substrates
AMP-tethered PU sheets were used for the cytotoxicity test due
to the limited surface area of catheter samples. The harvested
T24 cells (human bladder carcinoma cells) in McCoy's 5A medium
or firbroblast cells (3T3-L1) in Dulbecco's modified eagle medium
were aliquoted at ~3
105 well/500 mL, and seeded in 24-well
culture plates containing two sample groups: bare PU substrate
and AMP-tethered PU substrate. Each group comprised three
identical 1 cm
1 cm samples. At 1 and 3 days of culture, the
samples were transferred into empty wells, and washed with PBS
buffer (1 mL) for 2 times. Trypsin-EDTA solution (0.5 mL, 0.25%) was
added into each well to dislodge adherent cells. The plate was the
allowed to sit in an incubator for 3 min before adding 2 mL fresh
medium into each well. The cells were then transferred into new
tubes. After centrifugation at 70g for 5 min, the supernatant me-
dium was removed and the cell pellets were re-suspended into
200 mL PBS. Cell suspension (20 mL) was taken for cell number
counting using a hemocytometer (Bright-line, Hausser Scientific,
Horshan, PA, USA). Propidium iodide (PI) staining solution was
added into the tube to a final concentration of 10 mg/mL and PI-
stained cells were then transferred into flow cytometry assay
tubes and stored at 4  C in the dark. A BD FACSCanto II cell cy-
tometer was used to determine PI fluorescence of cells (PI only
enters into permenabilized, dead cells). The cell viability was
calculated using a pre-established gating protocol, and at same
time, data for unstained cells and single-color positive controls
were also acquired.
2.7.1. Statistical analysis
All the data values were expressed as mean ± standard deviation
(SD). Statistically significant value was set as p < 0.05 based on
Student's two-tailed unpaired t-test.
3. Results
3.1. Synthesis of AMP-brush coated PU catheters
An illustration of the functionalization of a catheter with AMP-
brush coating is given in Fig. 1. The polymer brush coating was
prepared using surface initiated atom transfer radical polymeriza-
tion (SI-ATRP) from PU catheter surface. The PU catheter was
initially modified with allylamine plasma treatment to modify the
PU surface via the addition of an amine layer, followed by the re-
action with 2-chloro-N-glycidyl propionamide to generate ATRP
initiating sites. The presence of a Cl (2p) peak in the XPS spectra of
modified catheter surfaces confirmed the successful coupling of the
initiator (Supplementary Fig. S1) on the surface. The ATRP initiating
groups were used to facilitate the copolymerization of DMA (N,N-
dimethylacrylamide) and APMA (N-(3-aminopropyl) meth-
acrylamide hydrochloride) to generate a PDMA-co-APMA polymer
brush layer on the catheter surface. The successful grafting of the
polymer was confirmed by ATR-FTIR measurements (Fig. 2A). The
 K. Yu et al. / Biomaterials 116 (2017) 69e81 73

Fig. 1. Functionalization of polyurethane catheter with AMP-brush coating. The catheter was initially treated with allylamine to generate a surface layer containing amine
group. This was followed by a reaction with epoxy containing ATRP initiator to generate polymerization initiating sites. PDMA-co-APMA brushes were grown from these initiator
modified PU catheter by SI-ATRP. The sulfhydryl group of C-terminal cysteine peptides E6 (sequence RRWRIVVIRVRRC) were used tether it to the PDMA-co-APMA brush func-
tionalized with iodoacetic acid N-hydroxysuccinimide ester.

Fig. 2. Surface characterization of catheter coating. (A) ATR-FTIR spectra of PDMA-co-APMA brush and AMP-brush coated PU catheter surface. The presence of absorption peaks
at 1627 and 1550 cm1 in the PDMA-co-APMA-Catheter spectrum is attributed to the C]O stretching and the N-H bending vibrations of the amide group of the polymers. The broad
peak at 3300 cm1 in the spectrum is due to the stretching of N-H in the amide and amine groups. The increase in peak intensity at 1627 and 1550 cm1, and 3300 cm1 in the
PDMA-co-APMA-E6-Catheter spectrum indicates that the peptide is successfully conjugated on to catheter surface. (B) Surface morphology of allylamine modified PU catheter (i),
PDMA-co-APMA brush coated PU catheter (ii) PDMA-co-APMA brush coating conjugated with AMP on PU catheter (iii). Representative AFM force approach curves for the initiator,
brush, and AMP tethered catheter surface (iv) confirm the formation of polymer brush on PU surface. The arrows indicate the jump to contact (JTC) interaction between the AFM tips
and the catheter surface.

appearance of peaks at 1627 and 1550 cm1 was attributed to the
C]O stretching and the N-H bending vibrations of the amide group
of the grafted polymers. The broad peak at 3300 cm1 in the
spectrum was due to the stretching of N-H in the amide group. The
modification was also evidenced from the water contact angle of
the polymer modified PU surface (48.9 ± 4.2 ), which was lower
than that of the parent initiator modified substrate (60.8 ± 3.5 ).
AFM measurements were employed to characterize the
morphology of grafted polymer on the catheter (with gauge size of
14G) surface. The brush conjugated catheter surface was smooth as
indicated by a roughness reading of 1.4 nm (Fig. 2Bii). The equi-
librium thickness (hydrated thickness) of the polymer layer on the
catheter surface as probed by AFM force spectroscopy was
75.8 ± 5.3 nm (Fig. 2Biv). The grafted polymer conformation on the
catheter surface are still in brush conformation based on the dis-
tance between the grafted chains (s1/2, 0.55 nm) being less than
the radius of gyration (Rg, measured by multi-angle laser light
scattering) of the chains in solution (2Rg ¼ 2
20.6 nm) [38].
The primary amine groups on the PDMA-co-APMA brush were
further modified with iodoacetic acid N-hydroxysuccinimide ester
linker for coupling with cysteine containing peptides. A high-
resolution XPS surface scan conducted on the modified substrate
showed the presence of iodine (3d) on the surface which confirmed
the modification of PDMA-co-APMA with the linker molecule
(Fig. 3Ci). The water contact angle of the linker conjugated sub-
strate increased to 62.1 ± 2.9 compared to the unconjugated brush
modified substrate (48.9 ± 4.2 ). Iodoacetyl groups in the linker
were then reacted with thiol groups of the cysteine residue at the C-
terminus of the AMPs to generate the antibacterial PU surface. A
cathelicidin-related peptide E6 (with cysteine at the C-terminus)
was chosen to construct the AMP-tethered polymer brush coating
on the catheter surface as it is biocompatible and has low MIC
values against urinary pathogens (<32 mg/mL) [26,39]. The increase
in peak intensity measured by ATR-FTIR spectroscopy at 1627 and
1550 cm1, and 3300 cm1 (Fig. 2A) indicated the presence of the
carbonyl and N-H groups of the peptide. The presence of the S(2p)
peak (Fig. 3B), the disappearance of the I (3d) peak (Fig. 3Cii) and an
increase in the N/C ratio from 0.11 to 0.19 (Fig. 3A) in the surface
elemental composition of the AMP modified surface confirmed the
covalent conjugation of E6 to the PU substrate. The successful
modification was further supported by the decrease in water con-
tact angle to 46.9 ± 2.1 following peptide immobilization and the
increase in surface roughness to 2.6 nm (Fig. 2Biii) after peptide
immbobilization. Unlike unmodified PDMA-co-APMA brush, AFM
force-distance measurements showed a weak jump-to contact (JTC)
force [29] when the AFM tip approached the peptide conjugated
catheter surface. The peptide density on the surface was 1.58 mg/
cm2 determined by comparing the increase in the peak intensity in
74 K. Yu et al. / Biomaterials 116 (2017) 69e81

Fig. 3. Surface characterization of modified catheter. (A) XPS survey scan of PDMA-co-APMA brush, brush with iodoacetamide linker and AMP-brush coating on PU catheter. (B)
High resolution XPS scan of S(2p) on AMP-brush coated PU catheter. (C) High resolution scan of I(3d) on PU catheter surface before (i) and after (ii) peptide conjugation. The
presence of S(2p) and disappearance of I(3d) after peptide conjugation confirmed the conjugation of peptide on catheter surface.

ATR-FTIR spectra at 1627 cm1 to that obtained for the nano- polymer brush alone was able to reduce the adhesion of
particulate system [26]. P. aeruginosa by about 67.7% (0.5 log, P < 0.05), compared to the
uncoated catheter, while the tethering of E6 significantly increased
the antifouling nature of the coating, reducing bacteria by 93.2%
3.2. In vitro antimicrobial efficiency of surface tethered AMPs
(1.2 log, P < 0.05)). The E6 tethered coating also appeared to have an
antimicrobial effect on planktonic bacteria, decreasing the number
The antimicrobial activity of E6 tethered catheter surfaces was
of bacteria by 27.2± 14.7% (0.14 log, P < 0.05) compared to the bare
tested in both LB medium and artificial urine (AU) against some of
catheter surface (Fig. 4B). The polymer brush tethered E6 coating
the most common uropathogens including Gram-negative and
showed greater efficiency against Gram-positive bacteria, since
positive bacteria (P. aeruginosa, S. aureus, S. saprophyticus). Pristine,
tethering E6 to the PU catheter surface reduced the adhesion of
brush coated and peptide conjugated brush coated catheter sur-
S. aureus and S. saprophyticus by approximately 98.4% and 96.6%
faces were tested by assessing the amount of adhered bacteria on
(1.8 log, and 1.5 log, P < 0.001) respectively (Fig. 4C, E) in compar-
the material surface and planktonic bacteria in solution following
ison to the control catheters. The brush modification alone showed
6 h of incubation (Fig. 4). Fig. 4A shows the adhesion of P. aeruginosa
66.9% and 78.6% (0.48 log and 0.67 log, P < 0.001) reduction in
on differently coated surfaces, while Fig. 4B shows the number of
bacterial adhesion respectively. The reduction in planktonic growth
planktonic bacteria left in solution. These results showed that the
 K. Yu et al. / Biomaterials 116 (2017) 69e81 75

Fig. 4. Broad spectrum activity of AMP conjugated PU catheter in vitro (in LB medium): Reduction of bacterial adhesion on catheter surface and planktonic growth of
P. aeruginosa (A, B), S. aureus (C, D) and S. saprophyticus (E, F) on brush and AMP-brush coated catheter surface. The experiment was carried out in LB medium. * indicates P  0.05, **
indicates P  0.01, and *** indicates P  0.001. 1 mL bacterial culture with initial count of ~5
105 CFU/mL was added to each sample in Eppendorf tubes and incubated at 37  C at 50
RPM for 6 h. The adhered bacteria were then detached from the catheter by sonication and spot plated for CFU counts. The concentration of planktonic growth of bacteria was
measured by CFUs as well.

of P. aeruginosa was not seen in the case of S. aureus and Even after 7 days, the E6 coated catheter effectively reduced bac-
S. saprophyticus (Fig. 4D, F) compared to control catheter for brush terial adherence by approximately 78.5% (0.67 log), indicating long-
modified or E6 tethered catheters. More study needed to access term efficacy against S. aureus. The planktonic growth of S. aureus in
how the tethered peptide interacted with planktonic bacteria, presence of E6 conjugated catheter was also decreased compared to
especially P. aeruginosa. Collectively these data indicated that the control catheter (Fig. 5D).
tethering E6 to the anti-adhesive polymer brush significantly
increased the antimicrobial potency of the coating.
3.3. Antimicrobial efficiency of the catheter coating in a mouse
To assess the antimicrobial efficacy of the coating in a more
urinary infection model
relevant environment, its activity was tested over time in the
presence of artificial urine, since this most closely mimics the
To show efficacy of the E6-coating in vivo, we utilized an
physiological environment for the study of biofilms formed inside
ultrasound-guided percutaneous model of catheter-associated
the bladder. The adherence of P. aeruginosa to coated PU catheter
urinary tract infections developed in our lab [40]. This model has
surfaces was decreased by 91.3% and 89.2% (1.1 log and 0.97 log,
proven to be very efficient with a 100% catheter implantation rate
P < 0.001) at 4 h and 24 h post-inoculation respectively (Fig. 5A)
and retention of catheters. Furthermore, it allows for the easy re-
compared to the uncoated control, similar to results in LB medium
covery of catheters following infection for further analysis. Given
(Fig. 4A). No significant difference in adhesion between coated and
that we have observed a more consistent infection rate in this
uncoated samples was observed after prolonged incubation times
model using P. aeruginosa, we opted to test our novel coating
(7 days). Aside from seeing an effect of the AMP-coating on bac-
against this pathogen. Following the implantation of the catheter
terial adhesion, a reduction in planktonic growth of P. aeruginosa by
pieces, bacterial instillation and the development of infection was
about 30.7% (0.16 log, P < 0.05) was observed at 4 h post-
visualized using IVIS® imaging (the bacteria bioluminesce due to
inoculation compared to the uncoated controls, which was not
expression of lux gene) (Fig. 6A), which showed a similar bacterial
present after 24 h or 7days of co-incubation (Fig. 5B). The polymer
load in the bladders of animals implanted with either untreated
brush-peptide coating was stable in artificial urine for 7 days as the
control catheters or E6 coated catheters as indicated by a similar
ATR-FTIR measurement (Supplementary Info, Fig. S4) revealed
average total flux on day 1 post bacterial instillation between the
there was no change of peak intensity at 1627 cm1, which is due to
two groups of animals (1.7
105 and 1.6
105 photons/s respec-
the amide bonds in the polymer and peptides.
tively). After 4 days post-instillation, the total photon flux
While the AMP-coating proved to be effective against
measured for mice implanted with E6 coated catheters averaged
P. aeruginosa in both LB medium and artificial urine, a greater
8.2
104, a 96.6% (P < 0.01) reduction compared to mice implanted
reduction in bacterial adhesion on the E6-coated catheter segments
with untreated control catheters. Impressively, IVIS measurements
was observed against S. aureus in artificial urine. The adhesion of
of mouse bladders on day 7 post-infection showed the average total
this Gram-positive uropathogen was observed to be reduced by
photon flux reading for mice bearing E6 coated catheters to be even
99.9% (3 log, P < 0.001) after 4 h and 24 h of co-incubation (Fig. 5C).
lower (6.7
104 photons/s) compared to their control counterparts
76 K. Yu et al. / Biomaterials 116 (2017) 69e81

Fig. 5. Broad spectrum activity of AMP conjugated PU catheter in vitro (in artificial urine): Reduction of bacterial adhesion on PU catheter surface and planktonic growth of
P. aeruginosa (A, B), S. aureus (C, D) on AMP-brush coated catheter surface. The experiment was carried out using artificial urine. * indicates P  0.05, ** indicates P  0.01, and ***
indicates P  0.001. Each sample was introduced to artificial urine containing approximately 1 mL of 5
105 CFU/mL bacteria at per sample in Eppendorf tubes and incubated at
37  C on a 360 rotator. The medium was refreshed every 24 h by replacing half of the existing medium with fresh, sterile AU. After post-incubation at 4 h, 24 h, and 7d, samples
were plated for both planktonic growth and bacterial adhesion of PU catheter surface.

(2.32
107 photons/s) indicating a reduction close to 99.7%
(P < 0.01). The reduction in bacterial numbers both on the catheter
sample as well as in the urine was confirmed by CFU counts on day
7 (Fig. 7). Specifically, the number of adherent bacteria on uncoated
control catheters was 1.2
106 CFU/mL, whereas that for catheters
coated with the polymer conjugated E6 was 5
101 CFU/mL,
demonstrating a greater than 4-log decrease in bacterial adherence
on coated catheters. In terms of bacterial growth in the urine on day
7, the average CFU counts for mice bearing untreated control
catheters was 1.1
107 CFU/mL while those implanted with cath-
eters bearing polymer-conjugated E6 was 1.47
104 CFU/mL
indicating a nearly 3-log reduction in the number of planktonic
bacteria in the bladders of animals from the two different groups.
3.4. Biocompatibility of E6 conjugated catheter coating
The biocompatibility of the coating is another important aspect
with regard to its potential clinical translation. To obtain an initial
indication of the biocompatibility of the coating, we utilized a cell
cytotoxicity assay using both human bladder epithelial cells (T24)
and fibroblasts, which are two cell types the catheter surface might
come into contact with while indwelling. The results are shown in
Fig. 8. Following 24 h of co-incubation, the number of cells grown in
wells containing substrate coated with peptide E6 and bare PU
substrate were observed to be similar for both cell types. However,
on day 3, wells containing peptide coated samples showed slightly
higher cell numbers compared to those containing control bare PU
samples, which was again consistent between the two cell types
(Fig. 8A, C). Collectively these results suggest that our novel poly-
mer conjugated E6 coating does not impart any toxic effects on the
T24 and fibroblast cells lines (Fig. 8B, D). Future studies will be
aimed at studying the cytotoxicity and genotoxicity of peptide
tethered substrates using primary urothelial cell lines to support
the biocompatibility.
4. Discussion
The use of antimicrobial peptide-based coatings on indwelling
urinary devices provides a promising approach to prevent CAUTIs.
While significant research into the development of new AMPs and
their conjugation to catheter biomaterial surfaces has been per-
formed, their efficacy has only been tested using in vitro conditions
to date [32e36] rather than in a relevant animal model. Using a
clinically relevant in vivo catheter-associated urinary tract infection
model developed in our laboratories, the work presented here
demonstrates the high efficacy of a novel anti-adhesive coating
consisting of polymer brush conjugated E6 antimicrobial peptide,
in significantly reducing bacterial biofilm formation on clinically
used polyurethane catheter surfaces over a 7 day period. Further-
more, the coating was shown to have excellent biocompatibility
 K. Yu et al. / Biomaterials 116 (2017) 69e81 77

Fig. 6. Antibacterial activity of AMP conjugated PU catheter in vivo in urinary infection model (A) IVIS images of mice bearing either uncoated (untreated) or AMP-Brush coated
(treated) PU catheter at day 1 (1 h after instillation with P. aeruginosa lux into bladder) and 7 days post-instillation with P. aeruginosa lux into bladder. (B) Bioluminescence readings
measured for untreated and treated mice using the IVIS® Lumina. Bioluminescence from the region of interest (ROI) was defined manually, and the data were expressed as total
photon flux (photons/s). All bioluminescent data were collected and analyzed using IVIS at day 1, 4 and 7 days post-bacterial-instillation into mice bladder. * indicates P  0.05, **
indicates P  0.01, and *** indicates P  0.001. P. aeruginosa (5
105 CFU/mL in 50 mL PBS) was percutaneously injected into the bladder lumen using a 30 gauge needle under
ultrasound guidance at day 1.

with relevant cell lines. The translation of these results to realistic
indwelling urinary devices is in the use of a clinically relevant
biomedical plastic, polyurethane, which is extensively used in
urological applications, in particular in urethral catheters and
ureteral stents [15] two of the most commonly used devices not
only in urology, but across other medical specialties.
The development of our novel coating involves an initial plasma
modification of the catheter surface with allylamine plasma, fol-
lowed by the generation of an antifouling PDMA-co-APMA polymer
brushes on the device surface. Interestingly this PDMA-co-APMA
brush coating was shown to prevent the accumulation of bacteria
by ~70% on the catheter surface in both LB medium and artificial
urine, likely by acting to repel planktonic bacteria (Fig. 4). These
results suggest that this component alone already imparts a sig-
nificant anti-adhesive characteristic to the surface. Given the
attractiveness of a coating that not only imparts anti-adhesive
characteristics but that also acts to kill bacteria, we chose to in-
crease the potency of our coating by modifying the coating further
through the immobilization of the antimicrobial peptide E6,
recently shown to have significant antimicrobial activity against
diverse uropathogens [39]. The E6 conjugated brush coatings
showed greater efficacy (>93%) in vitro in preventing bacterial
adhesion to the surface and subsequent colonization by both Gram-
positive and -negative bacteria, including S. saprophyticus, S. aureus
and P. aeruginosa. The additional decrease in bacterial adhesion and
colonization of the E6 conjugated surfaces could be attributed to
the direct killing of bacteria by the tethered peptide in combination
with the anti-adhesive characteristics of the PDMA-co-AMPA brush
[26]. Previous circular dichroism measurements [26,41] indicated
that tethered peptides assume a much more ordered peptide
structure upon interaction with biomembranes compared to their
soluble counterparts. Tethering E6 to PDMA brush resulted in the
greatest change in secondary structure upon interacting with
synthetic biomembranes which might relate to its potent antimi-
crobial activity [26]. The peptides actively killed bacteria adhered
on the catheter surface, while the brush layer protected the surface
by limiting the accumulation of bacterial debris (antifouling prop-
erty of the brush) thereby reducing the inactivation of AMP action
78 K. Yu et al. / Biomaterials 116 (2017) 69e81

Fig. 7. Antibacterial activity of AMP conjugated PU catheter in vivo in urinary infection model. Number of survived P. aeruginosa recovered from the PU catheter surface (A) and
in the urine (B) after 7 days in the mice percutaneous model. N ¼ 8 for the control (pristine PU catheter) and N ¼ 9 for the AMP brush coated catheter. * indicates P  0.05, **
indicates P  0.01, and *** indicates P  0.001. P. aeruginosa (5
105 CFU/mL in 50 mL PBS) was percutaneously injected into the bladder lumen using a 30 gauge needle under
ultrasound guidance at day 1.

Fig. 8. In vitro toxicity of AMP-brush coating on PU surface. The number and viability of T24 cells (A, B) and fibroblast (C, D) grown on the AMP-brush coating. Bare PU sheet and
AMP tethered PU sheet (1 cm
1 cm) were used for the cytotoxicity test. 1 mL cell culture with initial count of ~3.1
105/mL was added to each sample in the wells of cell-cultured
plate and incubated at 37  C for 1 day and 3 days. The adhered cells were then detached from the plate by Trypsin-EDTA solution and stained with PI for flow cytometery analysis.

and providing a regenerating surface to kill bacteria [29,42e44]. In
addition, the polymer brush allowed the active antimicrobial
component of our coating to act further away from the device
surface. This is an important characteristic in the urinary tract, since
previous coating technologies and materials that rely on the active
ingredient to be incorporated directly into the material, have been
rendered inactive by the deposition and accumulation of urinary
components onto the device surface to form a conditioning film
shortly following device insertion. Having the polymer brush
present the antimicrobial peptide away from the surface and
limiting the formation of a conditioning film due to its anti-
adhesive nature would increase the long term antimicrobial ac-
tivity of the polymer brush based coating. The fact that our coating
was still active in vivo following 7 days of infection strongly sup-
ports this hypothesis.
The AMP-brush coating showed slightly better antimicrobial
activity in artificial urine compared to LB medium. For example, the
reduction of bacterial adhesion for S. aureus was 99.9% (3 log) after
 K. Yu et al. / Biomaterials 116 (2017) 69e81
4 h and 24 h in artificial urine compared to a reduction of 98.4% (1.8
log) observed in LB. The difference in antimicrobial activity
observed could be due to differences in bacterial growth patterns in
the different media due to varied nutrient content. This is evident
from the fact that the CFU count of bacteria in the less nutrient
enriched artificial urine was approximately 1 log lower than that in
LB at similar incubation times. Nonetheless, the trends in terms of
anti-adhesive and antimicrobial activities of the coatings were
similar when tested in the different media suggesting that their
activities are consistent in different types of environments.
Interestingly, we observed different long-term effects of the
polymer conjugated E6 coating between P aeruginosa and S. aureus
in artificial urine, since the efficacy of the coating was higher over
the 7-day co-incubation period for S. aureus compared to
P. aeruginosa (no decrease in adhesion was observed for the latter
on day 7). This is likely due to the difference in growth rates be-
tween the two bacterial species, since the total number of bacteria
for P. aeruginosa samples was always significantly higher than
S. aureus. The decrease in efficacy over time against P. aeruginosa is
likely a reflection of the fact that the number of bacteria over-
whelms the capacity of the peptide surface to repel (polymer
brush) and/or kill the bacteria (E6 peptide). This is further sup-
ported by the experiments studying the effect of the coating on
planktonic growth, wherein the efficacy against P. aeruginosa was
observed at the 4 h time point where the number of bacteria were
similar to that for S. aureus at 24 h and efficacy was still observed for
the latter; however, when the bacterial density for either species
approached 108 CFU/mL (24 h and 7 days for P aeruginosa; 7 days
for S. aureus) there was no effect on planktonic growth. These re-
sults suggest that varying polymer brush/peptide densities may
need to be considered to broaden the efficacy against multiple
bacterial species. Given this, further studies into identifying a
density that shows broader spectrum efficacy is warranted.
To test the efficacy of our novel coating in a realistic environ-
ment, we utilized our mouse model of CAUTI. In this model, the
catheter material is introduced into the bladder percutaneously
using ultrasound guidance, which is minimally invasive and en-
sures correct placement and retention of the material within the
bladder over the 7-day indwelling period. Upon challenge with
P. aeruginosa (106 CFU/mL), the peptide coating was found to resist
bacterial adhesion and biofilm formation over a 7-day infection
period. Interestingly, aside from decreasing bacterial adhesion, the
peptide coating also had an effect on extra-catheter bacterial
growth as indicated by a significant decrease in CFU counts in the
urine of animals implanted with the E6-coated material. These
results were very impressive, and this study is the first to report
such high efficacy (>4 log reduction) against bacterial biofilm for-
mation on indwelling device surfaces in vivo [28,30,45,46]. Previous
animal studies using subcutaneous infection models [28,30] have
shown titanium implants coated with melamine and maleimide-
tethered Tet20 to reduce bacterial adhesion and colonization by
only 1.3 log and 0.9 logs respectively. Even though the AMP coating
developed in the current study showed greater efficiency in
combating indwelling device-associated biofilm formation and
infection, the bacterial strains and animal model used to test its
efficacy differed from those of the previous publications. Given that
the conditions of testing differ, a direct comparison can not be
made. Compared to the conventionally used silver alloy coated
catheter shown to be mostly affective against Gram-negative bacilli
only [47,48], the AMP conjugated catheter material developed here
shows broad-spectrum activity against different species. While
nitrofurazone coated catheters have shown better activity
compared to the AMP conjugated catheter materials in vitro [47,48],
the antimicrobial activity of the nitrofurazone coated material
decreased with time as its activity was diffusion-dependent.
79
Overall, the clinical use of these marketed products has not resul-
ted in a reduction in the incidence of symptomatic CAUTI or clini-
cally relevant urinary tract infection [49]. Further studies against a
variety of bacterial pathogens must be made in additional in vivo
models to give us an idea of whether our AMP-based coatings may
have superior activity compared to silver and nitrofurazone-
dependent coatings.
Our in vivo results were superior compared to the in vitro data in
terms of demonstrating the anti-adhesion and antimicrobial
properties of the AMP-tethered brush coating. Since the exchange
of urine in the bladder is constant given the frequency of urination
and production of urine by the kidneys, there is a good chance that
the passivating bacterial debris and some bacteria are being
constantly removed from the bladder as part of normal urination.
This phenomenon may contribute to the increased activity of the E6
tethered coating in the bladder as demonstrated. In addition to this,
the presence of an adaptive immune response in vivo that is able to
readily respond to infection [50,51], may also be contributing to the
increased bacterial clearance seen in the mouse model. Aside from
the conventional direct response to live bacteria, bacterial debris
formed in the bladder upon contact with E6 coated catheter might
evoke additional immune responses such as inflammation and the
recruitment of specific T- and B-cells to the site of infection by
activated antigen presenting cells, including macrophages [52e56].
Additionally, other mechanisms may include the phagocytosis of
soluble or tethered forms of antimicrobial peptides by host im-
mune cells, triggering further immunomodulatory effects that
result in fighting of infection [57e59]. Further studies will aid our
understanding of the specific mechanisms that contribute to
increased clearance of bacteria by polymer brush tethered E6 in the
in vivo environment.
In addition to challenging the peptide coated catheter materials
in vivo with P. aeruginosa, we also performed in vivo experiments
using S. aureus. In contrast to experiments using P. aeruginosa, we
found the majority of animals bearing both peptide-coated and
uncoated catheter materials were able to spontaneously and
rapidly clear the infection. As a result, it was challenging to evaluate
the efficacy of the coating against S. aureus in this model. For the
animals in which minimal infection (as indicated by low CFU counts
on catheter samples) was observed, there was a trend towards
fewer bacteria on the AMP coated catheter surface which may
suggest efficacy against S. aureus in vivo as well (Supplementary
Information, Fig. S5).
5. Conclusions
In conclusion, we reported the development of an anti-adhesive
antimicrobial peptide coating on clinically used biomedical plastic,
polyurethane and established the antimicrobial efficacy, both
in vitro and in vivo. The use of AMP E6 in combination with anti-
adhesive polymer brush coating conferred excellent antimicrobial
activity toward P. aeruginosa, S. aureus and S. saprophyticus while
providing strong biocompatibility. Importantly, the newly devel-
oped anti-adhesive coating showed a >4 log reduction in bacterial
adhesion on polyurethane catheter samples in a mouse CAUTI
model. The current study opens the way for the development of
AMP-immobilized catheter for preventing and limiting the occur-
rence of CAUTIs. The anti-adhesive, antimicrobial and biocompat-
ible properties make the polymer brush-AMP coating an excellent
candidate for further development as a coating for medical devices
susceptible to bacterial adhesion, colonization and infection.
Acknowledgements
This research was funded by the Canadian Institutes of Health
80 K. Yu et al. / Biomaterials 116 (2017) 69e81
Research and Natural Science and Engineering Research Council
(NSERC) of Canada. The authors thank the LMB Macromolecular
Hub at the UBC Centre for Blood Research for use of the analytical
facilities. The infrastructure facility is supported by Canada Foun-
dation for Innovation (CFI) and the British Columbia Knowledge
Development Fund (BCKDF). JNK is the recipient of a Career
Investigator Scholar award from Michael Smith Foundation of
Health Research. DK is the recipient of a New Investigator award
from CIHR. REWH holds a Canada Research Chair in new anti-
infective drug discovery. Dr. Deng Bo is acknowledged for
providing 2-chloro-N-glycidyl propionamide.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.biomaterials.2016.11.047.
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