Restriction of in vivo infection by antifouling coating on urinary catheter with controllable and sustained silver release: a proof of concept study
Catheter Associated Urinary Tract Infections are among the most common urological infections world-wide. Bacterial biofilms and encrustation cause significant complications in patients with urinary catheters. The objective of the study is to demonstrate the efficacy and safety of an anti-microbial and anti-encrustation silver nanoparticle (AgNP) coating on silicone urinary catheter in two different animal models.
Antifouling coating (P3) was prepared with alternate layers of polydopamine and AgNP and an outermost antifouling layer. Sixteen C57BL/6 female mice and two female PWG Micropigs® were used to perform the experiments. In mice, a 5 mm long silicone catheter with or without P3 was transurethrally placed into the urinary bladder. Micropigs were transurethrally implanted – one with P3 silicone catheter and the other with commercially available silver coated silicone catheter. Both models were challenged with E. coli. Bacteriuria was evaluated routinely and upon end of study (2 weeks for mice, 3 weeks for micropigs), blood, catheters and bladders were harvested and analysed for bacterial colonization and encrustation as well as for toxicity.
Lower bacterial colonization was seen on P3 catheters as well as in bladders of animals with P3 catheter. Bacteriuria was consistently less in mice with P3 catheter than with uncoated catheters. Encrustation was lower on P3 catheter and in bladder of micropig with P3 catheter. No significant toxicity of P3 was observed in mice or in micropig as compared to controls. The numbers were small in this proof of concept study and technical issues were noted especially with the porcine model.
Antifouling P3 coating reduces bacterial colonization on catheter and in animal bladders without causing any considerable toxicity for 2 to 3 weeks. This novel coating could potentially reduce the complications of indwelling urethral catheters.
KeywordsCAUTI Silver nanoparticles Urinary catheter Mouse model Porcine model Catheter associated urinary tract infections
Blood urea nitrogen
Catheter Associated Urinary Tract Infections
Colony forming unit
day(s) post inoculation
Institutional Animal Care and Use Committee
Inductive Coupled Plasma-Optical Emission Spectrometry
Anti-fouling coating prepared by coating PDA-AgNP-PDA-AgNP-PDA-poly(SBMA-co-AAm) layers on the inside as well as outside surfaces of the silicone tubing
Prestige BioResearch Pte Ltd., Singapore
Scanning electron microscopy
Uropathogenic Escherichia coli
Urinary catheters are among the most common medical devices in clinical use . However, catheter associated urinary tract infections (CAUTI) are among the most common hospital acquired infections [2, 3, 4]. The risk of CAUTI is reported to be between 3 and 7% daily and increases with the duration of catheterization .
Uropathogenic Escherichia coli (UPEC) are responsible for most CAUTI . Many UPEC strains are now resistant to multiple antibiotics, and thus difficult to treat, emphasizing the importance of preventative strategies [7, 8, 9].
Intraluminal and extraluminal biofilms, caused by the attachment of bacteria to the catheter, facilitate the entry and persistence of uropathogens in the bladder and thus promote the development of resistance . In addition, urease producing bacteria, especially P. mirabilis, contribute significantly to catheter encrustation through precipitation of mineral salts such as calcium phosphate and magnesium ammonium phosphate . These encrustations and biofilms are major contributors to morbidity and mortality in CAUTI [11, 12, 13, 14]. They are responsible for the failure of many medical devices . Novel approaches to preventing CAUTI have focussed on preventing the development of biofilms using various techniques including novel coatings .
We have previously developed an anti-fouling catheter with controllable and sustained silver release which is effective in resisting encrustation induced by Proteus mirabilis in vitro for up to 45 days . The coating comprises multiple layers of silver using polydopamine (PDA) as the surface anchor which reduces the silver ions into silver nanoparticles (AgNPs) and stabilizes and protects the AgNPs from oxidization and aggregation. Therefore, the amount of silver loaded, and its subsequent release profile is readily controllable by changing the number of silver-containing layers. The outermost layer is made of an antifouling polymer, poly (sulfobetaine methacrylate-co-acrylamide) [poly (SBMA-co-AAm)], which also allows free diffusion of silver. Thus, this novel coating is different in having a higher intrinsic silver release capability than the current commercially available silver coated urinary catheters.
Here, we used a previously described mouse model [18, 19] to study the safety and potential efficacy of this catheter against biofilm formation and colonization by UPEC in vivo. To further test the coating for human catheters, we chose a porcine model for our in vivo experiments as it provides a urinary tract that resembles the macroscopic human urinary tract fairly well and is a very accessible animal for laboratory testing . To our knowledge, this model has not been used for a long-term catheterization study. It was also important to compare the efficacy of our catheter to a silver coated catheter currently in use in patients. Therefore, we used an uncoated control in the mouse model whereas a commercially available silver coated catheter control was used in the porcine model.
This study was funded by Technology Enterprise Commercialisation Scheme-Proof-Of-Concept grant of SPRING Singapore (TI/TECS/POC/14/10).
Bacteria and growth conditions
E. coli UTI89, an uropathogenic strain known to be a good biofilm developer, isolated from a patient with uncomplicated cystitis, was kindly provided by Dr. Swaine Chen of Genome Institute of Singapore . E. coli was cultured in lysogeny broth (LB) (BD Difco, Singapore) at 37 °C with constant agitation at 180–200 rpm. LB agar was used to grow E. coli on solid medium and incubated at 37 °C for 12–18 h.
Catheters for mouse model of CAUTI
Small silicone tubing, SIL037 (3 French, 0.5 mm ID × 0.9 mm OD) was obtained from Braintree Scientific, Inc., USA. Anti-fouling coating (P3) was prepared by coating PDA-AgNP-PDA-AgNP-PDA-poly(SBMA-co-AAm) layers on the inside as well as outside surfaces of the silicone tubing as described previously . The method was modified to expedite the coating process as polydopamine was coated with oxygen bubbling at 60 °C for 20 min, instead of in air at room temperature for 24 h in our earlier work. Silver content in the coating was determined to be 33.98 ± 1.62 μg/cm2 using Inductive Coupled Plasma-Optical Emission Spectrometry (ICP-OES, iCAP 6200 duo, Thermo Scientific, USA) as previously described . The tubing was cut in 5 mm long segments, autoclaved and air-dried in a clean biosafety cabinet (BSC) before use in mice. Twelve mm long segments of SIL037 catheter tubing were fitted on sterile 25 G needles (Becton Dickinson). A 5 mm long, either coated or uncoated, sterile segment of SIL037 tubing was then placed on top of the 12 mm segment for implantation.
Catheters for porcine model of CAUTI
Uncoated silicone catheters of 14 Fr size were purchased from Promed Pte. Ltd., Singapore. P3 coated catheter was prepared by coating PDA-AgNP-PDA-AgNP-PDA-poly(SBMA-co-AAm) layers on pristine silicone catheter over the interior and exterior of the whole length as described previously with modification to obtain a similar silver content (30.30 ± 1.33 μg/cm2) as in the mouse work . For a 14 Fr Foley catheter (40 cm long), total silver amount was estimated to be approximately 3 mg per catheter. A commercially available, Dover™ silver coated 100% silicone Foley catheter was purchased from Covidien Pte. Ltd., Singapore.
Since the balloon inflation port of the silicone catheter was not autoclave-compatible, the coated catheters were disinfected by 70% ethanol followed by UV irradiation for 30 min and sealed before the pig study. The Dover™ catheter was used as received since it was in sterile package.
Murine model of CAUTI
All animal experiments were conducted in accordance with the university ethical regulations and were approved by the National University of Singapore Institutional Animal Care and Use Committee (IACUC). All animals were allowed an acclimatization period of at least five days in the animal testing facility after arrival, prior to experimentation. Laboratory feed and drinking water were provided ad libitum during entire duration, including before and during experimentation. The animal subjects were provided with environmental enrichment in accordance to species-specific standards. The temperature and humidity of the housing space was regulated as per international standards, and animals were provided with a cycle of 12 h of light followed by 12 h of darkness.
The mouse model was adapted from a previously published study with modifications mainly in the implant size and bacteria used . Briefly, 18–20 weeks old C57BL/6 female mice (from InVivos Pvt. Ltd., Singapore) were anesthetized and 5 mm long catheter pieces were delivered in the urinary bladders by catheterizing the mice with a covered 25 G needle via the urethra lumen. The mice were re-catheterized with IV Catheters (24 G) and 50 μL bacterial cultures (2 x 106 Colony Forming Units (CFU)/ml) were instilled in the urinary bladder. After 30 min dwelling, the bladder was gently pressed to expel the contents.
Urine cultures were collected daily in metabolic cages overnight. Metabolic cages were used so as to collect larger volumes of urine for measurement of silver release from the coating. However, this way of urine collection may increase the risk of contamination of the urine by faeces and feed which we attempted to mitigate by having multiple comparisons. At the end of 14 days, mice were euthanized with CO2 overdose (method approved by NUS-IACUC), and catheters were collected (if not passed out in urine). Bacterial biofilm was quantified from the collected catheters with a method using vortexing and sonication as reported recently . In addition, bacterial colonization in the urinary bladder was measured. Each bladder was cut in two halves – one for homogenization and bacterial count, the other for histopathology studies.
Porcine model of CAUTI
Female miniature pigs (PWG Micropig®) from Prestige BioResearch Pte Ltd., Singapore (PBR) were used for this study. The work was carried out at PBR in accordance with approval by PBR-IACUC. During acclimation, animals were group housed. After the Foley catheter implantation, the animals were individually housed in well labelled steel cages. A standard nutrient and micro-ingredient composition of laboratory animal pig diet ration (pellets - 400 g, Altromin 9029, Altromin Spezialfutter GmbH & Co.KG, Germany) was available to each animal daily. Ultraviolet-irradiated municipal tap water was provided ad libitum to the animals. The animal room environment was controlled: a temperature of 16–27 °C, humidity of 50–80% and approximately 12 h of the light/dark cycle with 150–300 lx.
The study was conducted in compliance with the principles specified in national and international regulatory authority test guideline: ISO 10993:2006 “Biological Evaluation of Medical Devices – Part 11: Tests for Systemic Toxicity”. This guideline largely deals with standardization of biological and clinical methods for evaluation of medical devices .
This proof of concept experiment comprised of two animals – one implanted with P3 coated catheter and the other animal with the commercially available silver coated Dover™ catheter for comparison. Implantation of the catheters was performed aseptically under general anaesthesia induced by isoflurane inhalation. The Foley catheter was inserted into the urinary bladder with long surgical forceps via the transurethral route with the animal in dorsal recumbency. The balloon was inflated with 5–6 ml of sterile distilled water according to the manufacturer’s instructions. Subsequently, the inner lumen and outer surface were inoculated with 20 μL each of E. coli (105 CFU/ml) in 1X PBS at the outlet. After waiting approximately 10 min for bacteria to attach to the catheter surface, the external part of the catheter was tied to the tail of the pig with suitable adhesives.
Initially a urine reservoir bag was attached to the catheter at all times to maintain a closed system of urine collection as is the case in humans. We were not successful in securing the bag to the animal and observed that the catheter was pulled out or dropped out due to additional pressure from the bag. Therefore, subsequently, this was modified to an open catheter system. The outlet of the Foley catheter was anchored via suturing to skin in a dependent position and the urine bag was used only for urine sample collection.
Animals were euthanized at the endpoint (20 days post inoculation (dpi) for P3 catheter and 21 dpi for Dover™ catheter) by exsanguination of the vena cava and thoracic vein/artery under deep barbiturate anaesthesia (pentobarbitone sodium) as approved by PBR-IACUC. Catheters and organs of urinary tract were collected for evaluation of bacterial colonization and encrustation.
Assays used for analysis
In mice, efficacy of the coating was analysed using bacterial quantification in urine, on catheters and in tissues, scanning electron microscopy (SEM) and by measuring IL-6 levels in urine. Moreover, safety of the coating was assessed by monitoring body weight, kidney function tests and histopathology examination. Additionally, in the porcine model, encrustation was qualitatively examined visually as well as quantified using ICP-OES to measure calcium and magnesium deposition on catheters and in tissues.
Bacteria in urine and from catheter or tissue samples were counted using Miles and Misra method . SEM was performed as published elsewhere  with some modifications and visualised using JEOL JSM-6701F Field Emission Scanning Electron Microscope. IL-6 levels were measured by ELISA to assess the degree of inflammation (Mouse IL-6, Cat# 88–7064-88, eBioscience Inc., USA).
Results and discussion
Euthanasia and harvesting
Out of 8 mice implanted with uncoated catheters, two mice were terminated early in the experiment due to bladder outlet obstruction as there was no urine output. An enlarged bladder was observed during post-mortem examination (Additional file 1: Figure S1). Catheters and bladders were collected and processed to assess bacterial colonization. One mouse was terminated 10dpi due to weight loss more than 25% and catheter and organs were collected and processed. The remaining 5 mice were euthanized at the endpoint (14 dpi). All 8 uncoated catheters were recovered.
Among the 8 mice implanted with P3 coated catheters, all mice survived till the endpoint. However, two catheters were lost, possibly, passed with urine from the bladder, hence only 6 catheters were recovered.
Dover™ catheter in the pig was collected after euthanizing the animal 21 dpi. The P3 catheter did drop out once at two dpi, which was replaced with a new catheter and reinoculated with E. coli and the catheter was collected at the endpoint for the animal (20 dpi). It should be noted the pig was implanted with the P3 catheter for 23 days in total.
Anti-biofilm activity of P3 coating
Catheter recovery, bladder harvesting and E. coli colonization
Up to 14dpia
Number of mice euthanized (total 8 mice per group)
Catheters collected (catheters lost)
Number of catheters processed for biofilm extraction assay (catheters processed for SEMb)
Number of catheters with viable biofilm
Median values of CFUc/catheter (range)
6.58 X 107 (0 to 1.52 X 108)
0 (0 to 1.18 X 108)
Number of urinary bladders collected with catheters
Number of bladders with viable bacteria
Median values of CFU/bladder (range)
5.0 X 102 (0 to 1.4 X 108)
0 (0 to 1.03 X 105)
The human catheter in the porcine model also showed promising results as average bacterial counts from the catheter collected from the pig with the P3 catheter (20 dpi) were 2.4 times lower than that on the silver coated Dover™ catheter (21 dpi). Biofilm extracted from three segments near tip of the catheters recovered from both animals yielded mostly E. coli colonies whereas two segments near the urethral exit yielded a polymicrobial biofilm.
Although an uncoated catheter would be an appropriate control to demonstrate efficacy in the pig model, the aim of this study was to develop a catheter which could be marketed for clinical use. However, by comparing the efficacy of our coating to a clinically tested silver coating which is licensed by the major regulatory agencies and in clinical use globally, the results of this proof-of-concept study would be more relevant as various reviews and meta-analyses have demonstrated ability of silver to reduce CAUTI [26, 27, 28]. We also do recognise that the proprietary nature of the commercial control catheter limits our ability to compare the details of the different coatings.
These results suggest that the P3 coating could inhibit E. coli biofilm formation both on catheter surface and in the bladder effectively in mice and in pigs.
Antibacterial activity in mice
The higher values of IL6 in uncoated group (Additional file 1: Figure S2) indicating more inflammation as compared to P3 group supporting the finding of low activity of pathogenic bacteria in P3 group mice compared with controls.
Reduction of encrustation in pigs
There were 1.8 times less calcium and magnesium precipitates in the bladder of the pigs with P3 coated catheter as compared to the silver coated Dover™ catheter (Additional file 1: Figure S3).
All mice lost up to 18% body weight in the first two dpi but recovered well 3rd day onwards (Additional file 1: Figure S4). Mice with the P3 coated catheter show slightly lower (~ 5%) weight loss as compared to mice with uncoated catheters indicating that silver does not appear to cause any general harm to the animals over a period of two weeks.
Silver levels could not be measured in urine of mice with P3 catheters due to undetectable (low) levels.
In this study, we have demonstrated, using a murine model and a novel porcine model the safety, and antimicrobial effect of the P3 coating. The results also suggest an anti-encrustation capability of P3 coating. In the porcine model, using a proof of concept with a single animal, we demonstrated the potential efficacy of P3 coating in comparison with the commercial Dover™ silver coating. The P3 coated catheter was safe for use as indicated by the absence of systemic clinical and laboratory signs in both animal models. This suggests low toxicity from the catheter coating material in line with ISO 10993–11:2006.
It should be noted that due to the small group size in the study and the limitations of the current pig model (e.g. difficulty in retaining the catheter), more extensive evaluations of the efficacy and safety of the P3 coated catheter are needed before proceeding for regulatory approval, human studies and widespread clinical use of the catheter.
This work was partly presented at Urofair 2017, Singapore (Oral presentation), at the 15th International Congress of Bacteriology and Applied Microbiology 2017, Singapore (Poster presentation) and at the 33rd Annual European Association of Urology Congress 2018, Copenhagen, Denmark (Moderated poster presentation). Abstracts were published in BJU International 119 (S4); 42, and in European Urology Supplements 17 (2); e481.
This work was supported by Technology Enterprise Commercialisation Scheme-Proof-Of-Concept grant of SPRING Singapore (TI/TECS/POC/14/10). The funding organization played a role in design of the study.
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
KGN, EC and PAT designed and supervised the experiments. KDM, RW and JNR performed the experiments and analysed the results. KDM and RW drafted the manuscript. KGN, EC and PAT critically reviewed the manuscript. All authors have read and approved the final manuscript.
All mouse experiments were conducted in accordance with the university ethical regulations and were approved by the National University of Singapore Institutional Animal Care and Use Committee (R14–0601). All pig experiments were conducted as approved by Prestige BioResearch Pte Ltd. Institutional Animal Care and Use Committee, Singapore (NP17027).
Consent for publication
Paul A Tambyah has received research grants from Sanofi-Pasteur, Fabentech and GSK and honoraria from Johnson and Johnson. All other authors declare that they have no competing interest.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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